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Nat Commun
Nat Commun
Nature Communications
2041-1723
Nature Publishing Group UK London

39294185
52542
10.1038/s41467-024-52542-9
Article
Timely lagging strand maturation relies on Ubp10 deubiquitylase-mediated PCNA dissociation from replicating chromatin
http://orcid.org/0009-0002-4013-1593
Zamarreño Javier 12
http://orcid.org/0000-0001-5512-2468
Muñoz Sofía 124
http://orcid.org/0000-0002-6326-5589
Alonso-Rodríguez Esmeralda 12
http://orcid.org/0009-0001-2702-9239
Alcalá Macarena 12
http://orcid.org/0009-0002-2762-6348
Rodríguez Sergio 12
http://orcid.org/0000-0002-2692-7045
Bermejo Rodrigo 3
http://orcid.org/0000-0003-2314-8430
Sacristán María P. msacristan@usal.es

12
http://orcid.org/0000-0002-0362-4391
Bueno Avelino abn@usal.es

12
1 https://ror.org/02f40zc51 grid.11762.33 0000 0001 2180 1817 Instituto de Biología Molecular y Celular del Cáncer (IBMCC), Universidad de Salamanca-CSIC, Campus Miguel de Unamuno, Salamanca, Spain
2 https://ror.org/02f40zc51 grid.11762.33 0000 0001 2180 1817 Departamento de Microbiología y Genética, Universidad de Salamanca, Campus Miguel de Unamuno, Salamanca, Spain
3 https://ror.org/04advdf21 grid.418281.6 0000 0004 1794 0752 Centro de Investigaciones Biológicas “Margarita Salas”, CSIC, Madrid, Spain
4 grid.11762.33 0000 0001 2180 1817 Present Address: Instituto de Biología Funcional y Genómica (IBFG), CSIC-Universidad de Salamanca, Salamanca, Spain
18 9 2024
18 9 2024
2024
15 81835 1 2024
11 9 2024
© The Author(s) 2024
2024
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Synthesis and maturation of Okazaki Fragments is an incessant and highly efficient metabolic process completing the synthesis of the lagging strands at replication forks during S phase. Accurate Okazaki fragment maturation (OFM) is crucial to maintain genome integrity and, therefore, cell survival in all living organisms. In eukaryotes, OFM involves the consecutive action of DNA polymerase Pol ∂, 5’ Flap endonuclease Fen1 and DNA ligase I, and constitutes the best example of a sequential process coordinated by the sliding clamp PCNA. For OFM to occur efficiently, cooperation of these enzymes with PCNA must be highly regulated. Here, we present evidence of a role for the K164-PCNA-deubiquitylase Ubp10 in the maturation of Okazaki fragments in the budding yeast Saccharomyces cerevisiae. We show that Ubp10 associates with lagging-strand DNA synthesis machineries on replicating chromatin to ensure timely ligation of Okazaki fragments by promoting PCNA dissociation from chromatin requiring lysine 164 deubiquitylation.

Synthesis and maturation of Okazaki fragments is crucial for lagging strand DNA replication. Here the authors find that the ubiquitin-specific protease Ubp10 works with the DNA synthesis machinery to promote Okazaki fragments joining by removal of PCNA through deubiquitylation of PCNA-K164.

Subject terms

DNA synthesis
DNA metabolism
Spanish Ministry of Science Grant reference number PID2019-109616GB-100 Junta de Castilla y León Grant reference number SA103P20Spanish Ministry of Science Grant reference number PID2020-116003GB-100issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

The POL30 gene of Saccharomyces cerevisiae encodes the sliding clamp PCNA (Proliferating Cell Nuclear Antigen), a conserved ring-shaped protein with essential roles in DNA metabolism as a crucial component of replication and repair machineries. PCNA forms an homotrimer that encircles DNA, where it interacts with a staggering number of proteins involved in every step required for DNA replication or repair. Thus, whereas it is devoid of enzymatic activity itself, PCNA exerts its function by recruiting and, in many instances, also activating numerous interactors1,2.

DNA polymerases require free 3ʹ-OH groups to initiate DNA synthesis, therefore, they can only synthesize DNA in a 5ʹ−3ʹ direction. As a result of this and the antiparallel nature of double stranded DNA, replication of one of the strands is discontinuous through the generation of Okazaki Fragments (OF). This strand is called the lagging strand (in contrast to the leading one). PCNA plays a crucial role in the synthesis and maturation of these OFs. Thus, PCNA is loaded on dsDNA at primer-template junctions to recruit Pol δ and enhance its processivity. When the Pol δ-PCNA complex collides with the 5ʹ-end of the preceding OF, it displaces a short flap that is cleaved off by the structure-specific flap endonuclease-1 Fen1, Rad27 in S. cerevisiae, upon binding to PCNA. This process generates a nick in the nascent DNA that is sealed by DNA ligase I, Cdc9 in S. cerevisiae, which is also recruited and catalytically activated through its interaction with PCNA3. All three subunits of DNA Pol δ, Fen1/Rad27, and DNA ligase I/Cdc9 harbor PIP (PCNA-interacting peptide)-boxes, through which they interact in a coordinated manner with the Inter-Domain Connecting Loop (IDCL) of PCNA, a major interaction site in the sliding clamp4–6. Although the mechanisms and factors involved in lagging strand maturation have been extensively studied key molecular details of OF maturation remain poorly understood.

To ensure the successful completion of DNA replication, particularly of the processive synthesis of the lagging strands, continuous recycling of chromatin-bound PCNA is required. The sliding clamp is loaded when required and actively unloaded when no longer needed in order to suppress illegitimate enzymatic reactions2,7,8. RFC (Replication factor C) and RFC-like complexes (RLCs) mediate the loading and unloading of PCNA (reviewed in ref. 9). There is a general consensus regarding Rfc1-RFC complex role as the main loader of PCNA on replicating chromatin10,11. One additional complex, Ctf18-RLC, also acts as a PCNA loader, although Ctf18 cannot substitute Rfc1. Moreover, Rfc1 and Ctf18 show a strand preference during replication, with a lagging strand bias for Rfc1 and a slight leading strand preference for Ctf1812–14. Both Rfc1 and Ctf18 also exhibit some ability to unload PCNA in vitro. Human Rfc1-RFC, but not yeast Rfc1, is able to unload PCNA in vitro in an ATP-dependent manner15,16 and yeast Ctf18-RLC promotes an in vitro PCNA unloading mechanism, that also requires ATP hydrolysis, in the presence of ssDNA coated with RPA17. Despite these in vitro results, PCNA unloading activities for Rfc1- or Ctf18- complexes in vivo have not been reported.

Elg1-RLC complex (ATAD5-RLC in mammals) is considered as the major PCNA unloader when the role of PCNA in DNA replication is completed8,18–22. However, given that ELG1 is a non-essential gene for cell division23–25 and that PCNA accumulated in Elg1-depleted cells ends up being removed from chromatin before M phase18,20, it is likely that additional PCNA unloaders are required during chromosome replication, at least in the absence of Elg1. Therefore, it remains unclear whether Elg1-RLC is the only in vivo PCNA unloader22,26.

PCNA functions are regulated by different post-translational modifications such as SUMOylation, ubiquitylation, phosphorylation or acetylation, which confer PCNA the necessary plasticity to interact with its different binding partners1,27,28. In the face of DNA lesions, PCNA is ubiquitylated to mediate damage-tolerance mechanisms that allow circumventing DNA lesions and prevent replication fork stalling29,30. PCNA is mono-ubiquitylated at K164 by the evolutionary conserved RAD6/RAD18 (E2/E3) ubiquitin ligase complex to switch its affinity from replicative polymerases to damage-tolerant translesion synthesis (TLS) DNA polymerases, which, although mutagenic, are capable to bypass damaged bases27,31,32. Furthermore, polyubiquitylation of the same residue by the Rad5/Mms2/Ubc13 PCNA-ubiquitin ligase complex leads to template switching (TS), the DNA damage tolerance (DDT) error-free pathway, to overcome the potentially lethal effects of replication fork stalling27,30. We and others reported that the precise regulation of these processes not only depends on writer enzymes, PCNAK164-Ubiquitin ligase complexes, but also on the erasers of these modifications. Thus, both TLS and TS pathways are limited by PCNA-deubiquitylation processes to minimize their deleterious cellular side effects. In mammals, deubiquitylating enzymes Usp1, Usp7, and Usp10 revert PCNA ubiquitylation caused in response to DNA damage28,33–35. Knockdown of USP1 induces aberrant PCNA monoubiquitylation, enhanced recruitment of error-prone TLS polymerases and increased mutagenesis levels in human cells33,35. In the case of the budding yeast S. cerevisiae, the PCNA-DUBs Ubp10 and Ubp1236,37 limit the extent of DDT processes during the progression of exogenously unperturbed S phase by reverting K164-ubiquitylation of the sliding clamp at replication forks37. Ubp10 ubiquitin-protease has also a role deubiquitylating histone H2BK123 working at genomics sites distinct of the SAGA-related histone H2BK123 DUB Ubp8 sites38–40. Moreover, Ubp10 activity is also involved in the regulation of RNA polymerase I stability41. Remarkably, despite the key roles of this PCNA-DUB, abrogation of the UBP10 gene is viable, even though ubp10 mutated cells have both growth and cell cycle progression defects36,37,41,42. Suppression of either growth or cell cycle defects can be accomplished by mutation of specific targets. Thus, multiple deletion of the TLS polymerases (REV1, REV3 and RAD30) rescues the cell cycle delay in S phase progression caused by the abrogation of Ubp1037, indicating that the role of Ubp10 in supporting normal replication rates through PCNA-K164 deubiquitylation is dependent, at least in part, on the TLS pathway.

A number of active roles in the regulation of key cellular mechanisms has been described for ubiquitin-signaling writers, but not so many for erasers. Along this line of thought, it is assumed that pivotal regulatory steps rest on ubiquitin writers while partly redundant erasers were considered to act automatically or spontaneously after the post-translational modification is added to a given substrate. For these reasons ubiquitin proteases were in general considered to play a minor role, if any, in regulatory controls. In this context, it is assumed that PCNA ubiquitylation is counteracted by constitutive deubiquitylation mediated by PCNA-DUBs43,44. However, recent work with yeast models concerning PCNA-DUBs role in DNA replication shows that something is amiss with this scenario both in fission and budding yeast37,43. Regarding this matter, Ubp10 has a remarkable slow S phase phenotype that we were very interested to understand in full.

PCNAK164 ubiquitylation has also been linked to the OFM process. Thus, loss of PCNA ubiquitylation seems to cause inefficient gap-filling which interferes with efficient OF ligation in fission yeast and human cells44,45. Moreover, PCNAK164 ubiquitylation suppresses replication stress resulting from Fen1/Rad27-defective flap processing during OFM46, and the PCNAK164R mutant shows inefficient OF processing in an in vitro DNA replication reconstitution assay using yeast proteins47. Here, we present evidence of the role of Ubp10 in the regulation of OFM. Mass spectrometry analysis of the Ubp10 interactome showed that this PCNA-DUB interacts with all major components of the OF metabolism. Based on this observation and on the cell cycle delay in S phase, characteristic of cells deficient in Ubp10, we focused on deciphering the potential link between Ubp10 and OFM processes. Ablation of Ubp10 leads to accumulation of unligated OFs and a markedly increase of chromatin-bound PCNA during S phase. POL30 mutants that conform unstable PCNA homotrimers on chromatin, particularly pol30R14E and pol30D150E alleles, counteract these ubp10∆-associated replication defects, as well as cell cycle delay. In addition, abrogation of ubp10 is strongly additive to elg1 depletion, resulting in substantial increase of the PCNA bound to chromatin during replication. Slow S-phase and PCNA accretion on chromatin in ubp10 cells is suppressed by non-ubiquitylatable K164 PCNA alleles. These data indicate that timely dissociation of PCNA during lagging strand synthesis requires action of the Ubp10 DUB to promote PCNA unloading. Collectively, this evidence reveals an important regulatory role for Ubp10 at the lagging strand synthesis.

Results

PCNA-DUB Ubp10 physically interacts with core components of the lagging-strand synthesis machinery

We first analyzed the interactome of the H2B- and PCNA-ubiquitin protease Ubp10 to reveal partners of this DUB during DNA replication by LC-MS-MS analysis. Mass spectrometry analysis of the proteome associated with Ubp10 in unperturbed S phase cells retrieved all major components of the lagging strand synthesis machinery (such as PCNA, Polα/primase, RFC replication clamp loader, DNA polymerase ∂) and Okazaki fragment metabolism (Fen1 flap endonuclease, Cdc9 ligase, RNase H2) (Table 1). A complementary proteomic analysis of DNA ligase Cdc9 also revealed Ubp10 as one of the DNA ligase interactors (to be published elsewhere). Relevant interactions were confirmed by direct co-immunoprecipitation (ChIP-CoIP) analyzes in epitope-tagged backgrounds, as shown for Pol3, catalytic subunit of Polδ, (Supplementary Fig. 1A), and Cdc9 (Supplementary Fig. 1B). Ubp10-PCNA and Ubp10-Fen1 ChIP-CoIP interactions have been described previously36,37. This evidence suggests the existence of a functional link between Polδ, Fen1, Cdc9, Ubp10 and PCNA, and, therefore, supports the hypothesis that Ubp10 works on the lagging strand during OFM. Likely significant, our Ubp10-GFP-trap proteomic approach did not detect RLC cofactor Elg1. In contrast, all Rfc1-RFC subunits were identified as Ubp10 unperturbed S phase interactors. Of interest, FACT subunits (Spt16 and Pob3) and RNA pol I subunits (Rpa190, 34, 43, 49 and 135), comprising known functional interactors of Ubp10 Spt16 and Rpa19041,48, were identified with high scores in our proteomic analyzes validating our experimental approach (Supplementary Fig. 1c).Table 1 PCNA-DUB Ubp10 interacts with core components of lagging-strand synthesis machinery

Selected Identified interactors	Gene names	Score	Unique peptides	
DNA primase small subunit	PRI1	12	5	
DNA primase large subunit	PRI2	21	4	
DNA polymerase α	POL1	26	6	
DNA polymerase α subunit	POL12	32	9	
DNA pol α-binding protein	CTF4	39	8	
Replication factor C subunit 1	RFC1	38	10	
Replication factor C subunit 2	RFC2	111	13	
Replication factor C subunit 3	RFC3	118	12	
Replication factor C subunit 4	RFC4	85	10	
Replication factor C subunit 5	RFC5	70	13	
PCNA	POL30	76	10	
DNA polymerase δ	POL3	32	9	
DNA polymerase δ subunit	POL31	23	5	
FLAP endonuclease I	FEN1/RAD27	34	8	
DNA ligase I	CDC9	22	7	
Replication factor A protein 1	RFA1	98	12	
Replication factor A protein 2	RFA2	38	5	
Replication factor A protein 3	RFA3	61	3	
Ribonuclease H2 subunit A	RNH201	22	6	
Ribonuclease H2 subunit B	RNH202	54	9	
DNA mismatch repair protein	MSH2	34	10	
DNA mismatch repair protein	MLH1	12	3	
Ubiquitin hydrolase 10	UBP10	323	35	

The PCNA-DUB Ubp10 acts downstream of the Fen1Rad27 FLAP-endonuclease during DNA replication

Our Ubp10-GFP-Trap proteomic approach shows that Ubp10 interacts physically with major components of the lagging strand synthesis machinery during S phase. Therefore, looking for a functional support of the proteomic evidence, a molecular analysis was designed to reveal a potential role of Ubp10 in the metabolism of OFs. Initially, we focused in discerning the potential interplay of FEN1 and UBP10 in the OFM pathway. We had recently shown that Ubp10 and the Fen1 Flap-endonuclease physically interact in early S phase, as detected by co-immunoprecipitation37. To further understand the functional relevance of this observation, we studied genetic interactions occurring in strains ablated for both replication proteins. We first monitored bulk DNA replication in synchronized cell cultures and observed that depletion of Fen1 suppressed the replication defect characteristic of ubp10 mutants, with ubp10 fen1 cells showing replication dynamics virtually indistinguishable from that of fen1 single mutants (Fig. 1a). We then tested fork transitions upon replication stress induction by dNTP pool depletion, in particular by examining the accumulation of anomalous small Ys in ubp10∆ cells37. Small Y-shaped replication intermediates are pathological molecules that accumulate as a consequence of non-canonical transitions from bubbles to large Ys during fork progression upon hydroxyurea (HU)-induced nucleotide depletion in Ubp10 ablated cells. In contrast to single UBP10 mutants, we found that replication intermediates accumulating in ubp10 fen1 double mutants are very similar to those of fen1 single mutant cells and clearly differ from those of ubp10∆ cells, lacking the characteristic small Y accumulation (Fig. 1b), indicating that FEN1 deletion prevents anomalous nascent strand transitions at stalled forks caused by Ubp10 absence. The functional nature of the genetic interaction was further confirmed by testing thermosensitivity and resistance to chronic exposure to HU, where we found that fen1∆ ubp10∆ cells phenocopy single fen1∆ mutants (Fig. 1c), indicating that Ubp10 and Fen1 support viability through a single genetically-related pathway and that Fen1 underlies ubp10∆ sensitivity to replication stress. Finally, chromatin fractionation assays in cells synchronously traversing S phase failed to show differences in Fen1 protein accumulation on chromatin in wild-type and ubp10 defective cells (Fig. 1d), suggesting that Ubp10 does not markedly influence Fen1 chromatin association during OFM. Taken together, these analyzes suggested that absence of the Fen1 endonuclease, required for Okazaki fragment flap-processing, bypasses a yet undefined replication related Ubp10-dependent function.Fig. 1 Flap-endonuclease FEN1/RAD27 is epistatic to PCNA-DUB UBP10 in the Okazaki fragment maturation pathway.

a S phase progression analysis of cells depleted for Fen1 and Ubp10. Wild-type (wt), ubp10∆, fen1∆ and fen1∆ ubp10∆ exponentially growing were synchronized in G1 with α-factor and release in fresh (rich) media. DNA content by FACS analysis of samples taken at indicated intervals is shown. Red arrows indicate approximated duration of bulk DNA replication for each strain. Note that fen1∆ ubp10∆ cells behave like single fen1∆ mutants. b Cells described in a were synchronized with α-Factor and released in the presence of 200 mM HU at 25 °C. Samples were taken after 60 min of treatment and processed for 2D-gel analysis of replication intermediates. Membranes were hybridized consecutively with probes spanning ARS305 and ARS306 replication origins. Note that fen1∆ ubp10∆ cells accumulated X-shaped replication intermediates (black arrows) comparably to fen1∆ single mutant cells and in clear contrast to abnormal small Ys intermediates (red arrows) observed in ubp10∆ cells (described in Álvarez et al. 37). A drawing of the normal replication intermediates and of the abnormal intermediates related to DNA replication fork collapse is shown. c Ten-fold dilutions of the strains indicated in a incubated in YPAD at 25 °C or 35 °C in the absence (unperturbed) or in the chronic presence of HU (25 mM or 50 mM as indicated). Data presented in a–c indicates that FEN1 is epistatic to UBP10. d S phase chromatin association of Fen1 in wild-type and ubp10∆ cells expressing Fen1-Flag tagged protein. Exponentially growing cultures of wild-type and ubp10∆ cells were synchronized with α-factor and released in fresh media to test S phase chromatin association of Flap-endonuclease Fen1/Rad27. Samples were taken at indicated intervals; chromatin-enriched fractions were prepared and electrophoresed in SDS-PAGE gels. Blots were incubated with α-Flag (to detect Fen1-Flag) or α-H2B antibodies. Blots from a representative experiment are shown. Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (p = 0.3250, two-way ANOVA test). This evidence suggests that chromatin association of Fen1 is not affected by depletion of Ubp10. Source data are provided as a Source Data file.

Ubp10 promotes Okazaki fragment ligation

In order to explore the role of the Ubp10 in lagging strand synthesis, we assayed chromatin binding of key OFM proteins during unperturbed S phase upon Ubp10 depletion. We show a control assay displaying a chromatin fractionation experiment in full (Supplementary Fig. 2), which allows analyzing the partitioning of proteins of interest into chromatin-free and chromatin-bound fractions. For clarity, we only show chromatin-bound fractions in further fractionation assays (including Fig. 1d), unless otherwise stated. PCNA is an abundant protein, detectable throughout the cell cycle, that temporarily associates to chromatin during S phase, in discrete but measurable amounts, in cycles of loading and unloading as the process of DNA synthesis requires1,8,26. Indeed, we observed that PCNA is readily detectable in WCE and chromatin-free fractions, and to a lesser extent on chromatin-bound fractions of cells synchronously undergoing genome replication (Supplementary Fig. 2). Therefore, during replication, a major fraction of PCNA is detectable in soluble forms while a small subset associates to chromatin with a quite reproducible periodicity.

In S. cerevisiae, CDC9 encodes the DNA ligase I, an essential enzyme acting downstream of Fen1 to seal Okazaki fragments during DNA replication49,50. Cdc9 has a human homolog, LIG1, also regulated by PCNA during the sealing of nicked DNA at lagging strands51. Remarkably, human LIG1 complements yeast cdc9 temperature-sensitive mutants at the restrictive temperature52. Having observed that in budding yeast PCNA-DUB Ubp10 physically interacts with Cdc9 during S phase, we were interested in understanding a possible functional interaction among them.

We studied genetic interactions of UBP10 with a conditional allele of the DNA ligase I by testing the viability of cdc9ts and ubp10Δ double mutant cells. In our study we used a cdc9-7 allele (from the National BioResource Project, NBRP Japan), a W303 derivative strain that we characterized and sequenced to find that it is synonymous to the cdc9-1 allele53. We found that depletion of Ubp10 aggravates dramatically the thermosensitivity of strains carrying this CDC9 temperature-sensitive mutant allele (Fig. 2a). Impaired ligation of Okazaki fragments leads to the accumulation of PCNA on chromatin as a consequence of poor PCNA unloading in the absence of replicative DNA ligase I Cdc921. We observed - by chromatin fractionation assays - that cdc9-7 cells transiently accumulated PCNA on chromatin when compared to wild-type cells during S phase even under permissive conditions (25 °C) (Fig. 2b). Similarly, under these conditions, ubp10∆ single mutants retained more, and for a longer time, PCNA on chromatin than wild-type cells (Fig. 2b). Furthermore, abrogation of UBP10 function in the cdc9-7 ts mutant cells lead to a significant accumulation of PCNA on chromatin throughout the time-course experiment (Fig. 2b). These observations indicate that the cdc9-7 allele, likely defective in nick ligation at 25 °C, impairs PCNA unloading, which is largely aggravated by Ubp10 ablation. In fact, the cdc9-7 mutation did not rescue the characteristic slow bulk DNA replication phenotype of ubp10Δ mutant, but instead further slowed down progression through S phase (Supplementary Fig. 3a, b). We also observed - by 2D-gel analysis of HU-treated cells - that deletion of UBP10 in cdc9-7 led to accumulation of unusual replication intermediates to a level equivalent to those observed in ubp10Δ single mutants (Supplementary Fig. 3c).Fig. 2 Abrogation of Ubp10 leads to accumulation of unligated Okazaki fragments.

Depletion of Ubp10 increases the thermosensitivity and PCNA accumulation defects of the cdc9-7 ts allele of DNA ligase I. a Ten-fold dilutions of equal number of cells of the indicated strains were spotted in Petri dishes and incubated either at 25 °C, 28 °C, 30 °C, or 35 °C for 60 h. b S phase chromatin association of PCNA in wild-type, ubp10∆, cdc9-7, and cdc9-7 ubp10∆ cells. Exponentially growing cultures of the indicated strains were synchronized in G1 with α-factor pheromone and released in fresh media to test S phase chromatin association of PCNA. Samples were taken at indicated intervals; chromatin-enriched fractions were prepared and electrophoresed in SDS-PAGE gels. Blots were incubated with α-PCNA or α-H2B antibodies. A blot from a representative experiment is shown. Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (wild-type vs ubp10∆ p = 0.0482; wild-type vs cdc9-7 p < 0.0006; wild-type vs cdc9-7 ubp10∆ p < 0.0001; ubp10∆ vs cdc9-7 p = 0.3329; ubp10∆ vs cdc9-7 ubp10∆ p < 0.0001; cdc9-7 vs cdc9-7 ubp10∆ p < 0.0001, two-way ANOVA test). c UBP10 mutants show a strong detectable lagging-strand replication phenotype that leads to the accumulation of unligated Okazaki fragments (OF). Exponentially growing cultures of cdc9-7 and cdc9-7 ubp10∆ cells incubated at 25°C were shifted to 29 °C. Aliquot samples were taken at the indicated intervals. Purified total genomic DNA was labeled with exonuclease-deficient DNA polymerase I (Klenow) fragment and α-32P-dCTP, separated by agarose denaturing electrophoresis, and visualized using a Phosphor Imager. A representative experiment of two biological replicates is shown. The relative amounts of 32P incorporated by end-labeling was quantitated in a Phosphor Imager and plotted. d In vitro analysis of ubp10Δ cumulative OFs reveals ligatable nick DNA. OFs were obtained from cdc9-7 and cdc9-7 ubp10∆ cells as in c. Where indicated (+) genomic DNA was treated in vitro with T4 DNA ligase before labeling with Klenow and α-32P-dCTP for quantifying the proportion of DNA fragments (OF) ready for nick ligation. A representative experiment of three replicates is shown. Values of means of these three replicates ± SD are plotted. Source data are provided as a Source Data file.

Okazaki fragments can be detected in vivo upon DNA ligase I CDC9 depletion or using conditional alleles of the ligase, which result in the accumulation of nicked DNA49,50. To test a potential accumulation of Okazaki fragments in cdc9-7 ubp10Δ cells and, particularly, after having observed that cdc9-7 cells grow poorly at 29 °C when combined with deletion of UBP10, we set up cultures of exponentially growing cells incubated at 25 °C to then shifted them to 29 °C. Okazaki fragments were end-labeled in samples taken at one-h intervals, separated by denaturing agarose gel electrophoresis, transferred to a nitrocellulose membrane, and visualized with a Phosphor Imager. We detected a transitory accumulation of Okazaki fragments in cdc9-7 cells upon sifting from 25 °C to 29 °C degrees cultures of cells growing exponentially. After end-labeling of DNA and denaturing electrophoresis, we observed the characteristic banding pattern of short and heterogenous nicked DNA that results from defects in the ligation at lagging strands50 (Fig. 2c). Remarkably, we found that cdc9-7 ubp10Δ cells accumulated OFs more abundantly than cdc9-7 control cells (Fig. 2c). We did not detect this characteristic banding pattern of Okazaki fragments in nick ligation competent wild-type cells or single ubp10Δ mutants tested (Supplementary Fig. 3d). In essence, all this evidence indicates that depletion of Ubp10 leads to a strong lagging-strand replication defect phenotype, downstream of Fen1 function. However, it is important to understand whether this strong phenotype is the cause or the consequence of the slow progression through S phase that characterizes the genetic depletion of Ubp10.

Cells with a defective Flap-endonuclease Fen1/Rad27 accumulate unprocessed Okazaki fragments with poorly ligatable ends due to the accumulation of flaps with different sizes54. Furthermore, impairment of PCNA unloader Elg1 function leads to the accretion of extended Okazaki fragments likely reflecting defective post-replicative nucleosome reposition55. However, in vivo depletion of Cdc9 DNA ligase I causes the accumulation of nicks that are, therefore, in vitro ligatable with no obvious defects in coupling with chromatin assembly50,54.

To characterize the molecular nature of the Okazaki fragments accumulating in cdc9-7 ubp10Δ double mutants, we examined their size by gel electrophoresis and tested the extent to which the isolated DNA fragments are competent for ligation after purification of total DNA. We observed that cdc9-7 ubp10Δ cells accumulate Okazaki fragments of normal length (Fig. 2c). In addition, these are ligated in vitro by T4 DNA ligase (Fig. 2d) with an efficiency equivalent to DNA ligase-deficient controls, such as Cdc9-deficient cells50,54. These results indicate that OFs accumulating upon Ubp10 ablation have DNA ends equivalent to those accumulated in cdc9ts mutants and suggest that OF accumulation is due to defects in the last steps of lagging strand maturation. The evidence shown so far suggests a role for Ubp10 in the timely maturation of Okazaki fragments, either in the regulation of nick ligation and/or in promoting chromatin disassociation of PCNA.

Ubp10 is required for timely ligase association to replicating chromatin

PCNA accumulates on replicating chromatin in the absence of Cdc9-mediated Okazaki fragment ligation21. We reasoned that the PCNA accretion observed in Cdc9 Ubp10 doubly depleted cells might be the consequence of Cdc9 function defects, due to either impaired DNA ligase activity or to reduced chromatin abundance of Cdc9. Therefore, we next tested a heterologous DNA ligase that can complement Cdc9 depletion56, and found that ubp10Δ cells overexpressing Chlorella virus DNA ligase exhibit a delay in S phase progression similar to that of controls (Supplementary Fig. 4a), strengthening the conclusion that ubp10Δ deficiency is not related with defects in overall DNA ligase activity.

We then monitored Cdc9 chromatin association in synchronously replicating cells and found that Ubp10 depleted cells show transiently reduced ligase levels compared to wild-type cells (Fig. 3), a decrease particularly marked at early time points after G1 release, coinciding with the slow progression of bulk DNA replication and in an inverse correlation with PCNA abundance (Supplementary Fig. 4b,c). Therefore, the accumulation or permanence of PCNA on chromatin in ubp10Δ mutants might be the consequence of a slow recruitment of DNA ligase Cdc9 on chromatin. To further understand whether the poor accretion of Cdc9 on chromatin was the cause or a consequence of the slow S phase phenotype of Ubp10 depleted cells, we tested ubp10Δ cells overexpressing CDC9, by means of a duplicated galactose-inducible allele, and found that high levels of chromatin-bound Cdc9 did not rescue the characteristic cell cycle defect, lengthy S phase, of ubp10 mutants nor rescued the accumulation of PCNA (Supplementary Fig. 4b,c). Therefore, we surmise that Ubp10 may directly regulate PCNA unloading from chromatin, indirectly impairing OF ligation.Fig. 3 Steady reduction of chromatin associated Cdc9 during replication in UBP10 defective cells.

a S phase chromatin association of Cdc9 in wild-type and ubp10∆ cells. Exponentially growing cultures of wild-type and ubp10∆ cells were synchronized with α-factor and released in fresh YPAD media to test S phase chromatin association of DNA ligase I Cdc9. Samples were taken at indicated intervals; chromatin-enriched fractions were prepared and electrophoresed in SDS-PAGE gels. Blots were incubated with α-Ha (to detect Cdc9-Ha), α -PCNA or α-H2B antibodies. A blot from a representative experiment is shown. Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (p = 0.0002, two-way ANOVA test). b Whole cell extract (WCE) aliquots from a were blotted to test Cdc9-Ha protein amounts. Again, a blot from a representative experiment is shown. Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (p = 0.2380, two-way ANOVA test). Source data are provided as a Source Data file.

Increased amounts of ubiquitylated PCNA leads to an increment in chromatin-bound PCNA

We have shown earlier that Ubp12 cooperates with Ubp10 to deubiquitylate PCNA37. Ubp12 depletion does not aggravate ubp10∆-associated slow S phase (ref. 37 and Supplementary Fig. 5a). However, in stark contrast to single mutants, the combined ablation of Ubp10 and Ubp12 accumulates ubiquitylated PCNA both in asynchronous cycling cells37 and during S phase progression (Supplementary Fig. 5b). We then tested the possibility that an increase of ubiquitylated-PCNA in S phase would retain the sliding clamp longer on chromatin and found that, indeed, Ubp10 and Ubp12 depleted cells persistently accumulate chromatin-bound PCNA (Supplementary Fig. 5c), suggesting a link between ubiquitylated PCNA and chromatin retention of the sliding clamp.

Defective deubiquitylation of K164-PCNA underlies the S phase progression defects of Ubp10 depletion

Apart from their roles acting on third substrates, and therefore focusing on nascent DNA, both Ubp10 and Ubp12 limit tolerance events during unperturbed S-phase by reverting ubiquitylation of PCNA at replication forks37,57. Having shown that abrogation of Ubp10 leads to increased levels of chromatin-bound PCNA in S-phase (Fig. 2b) and delays S phase progression (37 and Supplementary Fig. 3a), we tested the hypothesis that deubiquitylation of PCNA-K164 by Ubp10 promotes processive DNA synthesis. For this, we analyzed both phenotypes in ubp10∆ pol30K164R double mutant cells and observed that the non-SUMOylable non-ubiquitylable PCNA variant pol30K164R suppresses the steady PCNA accumulation as well as the slow progression through S phase that characterize Ubp10 depletion (Fig. 4a, b). This evidence implies that, K164-PCNA is the key target of Ubp10 in S phase and suggests that modification of this Lysine on the sliding clamp plays a main role in unperturbed DNA replication.Fig. 4 Processive S phase progression is ensured by dynamic ubiquitylation and deubiquitylation of PCNA Lysine 164.

a S phase chromatin association of PCNA in wild-type, ubp10∆ and ubp10∆ pol30K164R cells. Exponentially growing cultures of wild-type, ubp10∆ and ubp10∆ pol30K164R cells were synchronized in G1 with the α-factor pheromone and released in fresh (glucose-based) media to test S phase chromatin association of PCNA. Samples were taken at indicated intervals; chromatin-enriched fractions were prepared and electrophoresed in SDS-PAGE gels. Blots were incubated with α-PCNA or α-H2B antibodies. A blot from a representative experiment is shown. Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (wild-type vs ubp10∆ p = 0.0003; wild-type vs ubp10∆ pol30K164R p = 0.8376; ubp10∆ vs ubp10∆ pol30K164R p = 0.0011, two-way ANOVA test). b The characteristic ubp10∆ slow S phase progression is suppressed by non-ubiquitylable PCNAK164R variant form. DNA content analysis by FACS of wild-type, ubp10∆, and ubp10∆ ubp12∆ cells at the indicated time points (aliquot samples of the experiment in a). c Overexpression of the K164-ubiquitin ligase Rad18 leads to chromatin PCNA accumulation and a slow progression through S phase. S phase chromatin association of PCNA in wild-type, ubp10∆ and GAL1-10:RAD18 (opRAD18) cells. Exponentially growing cultures of the indicated strains incubated in YPAGalactose were synchronized in G1 with the α-factor pheromone and released in fresh (galactose-based) media to test S phase chromatin association of PCNA. Samples were taken at indicated intervals; chromatin-enriched fractions were prepared and electrophoresed in SDS-PAGE gels. Blots were incubated with α-PCNA or α-H2B antibodies. A blot from a representative experiment is shown. Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (wild-type vs ubp10∆ p = 0.0457; wild-type vs opRAD18 p = 0.0096; ubp10∆ vs opRAD18 p = 0.7701, two-way ANOVA test). d DNA content analysis by FACS of wild-type, ubp10∆, and opRAD18 cells at the indicated time points (aliquot samples of the experiment in c). Source data are provided as a Source Data file.

As pointed out above, Lysine 164 of the sliding clamp PCNA may be either SUMOylated or ubiquitylated27,32. To discern the importance of SUMO and Ubiquitin modifications, we tested whether overexpression of the Rad18 K164-ubiquitin ligase would mirror the S phase defects conferred by Ubp10 depletion. Indeed, we found that high-levels of Rad18 (opRad18) lead to a slow progression through S phase and a concurrent accumulation of chromatin-bound PCNA equivalent to that observed in ubp10 cells (Fig. 4c, d). By testing the overexpression of Rad18 in a pol30K164R variant background, we also found that both phenotypes depend on ubiquitylation of the K164 residue of PCNA as all opRad18 effects were suppressed by the K164R point mutation (Supplementary Fig. 6), in consistence with the previous observation (Fig. 4a, b). All this evidence indicates that dynamic ubiquitylation and deubiquitylation of PCNA at Lysine 164 takes place during unperturbed S phase and plays an important role ensuring processive lagging strand synthesis in yeast.

In the course of these experiments, we noticed that pol30K164R mutants of PCNA show reduced accumulation of chromatin-bound PCNA as compared to wild-type cells, somehow suggesting that modification (SUMOylation or ubiquitylation) of this residue may increase the stability of the sliding clamp on chromatin. This pol30K164R-associated phenotype is more evident in synchronous S phase (Supplementary Fig. 6) and, particularly, when synchronized cultures are incubated in galactose-based media, circumstance where yeast cells slow progression through S phase (Supplementary Fig. 6). A reduced stability of the sliding clamp in replicating DNA may explain the faulty processing of Okazaki fragments detected in vitro in DNA replication assays based on the use of yeast PCNA-K164R47.

PCNA disassembly-prone mutants pol30R14E and pol30D150E revert ubp10∆-associated replication defects

We reasoned that the excessive abundance of PCNA on chromatin in Ubp10 depleted cells might be related to defective unloading of the sliding clamp. If ubp10∆ phenotypes are related with the excessive retention of PCNA on chromatin, two predictions can be made. Firstly, cells depleted for Ubp10 should accumulate PCNA on chromatin during S phase. Secondly, a reduction in the amount of PCNA bound to chromatin would rescue ubp10 replication defects. Regarding this second prediction, there are a number of POL30 alleles that conform unstable PCNA homotrimers58. In particular, pol30R14E and pol30D150E alleles are described as PCNA trimer-disassembly-prone mutants due to their instability when bound to chromatin21,58. Hence, if the reason underlaying ubp10 defects is related to excessive PCNA on chromatin, the potential suppression by these disassembly-prone PCNA mutants might be easier to observe in a cdc9-7 ubp10∆ background due to the greater thermosensitivity as compared to single cdc9-7 cells. In fact, the growth defect of cdc9-7 ubp10∆ is suppressed by pol30R14E and pol30D150E mutant alleles, while they did not rescue cdc9-7 thermosensitivity (Fig. 5a). Furthermore, these point mutant alleles of PCNA also rescue abnormal replication intermediates accumulated in cdc9-7 ubp10∆ cells arrested in HU (Fig. 5b). In fact, these POL30 alleles rescue all tested Ubp10-depletion associated S phase defects, including those related with bulk genomic DNA replication in a CDC9 wild-type-like strain (Fig. 5c), implying that the slow S phase observed in Ubp10 depleted cells is directly linked to PCNA accumulation on replicating chromatin. Reduced replication intermediate levels in HU-treated UBP10 deleted cells were also efficiently suppressed by either pol30R14E or pol30D150E (Fig. 5d). This evidence links the replication phenotypes caused by Ubp10 ablation to a defective PCNA unloading mechanism during S phase.Fig. 5 PCNA disassembly-prone mutants pol30R14E and pol30D150E revert ubp10∆-associated replication defects.

a pol30R14E and pol30D150E alleles rescue ubp10∆-associated defects in cdc9-7 ubp10∆. Ten-fold dilutions of the cdc9-7 indicated strains incubated in YPAD at different temperatures for 60 h. Note that the increased lethality of cdc9-7 ubp10∆ is suppressed by pol30 mutant alleles that, in turn, do not rescue cdc9-7 thermosensitivity. b 2D-gel analysis of cells synchronized in early S phase with the ribonucleotide reductase inhibitor HU at 29 °C. Indicated cdc9-7 strains were grown to exponential phase at 25 °C, synchronized in G1 with α-factor, released in fresh media with 200 mM HU at 29 °C for 60 additional min. The membrane was hybridized to a probe spanning ARS305 early replication origin. Open red arrow points small Ys intermediates. Under these conditions, cdc9-7 ubp10∆ defects were suppressed by PCNA disassembly-prone mutants. Histogram plots of small/large Y-shaped replication intermediates ratios in cdc9-7, cdc9-7 ubp10∆, cdc9-7 ubp10∆ pol30R14E and cdc9-7 ubp10∆ pol30D150E mutants are shown. c DNA replication progression defects in ubp10∆ cells are abrogated by pol30R14E and pol30D150E alleles. DNA content analysis of wild-type, ubp10∆, pol30R14E, ubp10∆ pol30R14E, pol30D150E and ubp10∆ pol30D150E strains. Cells of the indicated strains (all cdc9td with CDC9 ON) were synchronized with α-factor and released in fresh YPAD at 25 °C. The progression of the bulk genome replication was monitored at the indicated time points. Open red arrows indicate approximate S phase duration in every strain where pol30R14E and pol30D150E suppression of the replication defect of ubp10∆ cells can be observed. d 2D-gel analysis of replication intermediates in cells synchronized in early S phase with HU. Indicated strains grown to exponential phase at 25 °C were pre-synchronized in G1 with α-factor, released in fresh media with 200 mM HU an incubated at the same temperature for one additional hour. Membranes were hybridized to a probe spanning ARS305 early replication origin. Open red arrows indicate small Ys intermediates in blots. Histogram plots of small/large Y-shaped replication intermediates ratios in wild-type and ubp10∆, pol30R14E, ubp10∆ pol30R14E, pol30D150E and ubp10∆ pol30D150E mutants are shown. Source data are provided as a Source Data file.

UBP10 mutants accumulate non-canonical small Y-shaped replication intermediates upon HU-induced fork stalling37. The accumulation of these non-canonical small Ys is characterized by a decrease in large Ys and is abated by mutating RAD5237. We re-examined the accumulation of these small Y-shaped molecules and found that is suppressed in pol30R14E and pol30D150E genetic backgrounds (Fig. 5b, d). These results strongly suggest that small Y-shaped molecules formed downstream of Fen1 function as a direct consequence of PCNA accumulation on replicating chromatin and, also, that these abnormal structures are generated as cells try to repair through a Rad52-dependent TS-like mechanism.

The disassembly-prone-mutant pol30D150E suppresses increased chromatin association of PCNA and OF accumulation in UBP10 defective cells

As mentioned, if ubp10 phenotypes are the consequence of a defective unloading of PCNA, cells depleted for Ubp10 should accumulate the sliding clamp on chromatin during S phase, indeed we have observed a chromatin-bound accretion of PCNA in ubp10∆ cells when testing cdc9-7 mutants and Cdc9 levels (Figs. 2b, 3a, 4a,c, and Supplementary Figs. 4c, 5c). We next re-evaluated how much and for how long the sliding clamp PCNA is bound to chromatin through a synchronized, otherwise unperturbed, S phase in wild-type and ubp10∆ cells. As expected from previous results (Fig. 5c), we found that the slow S phase progression in ubp10∆ cells correlates with an increased PCNA accumulation on chromatin (Fig. 6a, b). In parallel, we also evaluated the disassembly-prone pol30D150E point-mutant ability to suppress this chromatin-bound PCNA accretion phenotype and found that this PCNA mutant rescued chromatin retention of PCNA of ubp10∆ (Fig. 6a, Supplementary Fig. 7), as well as the bulk DNA replication defect (Fig. 5c).Fig. 6 PCNA retention on replicating chromatin underlies Okazaki fragments accumulation in Ubp10 abrogated cells.

a PCNA trimer-disassembly-prone mutant pol30D150E suppresses PCNA retention on chromatin phenotype of Ubp10 depleted cells. Exponentially growing cultures of wild-type, ubp10∆, and pol30D150E ubp10∆ cells were synchronized in G1 with α-factor and released in fresh media to test S phase chromatin association of PCNA. Samples were taken at indicated intervals; chromatin-enriched fractions were prepared and electrophoresed in SDS-PAGE gels. Blots were incubated with α-PCNA or α-H2B antibodies. A blot from a representative experiment is shown. Data in the graph represent the average of three biological replicates (and is expressed as means ± SD in triplicate) (wild-type vs ubp10∆ p < 0.0001; wild-type vs pol30D150E ubp10∆ p < 0.0004; ubp10∆ vs pol30D150E ubp10∆ p < 0.0001, two-way ANOVA test). b Slow S phase progression in PCNA-DUB UBP10 defective cells is a direct consequence of PCNA accumulation on replicating chromatin. DNA content analysis of wild-type, ubp10∆, and pol30D150E ubp10∆ cells at the indicated time points (aliquot samples of the experiment in a). c pol30D150E rescues ubp10∆ Okazaki fragments maturation timing defects. Exponentially growing cultures of cdc9-7, cdc9-7 ubp10∆, cdc9-7 pol30D150E and cdc9-7 pol30D150E ubp10∆ cells incubated at 25°C were shifted to 29°C. Aliquot samples were taken at the indicated one-hour intervals. Purified total genomic DNA was labeled with exonuclease-deficient DNA polymerase I, Klenow fragment and α−32P-dCTP, separated by agarose denaturing electrophoresis, and visualized using a Phosphor Imager. A representative experiment of two biological replicates is shown. Relative 32P incorporation by end-labeling in the Okazaki fragment test was quantitated and plotted. Source data are provided as a Source Data file.

Having shown that PCNA accumulation on replicating chromatin underlies not only the slow S phase progression but also the formation of anomalous small Ys in ubp10 defective cells, we next tested whether the PCNA disassembly-prone pol30D150E allele would be able to rescue the accumulation of unligated Okazaki fragments that can be evidenced in the ubp10 mutant using a cdc9-7 background. It has been shown that pol30R14E and pol30D150E PCNA trimer-disassembly-prone mutants alleviate the Okazaki fragment length extension problem described in elg1Δ cells55, in agreement with PCNA unloading being a key event in maturation of the lagging strand and nucleosome deposition. We predicted that pol30D150E would mitigate the OF accretion of cdc9-7 ubp10∆ cells. To test this hypothesis, exponentially growing cdc9-7, cdc9-7 ubp10∆, cdc9-7 pol30D150E and cdc9-7 pol30D150E ubp10∆ cells incubated at 25 °C were shifted to 29 °C, and samples were taken at one-hour intervals and processed for OF analysis. We observed that the pol30D150E point mutant suppresses ubp10∆ Okazaki fragment maturation timing defect (Fig. 6c), implying that the main problem caused by Ubp10 depletion during lagging strand replication is caused by PCNA retention on chromatin.

Ubp10 as a key regulator of a PCNA unloading mechanism likely distinct from Elg1-RLC

Thus far, our results argue that Ubp10 promotes timely PCNA unloading during lagging strand replication. One reasonable hypothesis is that Ubp10 promotes PCNA deubiquitylation to enhance Elg1-mediated PCNA unloading at the final steps of Okazaki fragment maturation. Fully aware of in vitro evidence showing that human ATAD5Elg1 is able to unload both ubiquitylated and deubiquitylated PCNA forms with similar efficiency59, we reasoned that in vivo, during unperturbed DNA replication, chromatin PCNA unloading would be enhanced by deubiquitylated forms of the sliding clamp in order to proceed smoothly and timely through lagging strand synthesis.

Yeast Elg1ATAD5 is an evolutionary conserved homolog of the replication factor C (RFC) subunit Rfc123,25. It has been shown that while Rfc1-RFC loads PCNA on replicating chromatin, the Elg1-RLC complex has a role in PCNA unloading in a molecular event preceded by Okazaki fragment ligation18,21. Elg1 forms an alternative RFC hetero-pentameric complex with all RFC2-5 subunits of RFC. Significantly, this alternative Elg1-RLC complex is important but not essential for DNA replication24. Elg1 interacts physically with PCNA and Fen1Rad27, has a role in PCNA unloading during Okazaki fragment maturation and is, therefore, important for efficient S phase progression, and likely has a role in proper nucleosome assembly18,25,55.

We have observed that, though Elg1 displays many phenotypes related to chromosome stability, depletion of ELG1 in budding yeast do not cause major replication delays as assayed in synchronous S phase (Supplementary Fig. 8a). In fact, bulk DNA replication timing in ELG1 mutants is equivalent to wild-type replication as tested by FACS DNA content analysis (Supplementary Fig. 8a). Moreover, deletion of ELG1 is viable while deletion of other RFC components is not (in particular RFC1)24,26. However, in 10-fold dilution assays we detected that elg1∆ strains show a poor growth rate particularly at high temperatures (Supplementary Fig. 8b). A defect exacerbated when single elg1∆ mutation is combined with the deletion of Ubp10 (ubp10∆ elg1∆) at any tested temperature, as compared to single mutants or wild-type cells (Supplementary Fig. 8b). This semi-lethality is indicative of a genetic interaction suggestive of a role for both factors in a common event, likely PCNA unloading.

To test chromatin-bound PCNA levels throughout S phase in wild-type, double mutant elg1∆ ubp10∆, and single mutants elg1∆ and ubp10∆, mid-log phase cultures of the indicated strains were synchronized in G1 with α-factor and released in fresh media. As in previous experiments, samples were taken at indicated time points and processed for chromatin pellet assays and DNA content analysis (Fig. 7). We initially expected elg1∆ ubp10∆ double mutants to behave like elg1∆ singles in accordance with the hypothesis that Ubp10 might regulate PCNA unloading through Elg1. Unexpectedly, we found that ubp10Δ and elg1Δ, when combined, are additive regarding PCNA accumulation on replicating chromatin (Fig. 7a, b), suggesting the existence of two parallel pathways of PCNA unloading. The simplest explanation for these observations is the existence of a PCNA chromatin disassociation mechanism regulated by Ubp10 separable from the Elg1ATAD5-dependent unloading.Fig. 7 Ubp10 is a key regulator of a PCNA unloading mechanism likely distinct from Elg1-RLC.

a Abrogation of Ubp10 is strongly additive to Elg1 depletion resulting in substantial increase of PCNA bound to chromatin during replication. S phase chromatin association of PCNA in wild-type, elg1∆, ubp10∆ and elg1∆ ubp10∆ cells. Exponentially growing cultures of the indicated strains were synchronized in G1 with α-factor and released in fresh media to test S phase chromatin association of PCNA and histone H2B. Samples were taken at indicated intervals; chromatin-enriched fractions were prepared and electrophoresed in SDS-PAGE gels. Blots were incubated with α-PCNA or α-H2B antibodies. Blots from representative experiments are shown. Data in the graphs represent the average of three biological replicates (and are expressed as means ± SD in triplicate) (wild-type vs elg1∆ p < 0.0001; wild-type vs ubp10∆ elg1∆ p < 0.0001; elg1∆ vs ubp10∆ elg1∆ p < 0.0001, two-way ANOVA test). Note that depletion of Elg1 results in transient accumulation of PCNA on chromatin during S phase. b S phase chromatin association of PCNA and histone H2B in ubp10∆, elg1∆ and elg1∆ ubp10∆ cells. The samples correspond to technical replicates of some of the samples shown in a, resolved in the same gel to better compare transient accumulation of PCNA (as well as SUMOylated-, and Ubiquitylated-PCNA modified forms) on replicating chromatin in ubp10∆ and elg1∆ single mutants in the same blot. c S phase progression analysis of wild-type, elg1∆, ubp10∆ and elg1∆ ubp10∆ cells. Progression of bulk genome replication was monitored at the indicated time points by FACS analysis. Source data are provided as a Source Data file.

Even though the proteomic analysis presented in this work revealed no interaction with the RFC cofactor Elg1, we examined whether the ablation of Ubp10 altered the chromatin binding pattern of Elg1 during a synchronized S phase. By chromatin fractionation assays, we found that depletion of the PCNA-DUB does not alter Elg1 interaction with chromatin (Supplementary Fig. 8c). Therefore, we concluded that UBP10 mutants do not deregulate Elg1 interaction with chromatin during S phase. Significantly, cells depleted for both UBP10 and ELG1 do not exacerbate the slow S phase phenotype conferred by ubp10∆ deletion (Fig. 7c), though they accumulate PCNA abundantly, far more than individual mutants (Fig. 7a, b). The fact that depletion of UBP10 alone has a replication progression defect underpins the importance of this Ubp10-dependent PCNA unloading mechanism for lagging strand synthesis.

Discussion

Okazaki fragment maturation is a complex, yet well understood, process in the synthesis of the lagging strand during DNA replication. Here, we unveil a role of the ubiquitin protease Ubp10 in the latest steps of maturation of Okazaki fragments in the model yeast S. cerevisiae. In yeast, the Ubp10 enzyme has been functionally related to RNA polymerase I through the stabilization of the Rpa190 subunit41. Ubp10 also cooperates with the FACT complex in the maturation of nucleosomes48. Furthermore, Ubp10 is involved in the reversal of histone H2BK123 ubiquitylation38–40. Significantly for this report, a key role of this deubiquitylase is to counteract futile bypass events at replication forks acting as a PCNAK164-DUB37. PCNA deubiquitylation is a requirement conserved throughout evolution as evidence for ScUbp10, SpUbp16 and HsUsp1 shows36,43. With this background in mind, the aim of this work was to understand the functional meaning of the interaction of the PCNA-DUB Ubp10 with proteins involved in Okazaki fragment synthesis and maturation, physical interaction described earlier for the Flap endonuclease Fen1Rad27,37. In this study we have presented ample evidence suggesting that this DUB regulates the dissociation of the sliding clamp PCNA from chromatin and that, by doing so, ensures proper maturation of the lagging strand.

One recent observation, Fen1Rad27-Ubp10 binding37, led us to the study of Ubp10’s S phase proteome. The analysis confirmed previous data regarding Ubp10 biology as the RNA polymerase I complex subunits Rpa190, Rpa34, Rpa43, Rpa49 and Rpa135 were among proteins trap with Ubp10. Spt16 and Pob3 FACT subunits were also found to bind Ubp10-GFP. Our approach confirmed Fen1-Ubp10 and PCNA-Ubp10 interactions and identified major components of synthesis and maturation of the lagging strand as feasible interactors of the PCNA-DUB. The proteomic studies were made in crosslinked protein samples from cells synchronized in S phase. We confirmed each observed interaction by individually testing Ubp10 ability to form a complex with each OFM complex component of interest in tagged strains during S phase.

A relevant point for this work is understanding the nature of the cell cycle defect of Ubp10 depleted cells, a defect we believed is poorly understood36,37,41,42. Growth and cell cycle defects are separable37,42. In our studies, we did not observe a G1 delay defect36,37. Further, the timing of entry into S phase is close to that of the wild-type, with equivalent timing in ARSs activation37. ubp10∆ characterization indicates that the defective cell cycle is a consequence of the slowdown in DNA replication progression. We also found here that this defect is based on the accretion of chromatin-bound PCNA during S phase and that, consistent with this evidence, it is efficiently suppressed by PCNA-disassembly-prone pol30R14E and pol30D150E mutant alleles (see below).

Fission yeast cells increase the amount of chromatin-associated PCNA when the K164 of this sliding clamp is ubiquitylated44. Based on their observations, Daigaku and coworkers proposed that in S. pombe an increase in Ub-PCNAK164 works to expand the time for PCNA-Pol δ binding to chromatin to allow the completion of OFs. Our findings in S. cerevisiae are consistent with a scenario where ubiquitylation of PCNA is a DNA retention signal for the sliding clamp at lagging strands in unperturbed replication. Indeed, the analysis of the rescue of the defective S phase parameters of Ubp10 depletion or Rad18 overexpression by pol30K164R, non-ubiquitylable point mutant of PCNA, proves that dynamic ubiquitylation and deubiquitylation of the Lysine 164 of the sliding clamp is a relevant event in PCNA unloading that ensures timely progression through S phase. In a cause-and-effect link (further discussed below), the increase in chromatin-bound PCNA likely slows down progression through S phase in budding yeast. This is consistent with previous evidence in S. pombe cells, where depletion of PCNA-DUBs leads to a cell cycle delay phenotype suppressed by abrogation of the PCNA-ubiquitin-ligase Rhp1843.

HsUsp1 in human cells, SpUbp16 in fission yeast S.pombe, and ScUbp10 in S.cerevisiae are orthologous genes that revert PCNA ubiquitylation33,37,43. Therefore, it can be expected that HsUsp1 and SpUbp16 might perform a similar role during lagging strand synthesis, as described here for ScUbp10, given that PCNA is also dynamically ubiquitylated and deubiquitylated during S phase in humans and fission yeast.

Of particular interest for our work were both the study of the cell cycle defect of Ubp10 depleted cells and the analysis of the genetic interactions that arise from individual mutants in the OFM pathway when combined with ubp10∆. One of these analyses has been made in a Cdc9 defective background. In this analysis, not only we found a semi-synthetic lethality among cdc9-7 and ubp10∆ but also created a tool and found a temperature for the OF accumulation tests. Indeed, cdc9-7 ubp10∆ results suggest that ablation of the PCNA-DUB Ubp10 leads to a strong lagging-strand replication defect phenotype, possibly owing to defects in Okazaki fragment ligation. On the other hand, we also show that the slow S-phase or OF accumulation phenotypes in ubp10∆ are unrelated to Cdc9 presence or activity. Therefore, we surmise that ubp10∆ cells have a genuine defect in the maturation of Okazaki fragments related to PCNA unloading.

We have performed a series of experiments to measure the length and molecular nature of the abundant OF accumulated in UBP10 mutant cells and, in summary, all observations indicate that only abundance is affected. Regarding the length, the detected OF in cdc9-7 ubp10Δ cells displayed the distinctive size periodicity evocative of the nucleosome repeat length and were very similar to those observed for cdc9-7. Moreover, we show here that Ubp10 accumulated OF are in vitro ligatable to the same extent as controls (cdc9-7 ubp10Δ versus cdc9-7), and controls are in accordance with published data54 again indicating that OFs are conventional nicked DNA. Since it has been demonstrated that lagging-strand synthesis in budding yeast is coupled with chromatin assembly on newly synthesized DNA50 we deduce as well that chromatin assembly is not affected in Ubp10 depleted cells.

Smith and Whitehouse have shown that rad9Δ and tof1Δ checkpoint mutants when combined with a cdc9td-degron allele accumulate abundant but normal length Okazaki fragments when Cdc9td is proteolyzed50. This observation is particularly strong, in terms of OF abundance, for tof1Δ mutants and is relevant for our work given the similarity with ubp10Δ data presented here. There is not such a strong accumulation in cdc9td rad9Δ case50. Tof1, named after topoisomerase I-interacting factor, is pertinent to this work because is a subunit of the Csm3-Mrc1-Tof1 replication pausing-mediator complex functionally associated with DNA replication forks60–63. On the other hand, the N-terminus of FACT-subunit Spt16 interacts with Tof1 to ensure chromatin replication in vitro64, supporting the hypothesis that FACT is recruited to replication forks by the Tof1-fork replication complex for parental nucleosomes removal. Given that the FACT complex interacts with Ubp10 likely to integrate transcription and DNA replication with nucleosome assembly48, a complex Tof1-FACT-Ubp10 connection emerges likely involved in robust DNA replication progression.

As mentioned before, an important conclusion here is that ubp10∆ S phase defects do not involve direct regulation of Cdc9 function. However, we observed that ubp10∆ cell cycle deficiency was assumably a consequence of increased residence time of PCNA during S phase and was directly related with a defective unloading of the sliding clamp in Ubp10 depleted cells. Accordingly, disassembly-prone mutant alleles of POL30, pol30R14E and pol30D150E, rescue ubp10∆ mutant defects. A corollary of these two remarkable observations is that during unperturbed S phase PCNA unloading may occur simultaneously with or even before Cdc9-mediated nick ligation and this may be in contrast with evidence published regarding Elg1 in the PCNA unloading subject21. However, this is a preliminary observation, and therefore, further work would be required to understand this conundrum.

Our observations do not exclude a functional interaction between Ubp10 and Cdc9 during OFM, as chromatin-bound Cdc9 levels remain low in Ubp10-ablated cells even when the rest of the cell cycle phenotypes are suppressed in PCNA disassembly-prone mutants. However, our results do not favor the hypothesis that Cdc9 loading defects underlie ubp10∆-associated cell cycle phenotypes, as we have found that deubiquitylation of the Lysine 164 of PCNA is the relevant event in Ubp10-mediated PCNA unloading that ensures timely progression through S phase.

Besides the slowdown in replication progression, the ablation of Ubp10 is characterized by the accumulation of non-canonical replication intermediates in HU-treated cells detected as small Ys by 2D-gel analysis. These intermediates are observed upon Ubp10-DUB depletion, suggesting that they are normally suppressed by PCNA deubiquitylation37. Canonical small Ys reflect passive replication by forks arising outside the probed fragment. We suggest that accumulation of small Ys may also reflect pathological features of lagging strand-associated replication fork defects (nick DNA that may generate breakage structures, following Fen1-mediated flap cleavage). Special consideration should be given to replication intermediates when testing drug-treated cells because fork progression is limited in HU, as elongation of DNA synthesis from ARSs is slow65, thus, all replication intermediates detected under our experimental conditions by 2D gel analysis belong to the closest origin of replication for any given restriction fragment tested. Consistent with the idea that non-canonical/small Ys might be pathological replication structures, both fen1rad27 and cdc9-7 mutants accumulate small Ys (comparable to top1 top2 double mutants66) compatible with increased accumulation of nicked DNA, therefore, fragile molecules.

The suppression of the accumulation of these non-canonical molecules in HU-treated cells by POL30 disassembly-prone mutant alleles, pol30R14E and pol30D150E, combined with the suppression of the slow bulk DNA replication, show that ubp10∆ mutation have an effect throughout the entire yeast genome. All this evidence indicates that this Ubp10 deubiquitylase plays a significant genome-wide role during the S phase of every unperturbed cell cycle.

PCNA trimer-disassembly-prone mutants pol30R14E and pol30D150E alleviate the Okazaki fragment length extension problem described in elg1Δ cells55. Further, combining pol30R14E with elg1Δ largely rescued the elevated mutation rate of the single elg1 mutant58. Although UBP10 mutant cells accumulate OFs, they do not show an elevated mutation rate, in fact their mutation rate is similar to that of wild-type cells36,43. Both pol30D150E and pol30R14E PCNA mutant alleles are excellent extragenic suppressors of UBP10 deletion. It has been described that due to their disassembly-prone nature of the homo-trimeric PCNA ring, both POL30 alleles accumulate low levels of PCNA on replicating chromatin. However, based in our own observations, pol30R14E and pol30D150E point mutant alleles differ between them in the total cellular amount of PCNA levels. In whole cell extracts, PCNAD150E levels mimics those of wild-type cells, while PCNAR14E shows significantly reduced levels as compared to PCNAwt. Thus, PCNAD150E behaves like a real prone-disassembly PCNA mutant. Nevertheless, PCNAR14E mirrors PCNAD150E low levels of PCNA bound to chromatin and, therefore, it is meaningful in our analysis.

In summary, disassembly-prone mutants pol30R14E and pol30D150E rescue the chromatin retention of PCNA phenotype of ubp10Δ (as shown in Fig. 6a), as well as cell cycle delay (Figs. 5c, 6b) and defective replication intermediate accumulation upon exposure to HU (Fig. 5d). Remarkably, the pol30D150E disassembly-prone mutant also rescues Okazaki fragments accumulation observed in DNA ligase I (cdc9ts) when combined with ubp10Δ (Fig. 6c). In other words, all major S phase phenotypes associated with defective Ubp10 (ubp10Δ) are relieved by reversion of accretion of PCNA by two different trimer instability PCNA/POL30 mutants (pol30R14E and pol30D150E)55,58. Together, this evidence indicates that a slow PCNA unloading underlies every replication defect in Ubp10 depleted cells.

The analysis of fen1∆ ubp10∆ mutants revealed the epistatic nature of their genetic interaction, where the loss of Fen1 suppresses the ubp10 phenotype (as evidenced by all tests, including the characteristic accumulation of PCNA on chromatin observed in fen1∆ mutants). This suggests that the flap endonuclease activity of Fen1Rad27 is necessary to generate an Okazaki fragment maturation intermediate upon which Ubp10 acts. In Fen1 depleted cells, the Ubp10-dependent mechanism would not unload the sliding clamp, potentially directing PCNA entirely to the Elg1-dependent pathway, thereby preventing its hyperaccumulation on chromatin. Conversely, Fen1’s activity on Okazaki fragments might commit PCNA for unloading by Ubp10, making it resistant to Elg1, which leads to the accumulation of PCNA on chromatin in FEN1 wild-type ubp10∆ cells.

Another relevant information here is that Elg1-depleted cells transiently accumulate PCNA on chromatin (Fig. 7). This transient nature of PCNA retention on chromatin is, on one hand, consistent with the viability of elg1∆ deleted strains23–25. However, on the other hand, it may also mean that in the absence of Elg1-RLC complex PCNA is steadily unloaded in vivo, as we show here (Fig. 7). Although it does not come as a total surprise, this unanticipated observation is coherent with the fact that Elg1 is not essential for DNA replication23–25. Nonetheless, as reported, Elg1 may be still important for efficient S phase progression or in the presence of replication stress18. However, by testing bulk DNA replication in unperturbed conditions at 25 °C, we observed no S phase delays in Elg1-depleted cells (in similar conditions where we detected a transient accretion of chromatin-bound PCNA). One possibility is that under these experimental circumstances an alternative complex to Elg1-RLC1 is unloading PCNA.

The transient accumulation of chromatin-bound PCNA in elg1∆ cells is, therefore, evocative of the existence of a PCNA unloading mechanism active during replication. We show here that only when elg1∆ is combined with ubp10∆ PCNA remains bound onto chromatin. Interestingly, we detected a robust accumulation and a very slow-paced PCNA unloading in elg1 ubp10 double-mutant cells in synchronized S phase, suggesting the existence of an Ubp10-regulated PCNA chromatin dissociation mechanism divergent from that of Elg1-RLC. Undeniably, while alternative interpretations are possible, all the evidence suggests that a Ubp10-dependent mechanism supports timely removal of PCNA from replicating chromatin during unperturbed S phase. Eventually, PCNA is unloaded in Elg1 and Ubp10 doubly-depleted cells, as chromatin-bound PCNA in G1 synchronized cells remains very low. Perhaps most significantly, S phase progression is slow in this elg1 ubp10 double mutant. The fact that depletion of Ubp10 has a similar slow S phase phenotype may be indicative of a default mechanism of PCNA unloading regulated by this PCNA-DUB in unperturbed cell cycle.

Although we cannot rule out alternative explanations, our results point at the existence of a PCNA chromatin dissociation route enabled and/or enhanced by Ubp10. If that were the case, it would be of interest to identify and functionally characterize additional factors involved in this novel, and likely Elg1-independent, mechanism.

Kubota et al., predicted Elg1-RLC alternative PCNA unloaders back in 201326. In fact, they suggested three models of PCNA unloading based on published evidence at the time and still valid today. A first model where Elg1-RLC would be the main unloader; a second model where Rfc1-RFC would work as a genome-wide unloader and Elg1-RLC would be the unloader of PCNA at specific genome localizations with emphasis in difficult to replicate sites; and a third model where Elg1-RLC complex would unload SUMOylated PCNA and Rfc1-RFC would unload unmodified PCNA rings to directly recycle them at lagging strands. Our findings are consistent with the last two models yet better support the third one where two different PCNA unloaders complexes would effectively recycle PCNA during the synthesis of lagging strands (model in Fig. 8). We suggest that the most likely alternative PCNA unloader is Rfc1-RFC, and that it may unload PCNA preferentially when deubiquitylated. Rfc1 is an essential subunit of the RFC complex that interacts with Ubp10 and PCNA. Ubp10 would eventually regulate the precise timing of PCNA unloading by Rfc1-RFC after Ub-PCNAK164 deubiquitylation. Our working model predicts that PCNA is ubiquitylated at K164 at lagging strands (Fig. 8), we hypothesize that such event would be a consequence of the collision of the PCNA-Pol ∂ complex with the preceding (5ʹ end) Okazaki Fragment. Prior to Ubp10 action and subsequent PCNA unloading, PCNA ubiquitylation would enhance Pol ∂ release by a collision-release mechanism already described67.Fig. 8 Model for Okazaki fragment maturation through dynamic ubiquitylation and deubiquitylation of PCNA Lysine 164.

Ubp10-mediated deubiquitylation of K-164-PCNA triggers a PCNA chromatin unloading mechanism at the final steps of the synthesis and ligation of DNA at lagging strands (see Discussion text for details).

A strong case can be made here that yeast cells coordinate the last steps of synthesis and maturation of Okazaki fragments through a Ubp10-regulated PCNA unloading mechanism (see model in Fig. 8). Our findings are consistent with the hypothesis that S. cerevisiae cells ubiquitylate and deubiquitylate PCNA during S phase in a dynamic, yet ordered, manner to ensure normal DNA replication, such that PCNA ubiquitylation is orderly followed by its deubiquitylation to strengthen PCNA unloading at lagging strands in a genome-wide scale, regulating and ensuring the time frame for Okazaki fragments maturation to generate a continuous lagging double-stranded DNA.

Methods

Yeast strains, growth conditions and media

All the budding yeast used in this study originate from a MATa W303 RAD5 bar1::LEU2 strain36 and are listed in Supplementary table 1. Budding yeast strains were grown in YPAD medium (1% yeast extract, 2% peptone supplemented with 50 μg/ml adenine) containing 2% glucose. For block-and-release experiments, cells were grown in YPAD with 2% glucose at 25 °C and synchronized in G1 with α-factor pheromone (40 ng/ml, 2.5 h). Cells were then collected by centrifugation (800 x g, 3 min) and released into fresh media (supplemented with 50 µg/ml of Pronase) in the absence or in the presence of HU (0.2 M, FORMEDIUM). Overexpression experiments with cells grown in YPAD medium with 2% raffinose at 25 °C were conducted by adding to the medium 2.5% galactose (to induce) or 2% glucose (to repress).

General experimental procedures

General experimental procedures of yeast Molecular and Cellular Biology, and generation of tagged alleles and specific gene deletions were performed as described61,68–70. Transformation was performed by lithium acetate protocol and transformants were selected by growing in selective mediums. Different selection markers were used (KANMX6, HphMX4, NatMX6, URA3, TRP1, HIS3), as indicated in Supplementary table 1. Constructs were confirmed by PCR and/or sequencing. The presence of tagged proteins was further confirmed by immunoblot. Moreover, strains with tagged alleles were carefully checked for growth rate and sensitivity to HU. No differences with untagged controls were found. Some mutant strains, such as the cdc9-7 background, are genetically unstable and can give raise to revertants, contributing to experimental variability. To avoid this, once strains are thawed, single colonies were isolated and tested for cdc9-7 in terms of its thermosensitivity phenotype, just before to perform the experiments. All reagent information (antibodies, chemical peptides, recombinant proteins and oligonucleotides) is detailed in the Supplementary Tables 1 and 2.

Flow Cytometry Analysis

For flow cytometry analysis, 107 cells were collected by centrifugation, washed once with water, fixed in 70% ethanol and processed for DNA analysis71. DNA content was determined by using SYTOX Green (Molecular PROBES) for DNA staining72,73. The DNA content of individual cells was measured using a Becton Dickinson Accuri C6 plus software. A minimum of 10.000 cells were scored per time-point.

HU and temperature sensitivity assays

Stationary cells were counted and serially diluted in YPAD media. Ten-fold dilutions of equal numbers of cells were plated onto YPAD (2% glucose) media (always supplemented with 50 μg/ml adenine), or YPAD containing HU, incubated at the indicated temperatures for 24, 48, 72 or 120 h and then scanned using Epson Easy Photo FixTM (v.3.9.2.0ES) software.

Identification of Ubp10 interactors by mass spectrometry

Ubp10-GFP expressing- and untagged control cells were synchronized with α-factor and released into fresh YPAD. 30 min, 40 min, 50 min and 60 min time point samples were fixed with formaldehyde and harvested. Cell pellets were resuspended in Lysis Buffer (50 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Tritón-X100, 0.1% Na-deoxycholate) and broken using glass beads in a fast-prep. Chromatin extracts were disrupted by sonication, cleared by centrifugation, and incubated for 4 h at 4 °C with agarose-conjugated GFP-Trap™ beads (Chromotek). Beads were washed once with lysis buffer, once with Wash Buffer (10 mM Tris pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% Na-deoxycholate) and once with TE. 30 min, 40 min, 50 min and 60 min samples corresponding to each strain (Ubp10-GFP expressing cells and untagged control cells) were pulled and the two resulting samples resuspended in Laemmli Buffer, resolved by SDS-PAGE and silver stained. Regions of interest were excised, distained and digested with modified porcine trypsin (Promega, Madison, Wis.)74. Tryptic peptides were recovered, dried in a speed vacuum system, and desalted by using C18-homemade microcolumns. A nano-UHPLC system (NanoElute, Bruker Daltonics, Germany) coupled to a hybrid trapped ion mobility-quadrupole time-of-flight mass spectrometer Tims TOF Pro (Bruker Daltonics, Germany) via a modified nano-electrospray ion source (Captive Spray, Bruker Daltonics, Germany) was used for reversed-phase LC-MS/MS analysis. Peptides were dissolved in 0.1%FA/2%ACN and loaded onto a trapping column (Trap AcclaimPepMap 100 C18, Thermo) and were separated on a C18 1.9um 75ID 15 cm column (nanoElute FIFTEEN, Bruker Daltonics) at 40 °C using a 60 min gradient (from 2% to 35% ACN/0.1 FA) at a flow rate of 300 nL/min. MS acquisition was run in data-dependent acquisition (DDA) mode with PASEF. The acquired data were submitted to the MaxQuant (1.6.17.0) quantitative proteomics software package for identification and relative quantification by iBAQ and MaxLFQ.

The UniProtKB database has been used using the reviewed sequences and isoforms from the Saccharomyces cerevisiae proteome (UP 000002311 download 2022-02-22).

Search parameters were as follows: fully tryptic digestion with up to two missed cleavages, 40 ppm and 20 ppm mass tolerances for precursor and product ions, respectively, oxidation of methionine and acetylation of the protein N-terminus were established as variable modifications and carbamidomethylation of cysteine as fixed modification, and seven amino acids minimum peptide length. One percent false discovery rate (1% FDR) using Target-Decoy database for both peptide and protein validation. For protein identification one unique peptide was considered as the minimum number. This experiment was performed once (n = 1). This proteomic analysis was performed in the Proteomics Facility of the Cancer Research Center (Salamanca, Spain). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE75 partner repository with the dataset identifier PXD048249.

Total protein extracts for Immunoblotting

Total protein extracts were prepared following cell fixation using trichloroacetic acid (TCA) and resolved by SDS-polyacrylamide gel electrophoresis before transfer to nitrocellulose membranes.

Fractioning and Immunoblotting

For chromatin-enriched fractions around 6 × 107 exponentially growing cells were harvested by centrifugation and resuspended in 1 ml of Buffer 1 (containing 150 mM Tris pH 8.8, 10 mM dithiothreitol (DTT), and 0.1% sodium azide), and incubated at room temperature for 10 min. Cells were pelleted, washed with 1 ml of Buffer 2 (50 mM KH2PO4/K2HPO4 pH 7.4, 0.6 M Sorbitol, and 10 mM DTT), resuspended in 200 μl of Buffer 2 supplemented with 40 μg Zymolyase-100T and incubated at 37 °C for 10 min with intermittent mixing. The resulting spheroplasts were washed with 1 ml of ice-cold Buffer 3 (50 mM HEPES pH 7.5, 100 mM KCl, 2.5 mM MgCl, and 0.4 M Sorbitol), followed by resuspension and a 5-min incubation in 100 μl of EBX buffer (50 mM HEPES pH 7.5, 100 mM KCl, 2.5 mM MgCl, 0.25% Triton100, 1 mM phenylmethylsulfonyl fluoride (PMSF), Protease inhibitor tablets (EDTA-free, Roche), Leupeptin 1 μg/ml, Pepstatin 2.5 μg/ml, and RNAse 10 μg/ml), with occasional mixing. Aliquots of 30 μl of these disrupted cell suspensions were collected as whole cell extract samples (WCE). Remaining volume was layered onto 70 μl of cold EBX-S buffer (EBX buffer supplemented with 30% Sucrose) and subjected to centrifugation at 13,500 x g for 10 min at 4 °C. Aliquots of 30 μl of the resulting supernatant layer (Chromatin-free fraction) were also collected. After discarding supernatant, chromatin pellets were washed with 200 μl of EBX-S buffer, resuspended in 70 μl of EBX buffer supplemented with 0.5 μl of Benzonase, and incubated on ice for 15 min (Chromatin fraction). SDS-PAGE loading buffer was added to each fraction.

The different protein extracts were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Antibodies used for detection are listed in the Supplementary table 2 and were visualized using ECL reagents (Amersham Pharmacia Biotech) and films (FujiFilm). The levels of proteins bound to chromatin were quantified using Quantity One (v.4.6.6) Software (BioRad) and normalized with their corresponding Histone H2B values. All data in the bar graphs are presented as means SD in triplicate. Statistical analyzes were conducted with GraphPad Prism (v.10.1.0). A two-way analysis of variance (ANOVA) test was used to determine the statistical significance.

Protein interaction analysis

Cells expressing tagged or untagged (control) proteins were fixed with 1% formaldehyde and harvested. Chromatin extracts were prepared in a Lysis Buffer containing 50 mM Hepes pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Tritón-X100, 0.1% Na-deoxycholate, and supplemented with Antiproteolytic Cocktail using glass beads. Extracts were cleared by centrifugation; soluble protein fractions were discarded, and chromatin pellets were sheared by sonication. Chromatin extracts were clarified and tagged proteins were enriched by immunoprecipitation with specific Tag antibodies previously bound to Protein G Dynabeads (5 h at 4 °C). Then, antibody-bound Protein G Dynabeads and controls were extensively washed with lysis buffer, and elution was carried out in SDS-PAGE loading buffer. Immunoprecipitates were resolved by SDS-PAGE gels, transferred to nitrocellulose membranes and analyzed with specific-HRP conjugated antibodies.

Okazaki fragment analysis

Cells were collected by centrifugation, washed in SCE buffer (1 M sorbitol, 100 mM sodium citrate, 60 mM EDTA, pH 7.0) and spheroblasted for 3 min with 5 mg zymolyase 20 T (Amsbio) per 50 ml culture. Spheroblast were washed with SCE, resuspended by gently pipetting in 490 μl lysis buffer (50 mM Tris-HCl, pH 8.0, 50 mM EDTA, 100 mM NaCl, 1.5% sarkosyl) containing 150 μg proteinase K (Sigma-Aldrich) and digested for 14 h at 37 °C. Residual proteins and peptides were precipitated by adding 200 μl 5 M KOAc and centrifugation at 16,000 x g for 30 min at 4 °C. Nucleic acids were obtained from the supernatant by precipitation with isopropanol and centrifugation at 16,000 x g for 10 min at 4 °C. Pellets were washed with 70% ethanol, air dried, resuspended in 200 μl STE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 100 mM NaCl) and digested with 25 μg RNase A (Sigma-Aldrich) at 37 °C for 1 h. DNA was precipitated by addition of 20 μl NaOAc (pH 5.2) and 800 μl ethanol followed by centrifugation at 5000 x g for 10 min at 25 °C. DNA pellets were washed with 70% ethanol, air dried and resuspended in 1 μl TE/ml original culture volume. For DNA labeling, 2 μl of the DNA obtained was used in 20 μl labeling reactions containing 1U Klenow (exo-)polymerase (NEB) and 0,4 μl of α-32P-dCTP (Perkin Elmer). Reactions were incubated at 37 °C for 30 min. Free label was removed using Illustra microspin G-50 columns (GE healthcare). Labeled DNA was resolved in 1.3% denaturing agarose gels (50 mM NaOH, 1 mM EDTA). After electrophoresis, the gel was neutralized, and DNA transferred to an uncharged nitrocellulose membrane (Hybond-N; GE healthcare) by capillary transfer. Membranes were exposed to phospho screens (Fujifilm BAS-MS). Images were acquired using a Molecular Imager FX (BioRad).

Two-dimensional DNA gels

Cell cultures were mixed with ice-cold AZ-STOP solution (0.5 M NaOH, 0.4 M EDTA, 0.2% Sodium Azide) in a 2:1 ratio, shaken vigorously, and collected by centrifugation at 2,300 x g. Cell pellets were washed once with ice-cold water and then frozen before further processing. The cells were resuspended in 5 ml of NIB buffer (17% Glycerol, 50 mM MOPS, 150 mM potassium acetate, 2 mM MgCl2, 500 mM Spermidine, 150 μM Spermine) and disrupted by vortexing with an equal volume of precooled glass beads (30 seconds at maximum power – 30 seconds on ice, 16–18 cycles). Breakage was confirmed by microscopy. The supernatant was recovered with a Pasteur pipette and centrifuged at 6000 x g for 10 min at 4 °C. Pellets were resuspended in 5 ml of G2 Buffer (Quiagen) using a 1 ml cut pipette tip. 100 μL of RNAse (10 mg/ml) were added, and the samples were incubated for 45 min at 37 °C. Next, 100 μL of Proteinase K (20 mg/ml) were added, and samples were incubated for 1 h at 37 °C. Samples were clarified by centrifugation (2300 x g, 5 min at 4 °C) and 5 ml of QBT buffer (Quiagen) was added. DNA samples were then purified using Genomic-Tip 100/G columns (Quiagen) and precipitated with Isopropyl alcohol. Samples were centrifuged (21,000 x g, 30 min at 4 °C), and DNA pellets were washed with cold EtOH 80%, left to air dry, and resuspended in TE. DNA was digested with the NcoI restriction enzyme and subjected to first-dimension electrophoresis (0.4% agarose in TBE buffer, 20 V for 36 h at room temperature). Gel slices containing samples were excised, rotated 90° counterclockwise with respect to the first dimension, and placed in a gel-casting tray for second-dimension electrophoresis (1% agarose in TBE buffer containing 500 ng/ml Ethidium bromide, 160 V for 4.5 h at 4 °C). After denaturing with 0.4 M NaOH for 20 min, samples were transferred to nitrocellulose membrane Hybond-XL (GE Healthcare) by capillary transfer and hybridized to radiolabeled probes spanning the ARS305 and ARS306 origins of DNA replication. For each origin of replication tested, the specific probe corresponds to the following coordinates (retrieved from SGD): ARS305 (39073-40557, Chr III) and ARS306 (73001-73958, Chr III). PCR oligonucleotides used to generate them are listed in the Resources Table (Supplementary Information). Images were acquired using a Molecular Imager FX (BioRad) and the different replication-associated DNA molecules were quantified using Quantity One (v.4.6.6) software (BioRad).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information

Peer Review File

Reporting Summary

Source data

Source Data

Supplementary information

The online version contains supplementary material available at 10.1038/s41467-024-52542-9.

Acknowledgements

We are grateful to members of 08 research group at the IBMCC for helpful discussions. We would like to particularly thank Professor Anne Donaldson and Takashi Kubota PhD (University of Aberdeen) for pol30 mutant and ChVLig1 strains. We are also grateful to the National BioResource Project, NBRP Japan for the cdc9-7 strain. This work was supported by the Spanish Ministry of Science (grants PID2019-109616GB-100 to A.B. and M.P.S. and PID2020-116003GB-100 to R.B.) and Junta de Castilla y León (grant SA103P20 to A.B). J.Z. was supported by a Predoctoral Fellowship from the Junta de Castilla y León (JCyL). S.M. was supported by a University of Salamanca Postdoctoral Fellowship and a MSCA Postdoctoral Fellowship (grant n° 101106007). E.A. was supported by a JCyL Postdoctoral Fellowship. A.B. and M.P.S. Institution is supported by the “Programa de Apoyo a Planes Estratégicos de Investigaciόn de Excelencia” cofunded by the Junta de Castilla y Leόn and the European Regional Development Fund (CLC-2017-01).

Author contributions

Conceptualization, A.B., with substantial inputs from M.P.S., J.Z., S.M. and RB; Investigation, J.Z., S.M., E.A., M.A. S.R. and M.P.S.; Supervision, A.B. and M.P.S.; Formal Analysis, A.B., M.P.S., S.M. and J.Z.; Writing & Editing, A.B. and M.P.S. with the help of R.B. and S.M.; Funding Acquisition, A.B., M.P.S. and R.B.

Peer review

Peer review information

Nature Communications thanks George-Lucian Moldovan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Data availability

Yeast strains generated in this study are available on request from the corresponding authors. The raw mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE75 partner repository with the dataset identifier PXD048249. Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Javier Zamarreño, Sofía Muñoz, Esmeralda Alonso-Rodríguez.
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