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J Biol Chem
J Biol Chem
The Journal of Biological Chemistry
0021-9258
1083-351X
American Society for Biochemistry and Molecular Biology

S0021-9258(24)02158-6
10.1016/j.jbc.2024.107657
107657
Research Article
Phosphoproteomic analysis of the response to DNA damage in Trypanosoma brucei
McLaughlin Emilia 12
Zavala Martinez Monica Gabriela 1
Dujeancourt-Henry Annick 1
Chaze Thibault 3
Gianetto Quentin Giai 34
Matondo Mariette 3
Urbaniak Michael D. 5
Glover Lucy lucy.glover@pasteur.fr
1∗
1 Institut Pasteur, Université Paris Cité, Trypanosome Molecular Biology, Department of Parasites and Insect Vectors, Paris, France
2 Sorbonne Université, Collège doctoral, Paris, France
3 Institut Pasteur, Université Paris Cité, Proteomics Platform, Mass Spectrometry for Biology Unit, Centre National de la Recherche Scientifique, UAR 2024, Paris, France
4 Institut Pasteur, Université Paris Cité, Bioinformatics and Biostatistics HUB, Paris, France
5 Division of Biomedical and Life Sciences, Faculty of Health and Medicine, Lancaster University, Lancaster, UK
∗ For correspondence: Lucy Glover lucy.glover@pasteur.fr
14 8 2024
9 2024
14 8 2024
300 9 1076572 7 2024
29 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
Damage to the genetic material of the cell poses a universal threat to all forms of life. The DNA damage response is a coordinated cellular response to a DNA break, key to which is the phosphorylation signaling cascade. Identifying which proteins are phosphorylated is therefore crucial to understanding the mechanisms that underlie it. We have used stable isotopic labeling of amino acids in cell culture-based quantitative phosphoproteomics to profile changes in phosphorylation site abundance following double stranded DNA breaks, at two distinct loci in the genome of the single cell eukaryote Trypanosoma brucei. Here, we report on the T. brucei phosphoproteome following a single double-strand break at either a chromosome internal or subtelomeric locus, specifically the bloodstream form expression site. We detected >6500 phosphorylation sites, of which 211 form a core set of double-strand break responsive phosphorylation sites. Along with phosphorylation of canonical DNA damage factors, we have identified two novel phosphorylation events on histone H2A and found that in response to a chromosome internal break, proteins are predominantly phosphorylated, while a greater proportion of proteins dephosphorylated following a DNA break at a subtelomeric bloodstream form expression site. Our data represent the first DNA damage phosphoproteome and provides novel insights into repair at distinct chromosomal contexts in T. brucei.

Keywords

phosphoproteomics
Trypanosoma brucei
DNA damage response
DNA break
Abbreviations

ACN acetonitrile

ATM ataxia-telangiectasia mutated

ATR ATM and Rad3-related kinase

BES bloodstream form expression site

BSA bovine serum albumin

DDR DNA damage response

DSB double-strand break

DSBR DSB repair

FA formic acid

FASP filter aided sample preparation

FBS fetal bovine serum

FDR false discovery rate

GO Gene Ontology

HR homologous recombination

MS mass spectrometry

RBPs RNA binding proteins

RPA replication protein A

SILAC stable isotopic labeling of amino acids in cell culture

TFA trifluoroacetic acid

VSG variant surface glycoprotein

Reviewed by members of the JBC Editorial Board. Edited by Patrick Sung
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pmcOne of the most toxic lesions to the genome is a DNA double-strand break (DSB), where breaks occur simultaneously in the phosphate backbone of two complementary DNA strands. DSBs in the DNA can arise due to endogenous processes in the cell, such as replication fork collapse or stalling, and can also result from exogenous agents, such as chemicals or ionizing radiation (1). DSB repair (DSBR) is a coordinated program of events initiated by a signaling cascade, with protein phosphorylation at its core. In eukaryotes the DSBR is a conserved process initiated by detection and processing by the MRE11, RAD50, and NBS1 (MRN) complex and leads to the recruitment of two key kinases, ataxia-telangiectasia mutated (ATM) and ATM and Rad3-related kinase (ATR), which are the master regulators of the DNA damage response (DDR) (2). Subsequently, a phosphorylation cascade is initiated with some 900 substrates modified (3). One of the key substrates of ATM is S139 of the histone variant H2AX, which when phosphorylated is termed γH2AX and is regarded as an early marker of repair in mammals (4, 5). γH2AX spreads along large regions of chromatin fiber bidirectionally from the break site (3) which aids the recruitment of chromatin remodeling factors, allows DNA damage proteins to access the break (6) and concentrates repair factors at the damaged site (7).

Understanding phosphorylation cascades have been driven by stable isotopic labeling of amino acids in cell culture (SILAC) (8) based quantitative phosphoproteomics (3, 9, 10, 11). In human cells, SILAC phosphoproteomics has identified over 900 phosphorylation sites associated with ionizing radiation induced DNA damage, revealing a series of interconnected networks in the DDR including proteins associated with DNA repair, replication, and chromatin modifications (3). Over 70% of the phosphorylation sites identified are targets of the ATM kinase, and many of these substrates are themselves kinases, highlighting the central role of the phosphorylation cascade in the DDR.

Human African trypanosomiasis is a fatal vector borne disease caused by the protozoan parasite Trypanosoma brucei. In the mammalian host the parasite is found in the bloodstream, adipose tissue (12) and skin (13, 14). Here, the parasite is exposed to attack by the host immune system and is protected by a dense variant surface glycoprotein (VSG) coat, which is periodically exchanged by antigenic variation (15, 16). VSGs are exclusively expressed from a subtelomeric bloodstream form expression sites (BESs) (17), of which there are approximately 15, with only one active (the active BES) and the rest silenced. The majority of VSG genes are located in arrays in the subtelomeric regions of the megabase chromosomes, and also occasionally at chromosome internal regions (18) and act as a repertoire for antigenic variation. There has been much debate into what drives antigenic variation, with DSBs (19, 20), replication-derived fragility from the early replication of the BES (21, 22) or the formation of RNA:DNA hybrids (23, 24) all being implicated. In T. brucei repair occurs predominantly via homologous recombination (HR) (25). Repair by microhomology-mediated end joining accounts for approximately 5% of repair, but up to 25% at the active BES (20). Antigenic variation occurs mainly by gene conversion events, where the active VSG is deleted and replaced by a silent donor (26, 27, 28, 29), but crossover switching events, where two VSGs are exchanged, have also been observed (28, 30, 31, 32).

Several DNA damage linked proteins directly influence repair and antigenic variation, and within the homologous repair pathway, sequence diversity among the genes facilitating repair suggests functional divergence within the pathway as well (33). ATR mediates signal transduction in trypanosomes (34) and loss leads to an increase in nuclear DNA damage and VSG switching (35). The RecQ-like helicase is required for genome repair and mutants show elevated VSG switching by telomere recombination and VSG gene conversion events (22). RAD51, the primary recombinase in DNA repair, is required for homology searching and DNA strand exchange and is loaded onto single-strand DNA by BRCA2 (36, 37). In Trypanosoma brucei, BRCA2 is essential for homologous recombination, DNA replication, cell division, and antigenic variation (38, 39), while RAD51 essential for HR and rad51 null mutants have impaired, VSG switching (40, 41, 42, 43). In trypanosomes, five RAD51-related proteins, RAD51 to 3, 4, 5, and 6 are important for DSBR, but only RAD51 to 3 contributes to VSG switching (44). Early recognition and processing of a DNA break via the MRE11, RAD50, and NBS1 complex is important for both detection and signaling of a DSB. In Leishmania, MRE11 maintains genomic integrity and although in T. brucei does not affect the rate of VSG switching (40, 45, 46, 47), both MRE11 and RAD50 promote recombination using longer stretches of homology which restricts the diversity of VSG genes used for antigenic variation (48). At a chromosomal internal locus, MRE11 is required for efficient resection (48). The RECQ/TOPO3/RMI1 (RTR) complex which includes the RecQ-family helicase, a topoisomerase IIIα, and RMI1/2 suppress these mitotic crossover and removes recombination intermediates (49). In trypanosomes, VSG gene conversion and cross over events can be suppressed by the RECQ/TOPO3/RMI1 complex components TbTOPO3α and TbRMI1 or act in concert with RAD51 and RMI1 (42, 50). During the DNA damage repair cycle, the G2/M checkpoint prevents division of unrepaired DNA and preserves genome integrity. Although trypanosomes do show cells arrested in G2/M following a DSB, some cells do continue to replicate and divide their DNA with a DNA break. This suggests a level of tolerance to DNA damage greater than that seen in other eukaryotes (51, 52), perhaps aiding homology searching for antigenic variation. Despite the importance of HR in evasion of the host immune system only one DNA damage associated phosphorylation site has been identified in T. brucei, that of γH2A (53), which is analogous to H2AX in mammalian cells (4). In T. brucei, H2A T131 is phosphorylated in response to a DSB, typically during S or G2-phases of the cell cycle.

Here, we use a quantitative single-locus phoshoproteomic approach to characterize changes in phosphorylation site abundance in response to a DSB at two distinct loci (i) a chromosome internal locus where classic HR is the dominate form of repair (25) and (ii) the active BES where repair facilitates antigenic variation (20). We found that there is a striking distinction between the proteins phosphorylated in response to a chromosome internal DSB and one at the active BES and identify two novel DNA damage associated phosphorylation sites on Histone H2A.

Results

Adaptation of the 1HR and VSGup cell lines to SILAC medium

SILAC experiments require the metabolic incorporation of stable isotope labeled amino acids present in the cell culture medium. Trypanosomatids are auxotrophic for arginine (R) and lysine (K) (54) and we therefore used cell culture medium lacking in both and supplemented with either the “heavy” isotope labeled L-Arginine U–13C6 and L-Lysine 4,4,5,5-2H4 (R6K4), or “light” labeled L-arginine and L-lysine (R0K0) (Fig. 1A). Trypanosome parasites have been shown to grow normally in SILAC HMI-9 and remain infective in mice (55). In order to study the cellular response to locus specific DSBs, we used two established cell lines, 1HR and VSGup, that contain the tetracycline inducible yeast I-SceI homing endonuclease which induces a DSB in approximately 95% of all cells (20, 25). The 1HR cell line contains an 18 bp I-SceI heterologous recognition sequence (SceR; Fig. S1A) at an intergenic chromosome internal polycistronic transcription unit on one homolog of chromosome 11 (25), and the VSGup strain harbors the SceR upstream of the actively expressed VSG on BES1 on chromosome 6a (Fig. S1B) (19, 20). In the 1HR cell line approximately 60% of the cells are able to repair the DSB and survive, 85% of repair uses RAD51-dependent allelic HR and 5% ectopic HR and RAD51-independent microhomology-mediated end joining (25). In contrast, only 5% of VSGup cells survive a DSB suggesting lesions at this locus are highly toxic, 60% use RAD51-dependent HR and 40% a RAD51-independent repair to resolve the DSB (20). Incorporation of labeled amino acids in the 1HR and VSGup cell lines was assessed by mass spectrometry (MS), and we observed 93.4% and 96.1% heavy label incorporation in the 1HR and VSGup cell lines, respectively (Fig. S2A). γH2A foci formation was also observed in cells grown in SILAC medium (Fig. S2B). These foci have been shown to form post DSB induction (20, 25) and an indicator of a robust DDR. The SILAC adapted cells lines respond to DNA damage as expected, we therefore proceeded to establishing the DNA damage phosphoproteome in T. brucei.Figure 1 The1HR and VSGupDSB proteome.A, proteomic SILAC strategy for Trypanosoma brucei to identify proteins phosphorylated in response to a single double-strand break. T. brucei cells were labeled with either heavy or light amino acids and subject to DNA damage. The protein extracts were mixed at a 1:1 ratio and analyzed by mass spectrometry. B, upper panel: schematic of chromosome 11 Tb927.11.4530/40 locus following modification to generate the 1HR chromosome-internal DSB cell line with the I-SceI recognition site, SceR, highlighted. The DSB site is flanked upstream by red fluorescent protein (RFP) and puromycin-N-acetyltransferase (PAC) downstream. The site is positioned at an intergenic region between Tb927.11.4530 and Tb927.11.4540, shown as “4530” and “4540”, respectively. Black boxes are tubulin intergenic sequences. Lower panel: total proteins identified in 1HR. The x-axis is the Log2 value of the ratio of each protein given as its presence in the DSB induced versus uninduced sample. The y-axis is the intensity of a given protein in the sample. Ribosomal proteins are highlighted in purple, all other proteins shown in dark gray. C, upper panel: schematic showing the VSGup cell line set up showing the modified BES1 on chromosome 6a. An I-SceI meganuclease recognition site is inserted upstream of the actively expressed VSG-2, shown with a green box. The SceR is flanked downstream by a puromycin-N-acetyltransferase gene (PAC). Arrow; native promoter of the expression site, white boxes; genes, solid black box; 70 bp repetitive sequence, black circles; telomere. Lower panel: The VSGup proteome, with details as described in B. Ribosomal proteins are highlighted in purple, all other proteins shown in dark gray. Schematics generated with BioRender. BES, bloodstream form expression site; DSB, double-strand break; HR, homologous recombination; SILAC, stable isotopic labeling of amino acids in cell culture; VSG, variant surface glycoprotein.

Analysis of the total proteome following a double-strand break

In bloodstream form trypanosomes, γH2A accumulation peaks at 12 h post DSB induction in both the 1HR and VSGup cell lines (53), and ssDNA accumulates between 9 and 12 h (20, 25). In addition, at this time point we do not see any cell death allowing us to capture phosphorylation events associated with the DDR. We therefore chose to carry out proteomic analysis at 12 h post DSB induction. For each sample, a label swap replicate was carried out (induced cells grown in “light” media and uninduced in “heavy” media). Analysis of the total peptide extract was carried out, and in the 1HR dataset, a total of 2457 proteins were identified (Fig. 1B), and only 12 of these showed a > 2-fold change in abundance following DSB induction. In the VSGup proteome, a total of 2646 proteins were identified (Fig. 1C) only 8 of which had a > 2-fold change in abundance following DSB induction, suggesting large changes at the protein level are not seen at 12 h post DSB induction. However, we did observe a notable downregulation of the ribosomal proteins in both the 1HR and VSGup proteomes (Fig. 1, B and C). Ribosomal proteins are important for the assembly of ribosomal subunits and also function as RNA chaperones (56). The specific downregulation of ribosomal genes observed here suggests that there may be a global inhibition of protein translation in response to DNA damage as has been reported following a CRISPR-Cas9-induced DSB in mammalian cells (57).

The DSB locus-specific phosphoproteome of the 1HR and VSGup strains

Within the total protein extract, phosphorylated peptides are of low abundance, and so we carried out an enrichment step using affinity purification on a TiO2 column (58). To identify significantly changing phosphorylation sites in the two datasets, we used significance B testing (59), which takes into account the intensity-weighted significance. Overall, there was no significant change in the distribution of phosphorylation events among phosphor serine (S) and threonine, (T) but our dataset included no phosphorylation of tyrosine (Y) (Fig. S3A) compared to published data. The majority of the phosphorylation events identified were on known proteins (Fig. S3B). In total, we identified 6905 phosphorylation sites in the 1HR phosphoproteome and 6540 for VSGup (Figs. 2, A and B, S3, and Dataset S3). Of these sites, 5991 were common to both the 1HR and VSGup datasets, and 914 and 549 sites were unique to the 1HR and VSGup respectively (Fig. S3C) and a core set of 211 that are significantly upregulated or downregulated. Using γH2A as a positive control for the phosphoproteome analysis, in the 1HR strain we report an average of 5.39 fold increase in phosphorylation of γH2A at T131 (previously annotated as T130 excluding the initiator methionine (53)) (Figs. 2A and 3A) and a 1.97 fold increase in the VSGup strain (Figs. 2B and 3A), confirming that we are able to detect DSB specific phosphorylation events using quantitative phosphoproteomics.Figure 2 Locus specific phosphoproteome. A, upper panel: schematic of the 1HR locus. Lower panel: quantification of changes in phosphorylation in the 1HR phosphoproteome. Black circles, nonsignificant change in phosphorylation; enriched phosphorylation-teal circles, significantly increased, orange circles significantly decreased; blue circles selected proteins in table inset. B, upper panel: schematic of the VSGup locus. Lower panel: quantification of changes in phosphorylation in the VSGup phosphoproteome, details as in (A). HR, homologous recombination; VSG, variant surface glycoprotein.

Figure 3 Phosphorylation of proteins involved DNA repair and recombination.A, quantification of phosphorylation at specific amino acid sites. B, upper panel: Tandem mass spectral data showing assignment of T131 and S133 phosphorylation sites on H2A (Tb927.7.2940) the MaxQuant localization score for each site is 1; The loss of a phosphate group (−98 Da) is indicated with a (∗). The fragmentation pattern of the peptide is shown above. The b5 and b6 ions, highlighted on the spectrum with blue circles, show specific phosphorylation of T131, while the y2 and y3 ions highlighted with red circles show the specific phosphorylation of S133. Lower panel: amino acid sequence alignment of the H2A C terminus among trypanosomatids. Alignments shown are the C terminus of H2A from Trypanosoma brucei (Tb927.7.2940), Trypanosoma evansi (TevSTIB805.7.2930-t26_1), Trypanosoma congolense (TcIL3000_7_2140.1), Trypanosoma vivax (TvY486_0702710), Trypanosoma cruzi (TcCLB.508321.11), Leishmania mexicana (LmxM.08_29.1720.1), Leishmania infantum (LINF_210016800-T1) and Leishmania donovani (LdCL_210016600-t42). Red asterisks denote the S113, T131, and S133 phosphorylation sites; S, serine; T, threonine. C, Western blotting of the peptide competition assay shows the specificity of the anti-S133P antibody to the phosphorylated peptide. Parasite lysates treated or nontreated with MMS/tet were separated by SDS-PAGE and probed with rat anti-S133P serum, previously incubated with the phosphorylated (S133P) or nonphosphorylated (S133) peptides. A preincubation no peptide is included as control, and a Coomassie stain is included as a loading control for the S133P blot. MMS, methyl methanesulfonate.

We identified 128 significantly altered phosphosites on 81 proteins in the 1HR phosphoproteome, 107 of which were upregulated and 22 downregulated (Fig. 2A and Dataset S2). In the VSGup phosphoproteome, 135 significantly altered sites were identified on 95 proteins, 65 of which were upregulated and 70 downregulated (Fig. 2B and Dataset S2). Within the phosphoproteomes, 26 sites were significantly enriched in both the 1HR and VSGup strains. Of these 26 phosphorylation sites, 21 were upregulated in the 1HR, and only 3 were upregulated in VSGup, with downregulation of phosphorylation making a bigger contribution to subtelomeric repair (Data S2). Among the 26 phosphosites that were significantly enriched in both the 1HR and VSGup datasets, a number of modifications on proteins involved in RNA binding, RNA processing and translation were identified (Dataset S1). We surveyed the 211 DSB responsive phosphorylation sites for categorical enrichment of Gene Ontology (GO) terms, using a Fisher’s exact test (false discovery rate (FDR) ≤ 0.05). The significantly enriched 1HR and VSGup phosphorylation sites were further divided into phosphoproteins that were significantly upregulated or downregulated in each dataset. In 1HR 6 significantly enriched GO terms were identified for upregulated phosphorylated proteins, which included histones and proteins that respond to DNA damage (Fig. S4A(i)), and one for VSGup (Fig. S4A(ii)). The reverse was seen with downregulated phosphorylated proteins; RNA binding was common to both, and five terms were enriched in the VSGup including chromatin organization (Fig. S4B(ii)) and one term enriched for 1HR, RNA processing (Fig. S4B(i)).

Phosphorylation of DNA repair proteins in the 1HR and VSGup strains

We next identified modifications on selected DNA repair proteins (Fig. 3A). In mammals, phosphorylation of histone H2B S14 on the C terminus is a late marker of DNA damage and is dependent on γH2AX (60). We detected a novel phosphorylation site at S39 on the C terminus of H2B (Tb927.10.10590) in 1HR strain (1.89-fold increase, p = 0.0339), and in VSGup (1.25-fold increase, p = 0.38) (Fig. 3A). In mammalian cells, three members of the NIMA (never in mitosis gene a)-related (NEK) kinase family, NEK1, NEK1, NEK10, and NEK11 are involved in the DDR and are implicated in check point control following DNA damage (61). We saw a significant enrichment of the phosphorylation of the serine/threonine protein kinase NEK17 (Tb927.10.5950) in response to an 1HR DSB with two sites on NEK17, S197, and T195, increase by 6.14-fold (p = 6.13622 × 10−8) (Figs. 2A, 3A and Dataset S1). NEK17 kinase is therefore a possible candidate for implementing phosphorylation marks that are specific to chromosomal internal regions.

The heterotrimeric replication protein A (RPA) complex, consisting of RPA-1, RPA-2, and RPA-3, is the major single-strand DNA binding protein in eukaryotes, and we identified three of phosphorylation sites on this complex in our dataset (Figs. 2A, 3A and Dataset S1). In mammals and yeast, the N terminus of RPA-2 is hyperphosphorylated by the ATR kinase in response to a DNA break (2, 62) which increases the affinity of RPA-2 for RAD51 (63) and promotes repair by HR (64). We identified one phosphorylation site on RPA-2, S4, which showed a moderate upregulation of 1.45 and 1.82-fold in response to 1HR and VSGup DSBs, respectively, suggesting that the N terminus of RPA-2 is not hyperphosphorylated at 12 h post DSB induction (Fig. 3A). Two sites on RPA-1 (Tb927.11.9130), the single-stranded DNA binding component of the complex (65), also had specific sites phosphorylated. The first site, S5 was previously identified in a global phosphoproteomics analysis (55), and we reported an average fold change in phosphorylation of 2.77 (p = 0.001) and 1.31 (p = 0.29) (Dataset S1) in response to DSBs induced in the 1HR and VSGup cell lines, respectively (Fig. 3A). The second site, S43 showed a 6.1-fold increase (p = 9.69 × 109) in response to an 1HR DSB (Fig. 3A), and 1.7-fold increase (p = 0.06) in the VSGup phosphoproteome (Dataset S1). Several non-DNA repair proteins were differentially phosphorylated in response to DNA damage in our two cell lines. We identified a single phosphorylation event on S517 of NUP-1 in response to a DSB at a chromosomal internal site (Fig. 2, A and B), suggesting nuclear pore complex components may be differentially required for DSBR in T. brucei depending on the chromosomal context. Our data also revealed that the FK506-binding protein is phosphorylated on T9, S13 in 1HR but dephosphorylated on S2, S3, S6, S7, T9, and S13 (Fig. 2, A and B). On FK506, only phosphorylation of S2 has previously been identified in a global phosphoproteomics analysis (55), suggesting that de/phosphorylation of S3, S6, S7, T9, and S13 are specifically in response to DNA damage. These data suggest a number of additional proteins previously unknown to be involved in the DNA repair process in trypanosomes and could be important for repair at either a chromosome internal or subtelomeric region (Fig. 2, A and B and Dataset S1).

Phosphorylation of S133 of histone H2A in response to DNA damage

The H2A phosphorylation on a conserved S/T-Q motif (4, 66) to give γH2AX, or γH2A in trypanosomes, is a widely studied early marker of DNA damage (53, 67). We observe robust phosphorylation of T. brucei γH2A (T131) in response to a DNA break (Fig. 2, A and B) and identified two additional modifications on the H2A C terminus: S113 and S133 (Fig. 3), with only T131 and S133 being conserved among trypanosomatids (Fig. 3B). Phosphorylation of S133 increased by 5.39-fold (p = 2.0283 × 10−6) in the 1HR strain and 1.97-fold (p = 1.3392 × 10−3) in the VSGup strain (Fig. 3A and Dataset S1). Individual phosphorylation events were detected on fragments harboring exclusively T131 or S133, confirming that both sites are phosphorylated (Fig. 3B). A third phosphorylation site was also identified, S113, that is located on the H2A tail. The S113 site was 3.16-fold (p = 0.000432) upregulated in response to a DSB in the 1HR cell line. Residues T131 and S133 are highly conserved, but S113 shows some variation in its conservation, not present in Leishmania infantum or Leishmania donovani (Fig. 3B) indicating that there are lineage specific modifications to the histone tail resulting in a difference to the DDR between trypanosomatid species.

To confirm that phosphorylation of H2A S133 was DNA damage responsive, we raised antibodies to the cognate phosphopeptide. We used a peptide competition assay to confirm the specificity of the antibody; here only the phosphorylated antibody was able to deplete the H2A S133 phosphorylated signal (Fig. 3C). We now refer to this antibody as H2A S133P. We next looked to see if H2A S133P accumulated at the site of DNA damage in a manner similar to γH2A in trypanosomes. Using the 1HR and VSGup cell lines, we induced DNA damage and assessed the H2A S133P signal over a 24-h period. As with γH2A, we see a more robust signal in the 1HR cell line as compared to VSGup, and in both cases a higher proportion of cells showed a pan-nuclear signal, rather than single foci (Fig. 4, A and C). Cells with single foci also had a diffuse nuclear signal that was not seen in the unperturbed cells (Fig. 4B, inset). In the 1HR cell line, the 48% of cells had a pan-nuclear signal at 12 h, and 10% with single foci 9 h (Fig. 4A). In VSGup, 17% of the cells had a pan-nuclear signal and 9% had distinct foci at 12 h (Fig. 4C). The H2A S133P signal was specific to the nucleus and characterized by a pan-nuclear signal in the 1HR cells line, where damage is within a megabase chromosome, while in VSGup cell line, where damage is localized to the sub-telomeric regions, both pan, and subfocal accumulation was seen.Figure 4 Phosphorylation of H2A S133 following DNA damage.Line graphs present the general percentage of 1HR (A) or VSGup (C) parasites showing S133 foci (black circles) or pan-nuclear (gray squares) immunofluorescent signal at 0, 3, 6-, 9-, 12-, and 24-h posttetracycline induction. Bar graphs present the percentage of 1HR (B) or VSGup (D) cells with S133 foci (light green bars) or pan-nuclear (teal bars) signal in the G1/S or G2 cell cycle-phases post tetracycline induction. An inset image in (B) demonstrates the type of immunofluorescent signal displayed by the cells. The scale bar represents 1 μm. n = 100 for all times points, n = 2 technical replicates, counts performed by two independent researchers. Error bars are the standard deviation of the mean. HR, homologous recombination; VSG, variant surface glycoprotein.

Accumulation of DNA damage proteins has been shown to be cell cycle regulated, as is the case with the RAD51 recombinase and γH2A in trypanosomes (53). We therefore quantified both the pan and focal H2A S133P signal at post DNA damage. Cell cycle position was defined using the nucleus and kinetoplast as cytological markers that were 4 to 6 diamidine-2-phenylindole-stained. The H2A S133P signal was confined to G1/S and G2 phase cells, similar to γH2A (Fig. 4, B and D). A reduced number of those cells going through mitosis or cytokinesis showed either a pan-nuclear signal or foci (Fig. S5). The 1HR line showed a stronger H2A S133P signal overall (Fig. 4, A and B), in line with the higher fold change seen - 5.39-fold (p = 2.0283 × 10−6) in the 1HR strain versus 1.97-fold (p = 1.3392 × 10−3) in the VSGup strain (Fig. 3A). In both the 1HR and VSGup cell lines, there was a stronger pan-nuclear signal in both G1-S and G2 cells (Fig. 4, B and D). Following a chromosome internal DSB, 29% of cells had a pan-nuclear signal at 12 h post DNA damage in G1-S phase cells, and in VSGup at 13% at 12 and 6 h post damage in G1-S and G2 cells, respectively. We then quantified the colocalization of the γH2A and H2A S133P signal in both 1HR and VSGup. For this, we selected cells with a single H2A S133P focus and determined whether they coincided with γH2A (Fig. 5). In 1HR, 47% of the H2A S133P foci colocalized with a γH2A, while only 32% did in VSGup. These data suggest that a nuclear-wide signal, not just focal accumulation, occurs in trypanosomes and that H2A S133P accumulates at the site of DNA damage.Figure 5 Colocalization between H2A S133Pand γH2A.A, representative immunofluorescent images showing colocalization (i) or no colocalization (ii) of the anti-S133P (green) and anti-γH2A (magenta) fluorescent signals (the scale bar represents 5 μm). B, 1HR and (C) VSGup cells, the bar graphs show the percentage of cells with S133P foci only (green bars) or colocalizing with γH2A (magenta bars). Each bar represents the average of three independent experiments (black dots). Experiments were performed in triplicate: 1HR, n = 230, 181, and 302 cells, and for VSGup, n = 180, 119, and 192 cells. A student’s t test showed that there is a statistically significant difference between the percentages in VSGup (p value = 0.0096). VSG, variant surface glycoprotein; HR, homologous recombination.

Discussion

Here, we report the use of SILAC quantitative phosphoproteomics to characterize the T. brucei DSB phosphoproteome in response to DSBs targeted at both chromosome internal and subtelomeric loci. The phosphorylation status of a given protein is a dynamic equilibrium balanced by the actions of protein kinases and protein phosphatases. In human cells, phosphoproteomic analysis of the DDR revealed that approximately one-third of the total sites identified are dephosphorylated in response to a DNA break (9, 10), and here dephosphorylation was highly represented in VSGup, accounting for 51% of significantly altered modifications, indicating its important role at the subtelomeric locus. In contrast, the majority of significantly altered phosphorylation sites following an 1HR are upregulated, again highlighting the disparity between chromosome internal and subtelomeric repair. The dependence on phosphorylation at a chromosomal internal locus may be due in part to the dominance of repair by allelic HR at this locus, while repair at a subtelomeric expression site favors both RAD51-dependent and independent repair. While the purpose of this study was to determine the specific phosphorylation events that govern the DDR in T. brucei, analysis of the total proteome revealed ribosomal proteins are downregulated following a DNA break. A similar phenomenon is seen in human cells where phosphorylation of eIF2a halts translation following a DNA break via ribosome remodeling (57), while mouse embryonic fibroblasts exposed to global DNA damage results in transcriptional silencing in the nucleolus (68). The downregulation of ribosomal proteins identified here alludes to cross talk between the nucleolus and the DDR, as has been observed in other organisms (69). In the kinetoplastid parasite Trypanosoma cruzi using more wide scale DNA damage, DNA damage by ionizing radiation led to a reduction in protein translation (70) while DNA damage by gamma irradiation revealed that active translation is critical for parasite recovery (71). Interestingly, DNA damage by ultraviolet B irradiation in mammalian cells results in an overall inhibition of protein synthesis and a translational reprogramming that facilitates the specific synthesis of DDR proteins, a response that is mediated by DNA-PKcs, a DNA damage checkpoint kinase, linking the DNA damage signaling pathway with repair (72). In T. cruzi a similar, specific, response is seen where DNA damage by ionizing radiation associated the ubiquitin-proteosome system with DNA repair (73). These data, including the downregulation of ribosomal proteins, shown here, point to a posttranslational regulation of gene expression in response to DNA damage that may, speculatively, aid in cellular recovery. In bloodstream form trypanosomes, an I-SceI generate DNA break did not lead to an increase in the expression of RAD51, rather it was suggested that a preexisting pool of RAD51 relocalizes to the site of DNA damage (25). The limited changes in total protein abundance seen here may also be a function of the damage system used—here a single DNA break versus more widespread damage cause by chemical or radiation damage. Analysis of the T. brucei heat shock phosphoproteome displayed limited changes to the proteome in comparison to the large-scale changes seen in the phosphoproteome (74)—analogous to what we report here. We suggest that larger changes in the phosphoproteome are more revealing than changes in absolute protein abundance due to phosphorylation signaling driving the DDR.

In our dataset, RPA-1 S43 was the highest upregulated phosphorylation site following a I-SceI break in 1HR, increasing by an average of 6.1-fold, suggesting that the protein is abundantly and specifically phosphorylated in response to a break. The second phosphorylation site identified on RPA-1, S5, showed a moderate increase in phosphorylation compared to that of S43. Phosphorylation of both sites was previously identified in global studies of the T. brucei phosphoproteome (55, 75) indicating that these modifications also play a role outside of the DDR. Histone modifications also play a key role in the DDR, regulating access to chromatin and signaling for DNA damage (6). Key to this is the phosphorylation of histone H2A at T131 to give γH2AX in trypanosomes. In our phosphoproteome, we identified two additional sites in histone H2A that are phosphorylated in response to a DNA break: S113 and S133. Given that these modifications were not previously annotated in global analysis of the T. brucei phosphoproteome (55, 75) they are likely specific to DSB repair. In mammals, the phosphorylation sites on the histone tail in addition to γH2AX, here S139, are involved in the mammalian response to DNA damage (66, 76), while in Saccharomyces cerevisiae, systematic mutation of the histone tail identified three sites that are important for efficient DNA repair (77). Antibodies raised against the phosphorylated H2A S133 indicated that both a pan-nuclear signal and subnuclear foci are formed following I-SceI induced damage at two distinct chromosomal loci, that predominate in G1-S and G2 phase cells. Neither type of signal persists throughout the cell cycle, declining in post mitotic cells. Colocalization with γH2A suggests phosphorylation of H2A S133 is concentrated around the site of damage but can also occur in an undamaged chromatin context. While a strong pan-nuclear signal is not seen with γH2A in T. brucei, it has been reported in mammalian cells (78). Although it is unclear the role the pan-nuclear γH2AX plays, it does not inhibit repair, nor does it lead to a DDR in undamaged chromatin. It remains to be seen what the pan-nuclear H2A S133P function is in trypanosomes, whether there is interdependency between the two sites.

Phosphoproteomic studies of the DDR in human cells have also identified enrichment of proteins with RNA binding capacity (3) and a number of RNA binding proteins (RBPs) have been shown to have a dual role in both RNA binding and the DNA damage response. Such is the emerging evidence for the roles of RBPs in the DDR that a new class of proteins has been defined, the DNA damage response RNA binding proteins (DDRBPs) (79). Some of these DDRBPs can also bind double strand or single strand DNA and are involved in regulating R-loop formation (79, 80) by coating the nascent RNA (81, 82). R-loop formation is associated with increased DNA damage and VSG switching in T. brucei (23) and it is possible that some of the RBPs identified here supress R-loop formation following DSB induction. However, it is of note that in yeast R-loop formation is an important part of efficient HR (83, 84), and therefore possible that RBPs assist in productive R-loop formation that contributes to specifically double stand break repair in trypanosomes.

Although not DNA repair proteins, both NUP-1 and FK506-binding protein (Fig. 2, A and B) have been shown to be directly involved in DNA repair and recombination in yeast and mammalian cells, respectively. Several components of the yeast nuclear pore complex are phosphorylated in response to DNA damage, including NUP-1 at S637 (11), and have been shown to associate with damaged DNA and influence repair. In T. brucei, NUP-1 (Tb927.2.4230; nucleoporin 1) is organized into a lattice-like network at the nuclear periphery and maintains both nuclear architecture and chromatin organization (85). In our dataset, the phosphorylation status of NUP-1 following DNA damage is chromosome context dependent: It is phosphorylated in 1HR and dephosphorylated in VSGup cells, perhaps playing a role in repair pathway choice. FK506-binding proteins alter protein conformation through cis-trans isomerization of prolyl-peptide bonds, and in mammalian cells directs repair by promoting HR, potentially through its catalytic activity remodeling the chromatin environment (86). In trypanosomes, multiple sites in the FK506-binding protein are differentially phosphorylated, and again this is dependent on the chromatin context—being phosphorylated in 1HR and dephosphorylated in VSGup cells.

Protein phosphorylation is a dynamic process (87) and our results show only a snapshot of the DDR capturing the response at 12 h post DSB induction. Other post translational modifications are also important to both the DDR (6, 88, 89) and VSG switching, with histone methylation and acetylation important for antigenic variation (41, 90, 91), and SUMOylated proteins enriched at the active BES (92). This study is the first DNA damage phosphoproteome in T. brucei, and we have identified an abundance of novel proteins involved in the DDR. We have also revealed an overall trend toward phosphorylation following a break in a chromosomal internal region (1HR) and dephosphorylation following a break at a subtelomeric expression site (VSGup). We speculate this may reduce the stringency of DNA repair via HR which may be advantageous for antigenic variation. This remains to be tested. Validation of candidate phosphorylation sites from this dataset will provide key insights into the protein modifications that govern both DSBR and antigenic variation in T. brucei.

Experimental procedures

Trypanosome strains and culturing

T. brucei Lister 427 cell lines were grown in HMI-11 medium at 37.4 °C (75) with 5% CO2 and the density of cell cultures measured using a hemocytometer. The VSGup cell line has been described previously (20) and the 1HR cell line in (25).

HMI-9 SILAC medium

For all proteomic analysis, HMI-9 SILAC medium (55) minus L-Arginine and minus L-Lysine (Gibco, reference 074–91211A) was used. A 17.91 g pot of powder medium was used to make up 1L of medium by the addition of 900 ml H2O, 2 g sodium bicarbonate (Sigma-Aldrich), and 14 μl of beta-mercaptoethanol, and the mixture stirred for 1 h at room temperature. The pH was adjusted to 7.3, and the medium filtered using a 0.2 μM filter. The following components were added to the filtered medium: 10 ml of GlutaMAX (Thermo Fisher Scientific), 100 ml dialyzed SILAC fetal bovine serum (FBS) (3 kda molecular weight cutoff) (DC Biosciences) Gibco, 5 ml Pen/strep (5000 U/ml penicillin and 5000 μg/ml streptomycin) and heavy or light labeled L-arginine and L-lysine to the concentrations stated in Table 1 to make heavy and light medium, respectively. The final concentration of L-arginine is 120 μM and L-Lysine 240 μM.Table 1 The concentration of light and heavy labeled L-Arginine and L-Lysine used to supplement both IMDM and HMI-9 SILAC medium

Amino acid	Label	Source	Mw	Final concentration	
L-Arginine.HCl (R0)	Light	Sigma	210.6	25.8 mg/l	
L-Arginine.HCl-U-13C6 (R6)	Heavy	CIL	216.6	25.9 mg/l	
L-Lysine.HCl (K0)	Light	Sigma	182.6	43.8 mg/l	
L-Lysine.2HCl-4,4,5,5-2H4 (K4)	Heavy	CIL	223.1	53.5 mg/l	
CIL – Cambridge Isotope Labs, UK.

Assessing incorporation of the stable isotope label

To assess incorporation of the isotope label, the 1HR and VSGup cell lines were seeded in “heavy”, and “light” labeled SILAC HMI-9 and after 7 days 1 × 108 cells harvested from each cell culture. Samples were extracted by filter aided sample preparation (FASP) and processed for MS as described below.

Phosphoproteomic experimental set up

For preparation of samples for proteomic and phosphoproteomic analysis, 1HR and VSGup cell lines, grown in SILAC media for 7 days as detailed in the section above, were seeded in SILAC “heavy” and “light” medium and cells grown in “heavy” medium were induced using 1 μg/ml of tetracycline for 12 h. For each experimental condition, a label swap replicate was carried (induced cells grown in “light” media and uninduced in “heavy” media; Dataset S3). Approximately 3 × 108 cells were harvested from each culture condition by centrifugation at 1000g for 10 min at 4 °C. The supernatant was removed, and the cell pellet resuspended in 200 μl ice cold PBS and transferred to a microcentrifuge tube, where it was centrifuged at 12,000g for 15s and the supernatant discarded. The cell pellet was lysed at 0.5 × 109 cells/ml in ice-cold lysis buffer (0.1 mM N-tosyl-L-lysine-chloromethyl ketone, 1 μg/ml leupeptin, 1× phosphatase inhibitor cocktail II tablet (Calbiochem), 1 mM PMSF, and 1 mM benzamidine) and incubated at room temperature for 5 min, and the cell lysis was verified by microscopy. Cell lysates were then stored at −80 °C before further processing.

FASP protocol

The preparation of peptides for MS analysis was carried out using FASP (93) that has been optimized for T. brucei (55). For the four samples generated for investigation of the DSB response, the total amount of protein concentrate was 1 mg and 2 mg of protein for each of the 1HR label swap replicates, and 0.89 and 0.62 mg of protein for each of the VSGup replicates. For digestion of peptides, the concentrated sample was removed from the ultracentrifugal filter and a 1:50 ratio of mass spectrometry grade Trypsin Gold (Promega) added to the sample which was incubated with shaking for > 12 h at 37 °C in a thermal heat block. Trypsin digestion was inhibited by adding 0.1% formic acid (FA) to the digest.

Peptide desalting

Peptides were desalted using a Sep-Pak C18 SPE 360 mg cartridge (Waters) using a vacuum manifold according to manufacturer instructions. All buffers were freshly prepared. Briefly, C18 phase (Sep-Pak, Waters) was activated in methanol, rinsed once in 80% acetonitrile (ACN) with 0.1% FA, washed thrice in 0.1% FA. The sample was then loaded onto the cartridge twice. Resin was washed thrice in 0.1% FA and peptides were eluted in 50% ACN with 0.1% FA. The resulting sample was dried in a SpeedVac vacuum concentrator (Thermo Fisher Scientific) until 50 μl remained and then transferred to a nano LC tube (Thermo Fisher Scientific) and dried by lyophilization.

Phosphopeptide enrichment

Phosphopeptide enrichment and MS was carried out at the Mass Spectrometry for Biology Utechs (MSBio) platform at Institut Pasteur. Phosphopeptide enrichment was performed using a GELoader spin tip using EmporeTM C8 (3M) prepared for StageTip (Rappsilber et al.2007) and washed sequentially with 100% MeOH and 30% ACN, 0.1% trifluoroacetic acid (TFA). Before the enrichment step, 10 mg/ml TiO2 slurry (Sachtopore-NP TiO2, 5 μm, 300 Å, Sachtleben) was prepared in 30% ACN, 0.1% TFA and introduced into the GELoader C8 spin tip. The spin column was packed by centrifugation at 100g and then equilibrated loading buffer (80% ACN, 6% TFA, 1 M glycolic acid) before loading lyophilized tryptic peptides resuspended loading buffer at a ratio of 1:5 peptides to beads. An aliquot (10 μg) of tryptic peptides was retained for proteome analysis. TiO2 spin tip was first washed with 80% ACN, 6% TFA, and then with 50% ACN, 0.1% TFA at 200g. Phosphopeptides were eluted from TiO2 beads by transfer to into a new microcentrifuge tube containing 20% FA, using 10% NH4OH solution via centrifugation at 100g. To prevent the loss of phosphopeptides retained by the C8 plug a second elution was carried out with 80% ACN, 2% FA via centrifugation at 100g. Eluate fractions were combined and lyophilized prior to mass spectrometry analysis.

Mass spectrometry analysis

Peptides were analyzed on a Q-Exactive HF instrument (Thermo Fisher Scientific) coupled with an EASY nLC 1200 chromatography system (Thermo Fisher Scientific). Samples were loaded at 900 bars on an in-house packed 50 cm nano-HPLC column (75 μm inner diameter) with C18 resin (3 μm particles, 100 Å pore size, Reprosil-Pur Basic C18-HD resin) and equilibrated in 98% solvent A (H2O, 0.1% FA) and 2% solvent B (ACN, 0.1% FA). For both proteome and phosphoproteome analysis, peptides were eluted using a three to 29% gradient of solvent B during 105 min, then a 29 to 56% gradient of solvent B during 20 min and finally a 56 to 90% gradient of solvent B during 5 min all at 250 nl/minute flow rate. The instrument method for the Q-Exactive HF was set up in the data dependent acquisition mode. After a survey scan in the Orbitrap (resolution 60,000), the 12 most intense precursor ions were selected for higher energy collisional dissociation fragmentation with a normalized collision energy set up to 26. Precursors were selected with a window of 2.0 Th. Tandem MS spectra were recorded with a resolution of 15,000. Charge state screening was enabled, and precursors with unknown charge state or a charge state of 1 and > 7 were excluded. Dynamic exclusion was enabled for 30 s. For phosphoproteomic analysis, technical replicates were carried out in which samples acquisition was repeated twice for each label swap replicate.

MS data processing

All raw data were searched using MaxQuant software version 1.6.10.43 (https://www.maxquant.org/) (59, 94), which incorporates the Andromeda search engine (95), against the T. brucei 927 genome downloaded from TritrypDB (http://www.tritrypdb.org/) (Version 47, 11,074 protein sequences) (96) supplemented with frequently observed contaminants (such as mammalian keratins, porcine trypsin, and bovine serum albumins (BSAs)). All SILAC features were selected by default using the appropriate heavy K and R amino acid to be detected. Modifications included carbamidomethylation (Cys, fixed), oxidation (Met, variable) and N-terminal acetylation (variable) and N-pyroglutamate (variable) and phosphorylation (S, T, and Y variable). The mass tolerance was set to 6 parts per million (ppm) and peptides were required to be minimum 7 amino acids in length. Matching between runs allows peptides that are present in one sample but not identified by tandem MS in all samples to be identified by similarities in retention times and mass. The FDRs of 0.01 was calculated from the number of hits against a reversed sequence database. Only phosphorylation sites with a MaxQuant localization probability > 0.95 were considered.

Statistical analysis of proteomic data

Statistical analysis was carried out using Perseus (94) version1.6.1.3 (https://maxquant.net/perseus/). SILAC ratios were transformed to Log2 and intensities to Log10. Values were subject to further quality filtering such that ratios with >100% variation between label swap replicates were removed, and the localization probability of each phosphorylation site was required to be ≥ 0.95. Significantly changing phosphorylation sites were identified using significance B testing (59) which takes into account the intensity-weighted significance and used a Benjamini-Hochberg correction (97) to set the FDR at ≤ 0.01. Categorical enrichment was calculated using a Fisher’s exact test with an FDR ≤ 0.01. GO term enrichment was carried out on the proteins with significantly enriched phosphosites for the 1HR and VSGup datasets, using a Fisher’s exact test (FDR ≤ 0.05). All other statistical analysis was carried out in Microsoft Excel and GraphPad Prism, version 10 (https://www.graphpad.com).

Anti-S133P antibody

Anti-S133P antisera was raised in rats using a keyhole limpet hemocyanin-conjugated phosphopeptide, C-KSGKHAKATP[pS]V (Davids Biotechnologie GmbH). Antiserum was affinity purified using the corresponding peptides.

Peptide competition assay

For the peptide competition assay, primary antibody Anti-S133P was preincubated with 40 ng ml−1 of the phosphopeptide KSGKHAKATP[pS]V, the peptide KSGKHAKATPSV or the equivalent volume in water, in PBS 0.01% Tween 20 with 3% BSA, for 1 h at room temperature prior to incubation with the immunoblot.

Immunoblotting

Approximately 1 × 107 parasites were treated with 1 μg mL-1 tetracycline for 12 h, or 0.0003% methyl methanesulfonate for 24 h. Western blotting was carried out according to standard protocols, except that 1× phosphatases inhibitor PhosSTOP (Roche) was added to the lysis buffer, and samples were separated on a 15% SDS-PAGE gel. Immunoblots were blocked in PBS 0.01% Tween 20 with 5% BSA. Primary T. brucei Anti-S133P antibody was used at a 1:250 dilution and secondary goat anti-rat IgG horseradish peroxidase (Bio-Rad) was used at a 1:10,000 dilution. Blots were revealed by chemiluminescence using the Amersham ECL Prime Western Blotting Detection Reagent Kit (GE HealthCare) and a ChemiDoc Touch Gel Imaging System.

Immunofluorescence analysis

Immunofluorescence was carried out according to standard protocols. In brief, cells were fixed in final 1% volume (v/v) formaldehyde, on ice for 30 min. Fixed cells were centrifuged for 1 min at 1000g and washed with 1 ml ice cold PBS, twice. Cells were settled onto poly-l-lysine treated slides for up to 30 min and washed 3 × 5 min in PBS. Blocking was carried out for 15 min in 50% FBS in PBS, and all antibody dilutions were in 3% FBS. Primary antibodies rat anti-S133P and rabbit anti-γH2A (53) were used at a concentration of 1:250. Secondary antibody, goat anti-rat AF488 (AlexaFluor plus, Invitrogen, Lot #XI350194) and goat anti-rabbit AF555 (AlexaFluor plus, Invitrogen, Lot #VC297826) was used at a concentration of 1:1000. Cells were mounted in Vectashield (Vectorlabs) containing 4 to 6 diamidine-2-phenylindole. Images were acquired using a ZEISS Axio Imager Z2 epifluorescence microscope combined with an Axiocam 506 mono camera. Acquisition software Zen 2.3 (blue edition) (https://www.zeiss.com), version 2.3.69.1005. Images were processed using Image J2, version 2.14/1.54f (98) (https://imagej.net/software/fiji/downloads). Statistical analysis was carried out in GraphPad Prism, Version 10 (https://www.graphpad.com).

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (99) partner repository with the dataset identifier PXD034455.

Supporting information

This article contains supporting information.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Supporting information

Figure S1

Figure S2

Figure S3

Figure S4

Figure S5

Supplementary dataset 1

Supplementary dataset 2

Supplementary dataset 3

Supporting information

Author contributions

E. M., M. G. Z. M., and T. C., investigation; E. M., M. G. Z. M., Q. G. G., and M. D. U. formal analysis; E. M., M. G. Z. M., M. M., M. D. U., and L. G. writing–review and editing; E. M., M. G. Z. M., and L. G. writing–original draft; A. D.-H. validation; M. M. and L. G. resources.

Funding and additional information

Work in the LG laboratory has received financial support from the 10.13039/501100003762 Institut Pasteur . EJM is part of the Pasteur-Paris University (PPU) International PhD Program. This project has received funding from the European Union's 10.13039/100010661 Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No 665807 and from the Foundation Recherché Médicale grant number FDT202012010602 . M. G. Z. M. is funded by 10.13039/501100001665 Agence Nationale de la Recherche (ANR, https://anr.fr/) through the ParaFrap "Laboratoire d’Excellence" (LabEx, https://www.enseignementsuprecherche.gouv.fr/cid51355/laboratoires-d-excellence.html) (ANR-11-LABX-0024). Funding for open access charge: Institut Pasteur core funding.

Present address for Emilia McLaughlin: kyron.bio SAS, Institut Pierre-Gilles de Gennes, 6 Rue Jean Calvin, 75,005, Paris.
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References

1 Mehta A. Haber J.E. Sources of DNA double-strand breaks and models of recombinational DNA repair Cold Spring Harb. Perspect. Biol. 6 2014 a016428
2 Marechal A. Zou L. DNA damage sensing by the ATM and ATR kinases Cold Spring Harb. Perspect. Biol. 5 2013 a012716
3 Matsuoka S. Ballif B.A. Smogorzewska A. McDonald E.R. 3rd Hurov K.E. Luo J. ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage Science 316 2007 1160 1166 17525332
4 Rogakou E.P. Pilch D.R. Orr A.H. Ivanova V.S. Bonner W.M. DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139 J. Biol. Chem. 273 1998 5858 5868 9488723
5 Xiao A. Li H. Shechter D. Ahn S.H. Fabrizio L.A. Erdjument-Bromage H. WSTF regulates the H2A.X DNA damage response via a novel tyrosine kinase activity Nature 457 2009 57 62 19092802
6 Van H.T. Santos M.A. Histone modifications and the DNA double-strand break response Cell Cycle 17 2018 2399 2410 30394812
7 Celeste A. Fernandez-Capetillo O. Kruhlak M.J. Pilch D.R. Staudt D.W. Lee A. Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks Nat. Cell Biol. 5 2003 675 679 12792649
8 Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics Mol. Cell Proteomics 1 2002 376 386 12118079
9 Bennetzen M.V. Larsen D.H. Bunkenborg J. Bartek J. Lukas J. Andersen J.S. Site-specific phosphorylation dynamics of the nuclear proteome during the DNA damage response Mol. Cell Proteomics 9 2010 1314 1323 20164059
10 Bensimon A. Schmidt A. Ziv Y. Elkon R. Wang S.Y. Chen D.J. ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage Sci. Signal. 3 2010 rs3
11 Zhou C. Elia A.E. Naylor M.L. Dephoure N. Ballif B.A. Goel G. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks Proc. Natl. Acad. Sci. U. S. A. 113 2016 E3667 E3675 27298372
12 Trindade S. Rijo-Ferreira F. Carvalho T. Pinto-Neves D. Guegan F. Aresta-Branco F. Trypanosoma brucei parasites occupy and functionally adapt to the adipose tissue in mice cell Host Microbe 19 2016 837 848
13 Caljon G. Van Reet N. De Trez C. Vermeersch M. Perez-Morga D. Van Den Abbeele J. The dermis as a delivery site of trypanosoma brucei for tsetse flies PLoS Pathog. 12 2016 e1005744
14 Capewell P. Cren-Travaille C. Marchesi F. Johnston P. Clucas C. Benson R.A. The skin is a significant but overlooked anatomical reservoir for vector-borne African trypanosomes Elife 5 2016 e17716
15 Cross G.A. Identification, purification and properties of clone-specific glycoprotein antigens constituting the surface coat of Trypanosoma brucei Parasitology 71 1975 393 417 645
16 Horn D. Antigenic variation in African trypanosomes Mol. Biochem. Parasitol. 195 2014 123 129 24859277
17 Hertz-Fowler C. Figueiredo L.M. Quail M.A. Becker M. Jackson A. Bason N. Telomeric expression sites are highly conserved in Trypanosoma brucei PLoS One 3 2008 e3527
18 Berriman M. Ghedin E. Hertz-Fowler C. Blandin G. Renauld H. Bartholomeu D.C. The genome of the African trypanosome Trypanosoma brucei Science 309 2005 416 422 16020726
19 Boothroyd C.E. Dreesen O. Leonova T. Ly K.I. Figueiredo L.M. Cross G.A. A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei Nature 459 2009 278 281 19369939
20 Glover L. Alsford S. Horn D. DNA break site at fragile subtelomeres determines probability and mechanism of antigenic variation in African trypanosomes PLoS Pathog. 9 2013 e1003260
21 Benmerzouga I. Concepcion-Acevedo J. Kim H.S. Vandoros A.V. Cross G.A. Klingbeil M.M. Trypanosoma brucei Orc1 is essential for nuclear DNA replication and affects both VSG silencing and VSG switching Mol. Microbiol. 87 2013 196 210 23216794
22 Devlin R. Marques C.A. Paape D. Prorocic M. Zurita-Leal A.C. Campbell S.J. Mapping replication dynamics in Trypanosoma brucei reveals a link with telomere transcription and antigenic variation Elife 5 2016 e12765
23 Briggs E. Hamilton G. Crouch K. Lapsley C. McCulloch R. Genome-wide mapping reveals conserved and diverged R-loop activities in the unusual genetic landscape of the African trypanosome genome Nucleic Acids Res. 46 2018 11789 11805 30304482
24 Nanavaty V. Sandhu R. Jehi S.E. Pandya U.M. Li B. Trypanosoma brucei RAP1 maintains telomere and subtelomere integrity by suppressing TERRA and telomeric RNA:DNA hybrids Nucleic Acids Res. 45 2017 5785 5796 28334836
25 Glover L. McCulloch R. Horn D. Sequence homology and microhomology dominate chromosomal double-strand break repair in African trypanosomes Nucleic Acids Res. 36 2008 2608 2618 18334531
26 De Lange T. Kooter J.M. Michels P.A. Borst P. Telomere conversion in trypanosomes Nucleic Acids Res. 11 1983 8149 8165 6324075
27 Myler P.J. Allen A.L. Agabian N. Stuart K. Antigenic variation in clones of Trypanosoma brucei grown in immune-deficient mice Infect. Immun. 47 1985 684 690 2579027
28 Pays E. Guyaux M. Aerts D. Van Meirvenne N. Steinert M. Telomeric reciprocal recombination as a possible mechanism for antigenic variation in trypanosomes Nature 316 1985 562 564 2412122
29 Robinson N.P. Burman N. Melville S.E. Barry J.D. Predominance of duplicative VSG gene conversion in antigenic variation in African trypanosomes Mol. Cell Biol. 19 1999 5839 5846 10454531
30 Aitcheson N. Talbot S. Shapiro J. Hughes K. Adkin C. Butt T. VSG switching in Trypanosoma brucei: antigenic variation analysed using RNAi in the absence of immune selection Mol. Microbiol. 57 2005 1608 1622 16135228
31 Pays E. Delauw M.F. Van Assel S. Laurent M. Vervoort T. Van Meirvenne N. Modifications of a Trypanosoma b. brucei antigen gene repertoire by different DNA recombinational mechanisms Cell 35 1983 721 731 6197182
32 Rudenko G. McCulloch R. Dirks-Mulder A. Borst P. Telomere exchange can be an important mechanism of variant surface glycoprotein gene switching in Trypanosoma brucei Mol. Biochem. Parasitol. 80 1996 65 75 8885223
33 Dobson R. Stockdale C. Lapsley C. Wilkes J. McCulloch R. Interactions among Trypanosoma brucei RAD51 paralogues in DNA repair and antigenic variation Mol. Microbiol. 81 2011 434 456 21615552
34 Marin P.A. Obonaga R. Pavani R.S. da Silva M.S. de Araujo C.B. Lima A.A. ATR kinase is a crucial player mediating the DNA damage response in trypanosoma brucei Front. Cell Dev. Biol. 8 2020 602956
35 Black J.A. Crouch K. Lemgruber L. Lapsley C. Dickens N. Tosi L.R.O. Trypanosoma brucei ATR links DNA damage signaling during antigenic variation with regulation of RNA polymerase I-transcribed surface antigens Cell Rep. 30 2020 836 851.e835 31968257
36 Browne A.J. Guerra C.A. Alves R.V. da Costa V.M. Wilson A.L. Pigott D.M. The contemporary distribution of Trypanosoma cruzi infection in humans, alternative hosts and vectors Sci. Data 4 2017 170050
37 Wong A.K. Pero R. Ormonde P.A. Tavtigian S.V. Bartel P.L. RAD51 interacts with the evolutionarily conserved BRC motifs in the human breast cancer susceptibility gene brca2 J. Biol. Chem. 272 1997 31941 31944 9405383
38 Hartley C.L. McCulloch R. Trypanosoma brucei BRCA2 acts in antigenic variation and has undergone a recent expansion in BRC repeat number that is important during homologous recombination Mol. Microbiol. 68 2008 1237 1251 18430140
39 Trenaman A. Hartley C. Prorocic M. Passos-Silva D.G. van den Hoek M. Nechyporuk-Zloy V. Trypanosoma brucei BRCA2 acts in a life cycle-specific genome stability process and dictates BRC repeat number-dependent RAD51 subnuclear dynamics Nucleic Acids Res. 41 2013 943 960 23222131
40 Conway C. McCulloch R. Ginger M.L. Robinson N.P. Browitt A. Barry J.D. Ku is important for telomere maintenance, but not for differential expression of telomeric VSG genes, in African trypanosomes J. Biol. Chem. 277 2002 21269 21277 11919193
41 Glover L. Horn D. Locus-specific control of DNA resection and suppression of subtelomeric VSG recombination by HAT3 in the African trypanosome Nucleic Acids Res. 42 2014 12600 12613 25300492
42 Kim H.S. Cross G.A. TOPO3alpha influences antigenic variation by monitoring expression-site-associated VSG switching in Trypanosoma brucei PLoS Pathog. 6 2010 e1000992
43 McCulloch R. Barry J.D. A role for RAD51 and homologous recombination in Trypanosoma brucei antigenic variation Genes Dev. 13 1999 2875 2888 10557214
44 Proudfoot C. McCulloch R. Distinct roles for two RAD51-related genes in Trypanosoma brucei antigenic variation Nucleic Acids Res. 33 2005 6906 6919 16326865
45 Laffitte M.C. Genois M.M. Mukherjee A. Legare D. Masson J.Y. Ouellette M. Formation of linear amplicons with inverted duplications in Leishmania requires the MRE11 nuclease PLoS Genet. 10 2014 e1004805
46 Laffitte M.C. Leprohon P. Hainse M. Legare D. Masson J.Y. Ouellette M. Chromosomal translocations in the parasite Leishmania by a MRE11/RAD50-independent microhomology-mediated end joining mechanism PLoS Genet. 12 2016 e1006117
47 Robinson N.P. McCulloch R. Conway C. Browitt A. Barry J.D. Inactivation of Mre11 does not affect VSG gene duplication mediated by homologous recombination in Trypanosoma brucei J. Biol. Chem. 277 2002 26185 26193 12011090
48 Mehnert A.K. Prorocic M. Dujeancourt-Henry A. Hutchinson S. McCulloch R. Glover L. The MRN complex promotes DNA repair by homologous recombination and restrains antigenic variation in African trypanosomes Nucleic Acids Res. 49 2021 1436 1454 33450001
49 Mankouri H.W. Hickson I.D. The RecQ helicase-topoisomerase III-Rmi1 complex: a DNA structure-specific 'dissolvasome'? Trends Biochem. Sci. 32 2007 538 546 17980605
50 Kim H.S. Cross G.A. Identification of Trypanosoma brucei RMI1/BLAP75 homologue and its roles in antigenic variation PLoS One 6 2011 e25313
51 Glover L. Alsford S. Beattie C. Horn D. Deletion of a trypanosome telomere leads to loss of silencing and progressive loss of terminal DNA in the absence of cell cycle arrest Nucleic Acids Res. 35 2007 872 880 17251198
52 Glover L. Marques C.A. Suska O. Horn D. Persistent DNA damage Foci and DNA replication with a broken chromosome in the african trypanosome mBio 10 2019 10.1128/mBio.01252-19
53 Glover L. Horn D. Trypanosomal histone gammaH2A and the DNA damage response Mol. Biochem. Parasitol. 183 2012 78 83 22353557
54 Marchese L. Nascimento J.F. Damasceno F.S. Bringaud F. Michels P.A.M. Silber A.M. The uptake and metabolism of amino acids, and their unique role in the biology of pathogenic Trypanosomatids Pathogens 7 2018 36 29614775
55 Urbaniak M.D. Martin D.M. Ferguson M.A. Global quantitative SILAC phosphoproteomics reveals differential phosphorylation is widespread between the procyclic and bloodstream form lifecycle stages of Trypanosoma brucei J. Proteome Res. 12 2013 2233 2244 23485197
56 Xu X. Xiong X. Sun Y. The role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity Sci. China Life Sci. 59 2016 656 672 27294833
57 Riepe C. Zelin E. Frankino P.A. Meacham Z.A. Fernandez S.G. Ingolia N.T. Double stranded DNA breaks and genome editing trigger loss of ribosomal protein RPS27A FEBS J. 289 2021 3101 3114
58 Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips Nat. Protoc. 2 2007 1896 1906 17703201
59 Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification Nat. Biotechnol. 26 2008 1367 1372 19029910
60 Fernandez-Capetillo O. Allis C.D. Nussenzweig A. Phosphorylation of histone H2B at DNA double-strand breaks J. Exp. Med. 199 2004 1671 1677 15197225
61 Chen Y. Chen C.F. Chiang H.C. Mutation of NIMA-related kinase 1 (NEK1) leads to chromosome instability Mol. Cancer 10 2011 5 21214959
62 Vassin V.M. Anantha R.W. Sokolova E. Kanner S. Borowiec J.A. Human RPA phosphorylation by ATR stimulates DNA synthesis and prevents ssDNA accumulation during DNA-replication stress J. Cell Sci. 122 2009 4070 4080 19843584
63 Wu X. Yang Z. Liu Y. Zou Y. Preferential localization of hyperphosphorylated replication protein A to double-strand break repair and checkpoint complexes upon DNA damage Biochem. J. 391 2005 473 480 15929725
64 Shi W. Feng Z. Zhang J. Gonzalez-Suarez I. Vanderwaal R.P. Wu X. The role of RPA2 phosphorylation in homologous recombination in response to replication arrest Carcinogenesis 31 2010 994 1002 20130019
65 Byrne B.M. Oakley G.G. Replication protein A, the laxative that keeps DNA regular: the importance of RPA phosphorylation in maintaining genome stability Semin. Cell Dev. Biol. 86 2019 112 120 29665433
66 Redon C. Pilch D. Rogakou E. Sedelnikova O. Newrock K. Bonner W. Histone H2A variants H2AX and H2AZ Curr. Opin. Genet. Dev. 12 2002 162 169 11893489
67 Foster E.R. Downs J.A. Histone H2A phosphorylation in DNA double-strand break repair FEBS J. 272 2005 3231 3240 15978030
68 Kruhlak M. Crouch E.E. Orlov M. Montano C. Gorski S.A. Nussenzweig A. The ATM repair pathway inhibits RNA polymerase I transcription in response to chromosome breaks Nature 447 2007 730 734 17554310
69 Ogawa L.M. Baserga S.J. Crosstalk between the nucleolus and the DNA damage response Mol. Biosyst. 13 2017 443 455 28112326
70 Grynberg P. Passos-Silva D.G. Mourao Mde M. Hirata R. Jr. Macedo A.M. Machado C.R. Trypanosoma cruzi gene expression in response to gamma radiation PLoS One 7 2012 e29596
71 Vieira H.G. Grynberg P. Bitar M. Pires Sda F. Hilario H.O. Macedo A.M. Proteomic analysis of Trypanosoma cruzi response to ionizing radiation stress PLoS One 9 2014 e97526
72 Powley I.R. Kondrashov A. Young L.A. Dobbyn H.C. Hill K. Cannell I.G. Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes Genes Dev. 23 2009 1207 1220 19451221
73 Cerqueira P.G. Passos-Silva D.G. Vieira-da-Rocha J.P. Mendes I.C. de Oliveira K.A. Oliveira C.F. Effect of ionizing radiation exposure on Trypanosoma cruzi ubiquitin-proteasome system Mol. Biochem. Parasitol. 212 2017 55 67 28137628
74 Ooi C.P. Benz C. Urbaniak M.D. Phosphoproteomic analysis of mammalian infective Trypanosoma brucei subjected to heat shock suggests atypical mechanisms for thermotolerance J. Proteomics 219 2020 103735
75 Benz C. Urbaniak M.D. Organising the cell cycle in the absence of transcriptional control: dynamic phosphorylation co-ordinates the Trypanosoma brucei cell cycle post-transcriptionally PLoS Pathog. 15 2019 e1008129
76 Xie A. Odate S. Chandramouly G. Scully R. H2AX post-translational modifications in the ionizing radiation response and homologous recombination Cell Cycle 9 2010 3602 3610 20703100
77 Moore J.D. Yazgan O. Ataian Y. Krebs J.E. Diverse roles for histone H2A modifications in DNA damage response pathways in yeast Genetics 176 2007 15 25 17028320
78 Meyer B. Voss K.O. Tobias F. Jakob B. Durante M. Taucher-Scholz G. Clustered DNA damage induces pan-nuclear H2AX phosphorylation mediated by ATM and DNA-PK Nucleic Acids Res. 41 2013 6109 6118 23620287
79 Dutertre M. Vagner S. DNA-damage response RNA-binding proteins (DDRBPs): perspectives from a new class of proteins and their RNA targets J. Mol. Biol. 429 2017 3139 3145 27693651
80 Aguilera A. Garcia-Muse T. R loops: from transcription byproducts to threats to genome stability Mol. Cell 46 2012 115 124 22541554
81 Nishida K. Kuwano Y. Nishikawa T. Masuda K. Rokutan K. RNA binding proteins and genome integrity Int. J. Mol. Sci. 18 2017 1341 28644387
82 Sollier J. Stork C.T. Garcia-Rubio M.L. Paulsen R.D. Aguilera A. Cimprich K.A. Transcription-coupled nucleotide excision repair factors promote R-loop-induced genome instability Mol. Cell 56 2014 777 785 25435140
83 Keskin H. Shen Y. Huang F. Patel M. Yang T. Ashley K. Transcript-RNA-templated DNA recombination and repair Nature 515 2014 436 439 25186730
84 Ohle C. Tesorero R. Schermann G. Dobrev N. Sinning I. Fischer T. Transient RNA-DNA hybrids are required for efficient double-strand break repair Cell 167 2016 1001 1013.e1007 27881299
85 DuBois K.N. Alsford S. Holden J.M. Buisson J. Swiderski M. Bart J.M. NUP-1 Is a large coiled-coil nucleoskeletal protein in trypanosomes with lamin-like functions PLoS Biol. 10 2012 e1001287
86 Dilworth D. Gong F. Miller K. Nelson C.J. FKBP25 participates in DNA double-strand break repair Biochem. Cell Biol. 98 2020 42 49 30620620
87 Gelens L. Qian J. Bollen M. Saurin A.T. The importance of kinase-phosphatase integration: lessons from mitosis Trends Cell Biol. 28 2018 6 21 29089159
88 Cremona C.A. Sarangi P. Zhao X. Sumoylation and the DNA damage response Biomolecules 2 2012 376 388 24926426
89 Lee N.S. Kim S. Jung Y.W. Kim H. Eukaryotic DNA damage responses: homologous recombination factors and ubiquitin modification Mutat. Res. 809 2018 88 98 28552167
90 Figueiredo L.M. Janzen C.J. Cross G.A. A histone methyltransferase modulates antigenic variation in African trypanosomes PLoS Biol. 6 2008 e161
91 Wang Q.P. Kawahara T. Horn D. Histone deacetylases play distinct roles in telomeric VSG expression site silencing in African trypanosomes Mol. Microbiol. 77 2010 1237 1245 20624217
92 Lopez-Farfan D. Bart J.M. Rojas-Barros D.I. Navarro M. SUMOylation by the E3 ligase TbSIZ1/PIAS1 positively regulates VSG expression in Trypanosoma brucei PLoS Pathog. 10 2014 e1004545
93 Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis Nat. Methods 6 2009 359 362 19377485
94 Tyanova S. Temu T. Cox J. The MaxQuant computational platform for mass spectrometry-based shotgun Proteomics Nat. Protoc. 11 2016 2301 2319 27809316
95 Cox J. Neuhauser N. Michalski A. Scheltema R.A. Olsen J.V. Mann M. Andromeda: a peptide search engine integrated into the MaxQuant environment J. Proteome Res. 10 2011 1794 1805 21254760
96 Amos B. Aurrecoechea C. Barba M. Barreto A. Basenko E.Y. Bazant W. VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center Nucleic Acids Res. 50 2022 D898 D911 34718728
97 Benjamini Y. Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing the J. R. Stat. Soc. Ser. B 57 1995 289 300
98 Schindelin J. Arganda-Carreras I. Frise E. Kaynig V. Longair M. Pietzsch T. Fiji: an open-source platform for biological-image analysis Nat. Methods 9 2012 676 682 22743772
99 Perez-Riverol Y. Bai J. Bandla C. Garcia-Seisdedos D. Hewapathirana S. Kamatchinathan S. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences Nucleic Acids Res. 50 2022 D543 D552 34723319
