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Microb Biotechnol
Microb Biotechnol
10.1111/(ISSN)1751-7915
MBT2
Microbial Biotechnology
1751-7915
John Wiley and Sons Inc. Hoboken

10.1111/1751-7915.70008
MBT270008
MICROBIO-2024-173-RAR.R1
Research Article
Research Article
A novel DNA damage detection method based on a distinct DNA damage response system
A novel DNA damage detection method
Zhong et al.
Zhong Shitong 1
Song Shuang 1
Wang Linjia 1
Liu Yufeng 1
Xu Hong 1 2
Wang Liangyan 1
Lu Huizhi https://orcid.org/0000-0002-2408-4427
1 huizhilu@zju.edu.cn

Hua Yuejin https://orcid.org/0000-0001-6743-0919
1 2 yjhua@zju.edu.cn

1 MOE Key Laboratory of Biosystems Homeostasis & Protection Institute of Biophysics, College of Life Sciences, Zhejiang University Hangzhou Zhejiang China
2 Cancer Center Zhejiang University Hangzhou Zhejiang China
* Correspondence
Yuejin Hua and Huizhi Lu, MOE Key Laboratory of Biosystems Homeostasis & Protection, Institute of Biophysics, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, China.
Email: yjhua@zju.edu.cn and huizhilu@zju.edu.cn

17 9 2024
9 2024
17 9 10.1111/mbt2.v17.9 e7000826 3 2024
14 8 2024
© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc-nd/4.0/ License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

Abstract

DNA damage occurs when cells encounter exogenous and endogenous stresses such as long periods of desiccation, ionizing radiation and genotoxic chemicals. Efforts have been made to detect DNA damage in vivo and in vitro to characterize or quantify the damage level. It is well accepted that single‐stranded DNA (ssDNA) is one of the important byproducts of DNA damage to trigger the downstream regulation. A recent study has revealed that PprI efficiently recognizes ssDNA and cleaves DdrO at a specific site on the cleavage site region (CSR) loop in the presence of ssDNA, which enables the radiation resistance of Deinococcus. Leveraging this property, we developed a quantitative DNA damage detection method in vitro based on fluorescence resonance energy transfer (FRET). DdrO protein was fused with eYFP and eCFP on the N‐terminal and C‐terminal respectively, between which the FRET efficiency serves as an indicator of cleavage efficiency as well as the concentration of ssDNA. The standard curve between the concentration of ssDNA and the FRET efficiency was constructed, and application examples were tested, validating the effectiveness of this method.

FRET state diagram before and after cleavage. Before cleavage, the eCFP and eYFP groups in YDC approach each other, allowing emitted light of eCFP to excite eYFP. After cleavage by activated PprI‐D91A, the two groups separate, making it difficult for the eCFP's emission light to excite eYFP, resulting in a decrease in FRET efficiency.

National Natural Science Foundation of China 10.13039/501100001809 32370028 32200016 Natural Science Foundation of Zhejiang Province 10.13039/501100004731 LQ23C010002 source-schema-version-number2.0
cover-dateSeptember 2024
details-of-publishers-convertorConverter:WILEY_ML3GV2_TO_JATSPMC version:6.4.8 mode:remove_FC converted:17.09.2024
Zhong, S. , Song, S. , Wang, L. , Liu, Y. , Xu, H. , Wang, L. et al. (2024) A novel DNA damage detection method based on a distinct DNA damage response system. Microbial Biotechnology, 17 , e70008. Available from: 10.1111/1751-7915.70008
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pmcINTRODUCTION

DNA carries genetic information and instructions for protein synthesis, playing a critical role in life processes by regulating several pathways, including energy metabolism, DNA replication and repair, stress response, transcriptional regulation, signal transduction, protein turnover and so on. Once DNA damage occurs and is not repaired timely, the generated mutations accumulate till the cell loses control of normal metabolism and turns to apoptosis or cancer.

To understand the cellular DNA damage landscape, scientists have developed various detection techniques. The most used comet assay (single cell gel electrophoresis, SCGE) is designed to effectively determine the extent of DNA single or double‐strand nick damage in cells, based on changes in DNA physicochemical properties (Ostling & Johanson, 1984; Singh et al., 1988). Damaged DNA fragments with broken ends migrate away from the nucleus, resembling a ‘comet’ with a brightly stained head and a tail during electrophoresis. It allows the assessment of various types of DNA damage, including single‐strand breaks, double‐strand breaks and alkali‐labile sites, while it requires expertise in image analysis and its interpretation can be subjective. DNA break detection fluorescence in situ hybridization (DBD‐FISH) uses fluorescent DNA probes that target repetitive sequences or specific genes to detect and localize DNA damage (Cortes‐Gutierrez et al., 2012). It allows the examination of specific genomic regions or particular chromosomes, enabling the investigation of DNA damage hotspots or vulnerable regions. However, it requires a combination of immunostaining and fluorescence in situ hybridization (FISH) techniques, making it a complex and time‐consuming method. Relying on the phosphorylation of the histone variant H2AX at serine 139 (forming γ‐H2AX) at double‐strand break (DSB) sites, γ‐H2AX assay allows for sensitive and specific detection of DSBs in individual cells and provides a quantitative readout of the level of DNA damage within a cell population (Ismail et al., 2007). It cannot distinguish between different sources or types of DSBs, as γ‐H2AX formation is a response to any DSB. Some factors, such as DNA replication or cellular senescence, can lead to increased baseline levels of γ‐H2AX, making it important to carefully interpret assay results (Chowdhury et al., 2008; Lou & Chen, 2006; Moeglin et al., 2019).

Circulating cell‐free DNA (cfDNA) level is used as a biomarker for ailments such as cancer, in which DNA damage occurs frequently (Butt & Swaminathan, 2008; Casadio et al., 2013; Huang et al., 2020). Assays for measuring cfDNA content have been developed, such as Qubit assays, which could quantify ssDNA or double‐stranded DNA (dsDNA) in vitro (Mardis & McCombie, 2017; Ponti et al., 2018). However, the Qubit assays cannot distinguish injured DNA from undamaged DNA, as well as ssDNA from dsDNA.

Deinococcus, as a gifted model for studying DNA damage response, is one of the most robust organisms that can tolerate extreme environments, such as high doses of ionizing radiation, UV radiation, oxidation, mitomycin C and long periods of desiccation (Battista et al., 1999; Krisko & Radman, 2013; Slade & Radman, 2011). PprI (also called IrrE) is identified as an essential protein for Deinococcus that contributes to resistance as well as normal metabolism, and it regulates a series of DNA damage response genes (Hua et al., 2003; Wang et al., 2015). In coordination with the transcriptional repressor DdrO, which specifically binds to the RDRM (radiation/desiccation response motif)‐containing promoters, PprI mediates a distinct DNA damage response system (Blanchard et al., 2017; Lu et al., 2012; Ludanyi et al., 2014; Wang et al., 2015). The protease PprI cleaves the last α‐helix and destabilizes the C‐terminal hydrophobic core of transcriptional repressor DdrO, leading to the disruption of DdrO dimer formation and promoter binding capability (Lu et al., 2024; Ludanyi et al., 2014; Wang et al., 2015). Unlike most DNA damage response genes that are up‐regulated after irradiation, the transcriptomic analysis by Liu in 2003 stated that the transcription level of pprI remained constant during the early, middle and late phases of genome recovery (Liu et al., 2003), indicating that PprI can be activated by some kinds of undefined mechanisms. It is recently reported that ssDNA can directly bind to PprI and dramatically activate its protease activity, which guarantees the efficient response to DNA damage (Lu et al., 2024).

In this article, we designed a novel DNA damage detection method in vitro based on the PprI‐DdrO response system with the FRET method which can measure damaged DNA quantitatively. Fluorescence‐labelled (eYFP and eCFP) DdrO was constructed as the substrate of PprI and a specific site of PprI was mutated to enhance the detection efficiency. The FRET efficiency between the fused eYFP and eCFP on DdrO serves as an indicator of cleavage efficiency as well as the concentration of ssDNA. The standard curve was constructed to precisely calculate the concentration of ssDNA and cases were used to verify the feasibility of the method. With this developed method, researchers can eliminate the interference from dsDNA and quickly measure ssDNA content in solution, facilitating the detection of cfDNA, thereby contributing to the diagnosis of cancers.

EXPERIMENTAL PROCEDURES

Bacterial strains, media, chemical reagents and primers

The plasmids and strains used in the study are shown in Table S1, and the primers used for vector construction are shown in Table S2. E. coli Trans5a and E. coli BL21(DE3) were obtained from Beijing TransGen Biotech Co., Ltd. Unless otherwise specified, chemical reagents used in this study were purchased from Sangon Biotech (Shanghai) Co., Ltd. The primers were synthesized by Hangzhou Tsingke Biology Co., Ltd. E. coli was cultured in LB medium containing 0.5% yeast extract, 1% tryptone and 1% NaCl. LB solid medium was supplemented with 1.5% agar.

Construction of plasmids

We fused eyfp and ecfp with complete ddro gene (dr_2574) from Deinococcus radiodurans and constructed them in pET‐28a(+) vector. Similarly, we amplified the complete gene of the PprI protein (DR_0167), DdrO protein (DR_2574), eYFP and eCFP and also constructed them in the pET‐28a(+) vector, respectively. pET‐28a‐YDC was constructed by sequentially recombining eyfp, ddro and ecfp fragments onto pET‐28a using Exnase II from Nanjing Vazyme Biotech Co., Ltd. pET‐28a‐PprI was constructed by recombining the ppri gene onto pET‐28a using Exnase II. Using the same method as that employed for constructing pET‐28a‐PprI, the pET‐28a‐DdrO, pET‐28a‐eYFP and pET‐28a‐eCFP were also constructed together. pET‐28a‐PprI‐D91A, pET‐28a‐PprI‐R111A and pET‐28a‐PprI‐N153A were obtained by introducing point mutation into pET‐28a‐PprI, respectively. Each protein possessed a 6 × His tag at the N‐terminus for protein purification, and the tag was not removed throughout the experimental process. Plasmid construction was performed using E. coli Trans5a competent cells and the working concentration of the antibiotic kanamycin was 40 μg/mL. All the insert fragments were amplified using TransStart® FastPfu DNA Polymerase and the primers listed in Table S2.

Expression and purification of proteins

The plasmids were transformed into E. coli BL21 (DE3). The transformed strains were inoculated into LB liquid medium and cultured at 37°C until OD600 was about 0.6. Then, isopropyl‐β‐D‐thiogalactoside (IPTG) was added to a final concentration of 0.2 mM to induce the expression of the target proteins. The proteins were induced to express at 30°C for 6 h before the cells were harvested by centrifugation.

For protein purification, the cells were first washed once with phosphate‐buffered saline (PBS) and resuspended in buffer A (5% glycerol, 1 M NaCl, 20 mM Tris–HCl, pH 7.5). Then cells were then disrupted using a High Pressure Cell Cracker from JNBio. After centrifuging the cell lysate at 12000 rpm for 30 min, purification was performed using the AKTA pure25 purification system from GE Healthcare (Pittsburgh, PA, USA). The supernatant of the cell lysate was loaded onto a Ni‐NTA column and the column was equilibrated with buffer A. After equilibration, 5% buffer B (5% glycerol, 1 M NaCl, 500 mM imidazole, 20 mM Tris‐HCl, pH 7.5) was used to wash away interfering proteins and 30% buffer B was used to elute the target protein. The buffer of YDC was changed to buffer C (5% glycerol, 150 mM NaCl, 1 mM EDTA, 20 mM Tris‐HCl, pH 7.5), and the buffer of eYFP, eCFP, DdrO, PprI and mutants of PprI was changed to buffer D (5% glycerol, 150 mM NaCl, 20 mM Tris‐HCl, pH 7.5) using a 50 mL desalting column. YDC protein was further purified using a heparin column equilibrated with buffer C and eluted with 30% buffer A. Finally, the purification results of the proteins were analysed by SDS‐PAGE. Proteins intended for use within a few weeks were stored at 4°C. For long‐term storage, proteins were rapidly frozen with liquid nitrogen before being placed at −20°C or −80°C.

Size exclusion chromatography

The size exclusion chromatography of YDC was performed using Superdex 200 Increase 10/300 GL columns with AKTA pure25 purification system under the conditions of buffer E (5% glycerol, 250 mM NaCl, 20 mM Tris‐HCl and pH 7.5). The protein concentration was approximately 1 mg/mL, and the flow rate was set at 0.4 mL/min.

Scanning of emission spectrum and determination of salt concentration

After adjusting the purified YDC to 3 μM with buffer E, the emission spectra of YDC were measured using multimode plate reader SpectraMax M5 from Molecular Devices (California, USA) under excitation light of 440 and 480 nm, with emission wavelengths ranging from 460 to 600 nm and 500 to 600 nm, respectively, with a step size of 5 nm. Following the same procedure, the emission spectra of 3 μM eYFP, 3 μM eCFP, and their mixture (with both eYFP and eCFP at a final concentration of 3 μM) were measured under excitation lights of 440 and 480 nm. Afterwards, the emission light of YDC (ranging from 460 to 600 nm) was measured separately at excitation wavelengths of 430, 435, 440, 445 and 450 nm. Subsequently, YDC solutions were prepared to a final concentration of 3 μM, and the salt concentrations were adjusted to 50, 100, 250, 500 mM and 1 M, respectively. The emission of YDC (ranging from 460 to 600 nm) at different salt concentrations was measured under 440 nm excitation light. The emission spectra (ranging from 460 to 600 nm) for the other samples in this article were uniformly assessed at an excitation wavelength of 440 nm. The Relative Fluorescence Units (RFU) obtained by the machine were used to characterize the intensity of the emission light.

Confirmation of enzyme activity

The cleavage experiment of PprI on YDC was conducted in buffer E. The final concentrations of PprI, the substrates (DdrO or YDC) and MnCl2 were 1 μM, 3 μM and 1 mM, respectively, and the reaction was carried out at 37°C for 30 min. For the time‐course cleavage on YDC, the emission spectrum scanning was performed according to the above method after 0, 15 and 30 min of reaction.

Enzyme cleavage experiment to determine the activation effect of ssDNA on PprI was conducted using oligo‐dT (37 nt) and the oligonucleotide fp‐pet‐f as ssDNA. The cleavage reaction was performed at 37°C for 30 min in buffer E, with final concentrations of PprI, DdrO, MnCl2, and ssDNA at 0.3 μM, 3 μM, 1 mM and 0.1 μM, respectively.

Cleavage assays were also performed under buffer E conditions to identify suitable enzyme mutants with the final concentrations of the enzymes (PprI, PprI‐D91A, PprI‐R111A or PprI‐N153A), DdrO, and MnCl2 being 1 μM, 3 μM and 1 mM, respectively. The ssDNA (oligonucleotide fp‐pet‐f) was added as required to a final concentration of 0 μM or 0.5 μM, and the reaction was carried out at 37°C for 30 min.

To verify the enzymatic cleavage effect on YDC, 100 μL of samples were prepared by adding enzyme (PprI or PprI‐D91A), YDC and MnCl2. The concentration of NaCl was adjusted to 250 mM using buffers and ddH2O. Buffer E was used to make up the volume. The final concentrations of enzyme, YDC, and Mn2+ were 1 μM, 3 μM and 1 mM, respectively. ssDNA (oligonucleotide fp‐pet‐f) was added as needed to final concentrations of 0, 0.05 and 0.5 μM, and the reaction system was incubated at 37°C for 30 min. Three ssDNAs (ssDNA‐I, ssDNA‐II and ssDNA‐III) were used to prepare different DNA samples. The ssDNA sample was prepared using ssDNA‐I directly. The blunt‐end sample was obtained by annealing ssdna‐I and ssdna‐II. The 5′‐overhang sample was obtained by annealing ssdna‐I and ssdna‐III. The concentration of the DNA sample was 5 μM, and 5 μL sample was added to 100 μL reaction system (DNA final concentration at 0.25 μM).

Treatment and preparation of experimental samples

To ensure the activation effect of ssDNA, we chose oligonucleotide fp‐pet‐f as the preparation of a standard ssDNA sample. The initial ssDNA concentrations were 5, 2, 1 μM, 500, 200, 100, 50, 20, 10, 5, 2 and 1 nM. The genome was obtained from E. coli BL21(DE3) strain, and extracted using a bacterial genomic DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd. The genome concentration was adjusted to approximately 30 ng/μL and the sample was divided into four parts. Each part was subjected to different treatments: stored at 4°C for 10 days, incubated at 37°C for 5 days and then stored at 4°C, incubated at 37°C for 10 days and incubated at 70°C for 10 days, respectively.

For the ssDNA samples in different solutions, the ssDNA dry powder was dissolved separately in PBS, LB liquid medium, foetal bovine serum (FBS), and Dulbecco's modified eagle medium (DMEM, supplemented with 10% FBS) and prepared into ssDNA solutions of concentrations 0 μM, 0.1 μM and 1 μM. For the analysis of the extracellular solutions from E. coli BL21(DE3), after overnight culture in LB liquid medium, 2 mL of the bacterial culture was spread on a clean plate with a diameter of 10 cm. The samples were then subjected to UV irradiation at an energy density of 100 J/m2 or were maintained without exposure. Afterwards, 1.6 mL of the bacterial culture was transferred to a clean Eppendorf tube and centrifuged at 5000 rpm for 3 min to remove the supernatant. The resulting pellet was washed twice with PBS to eliminate residual LB medium. Finally, the bacterial cells were resuspended in 1.6 mL of PBS and incubated with shaking at 37°C. Samples of 60 μL were taken at 0, 0.5, 1, 2 and 4 h, and the samples were centrifuged at 12,000 rpm for 1 min to collect the supernatant for subsequent ssDNA detection experiments. The human cervical cancer cell line HeLa (ATCC) was cultured in 6 mL of DMEM containing 10% FBS in a 10‐cm culture dish, and incubated at 37°C with 5% CO2 for 48 h. The cells were then subjected to UV irradiation at an energy density of 100 J/m2 or left untreated. After removing the original medium, 6 mL of fresh medium was added and the culture was continued in the incubator. At 0, 0.5, 1, 2 and 4 h, 200 μL of the culture supernatant was collected for ssDNA detection experiments.

Qubit ssDNA assay

The ssDNA rapid quantification kit for Qubit was purchased from Sangon Biotech (Shanghai) Co., Ltd. The ssDNA sample, blunt‐end sample, 5′‐overhang sample and gradient DNA samples (from 1 nM to 5 μM) were prepared as described above. 5 μL of the DNA sample was added to 195 μL of the ssDNA detection working solution to make a 200 μL reaction solution. After reacting in the dark for 2 min at room temperature, the fluorescence intensity was measured using Qubit™ 4 Fluorometer (Thermo Fisher Scientific Inc.), and RFU were used to characterize the intensity of the fluorescence.

Application of detection system

A total of 5 μL of each DNA sample or test sample was added to the detection system. The final concentrations of PprI‐D91A and YDC were adjusted to 0.1 and 3 μM, respectively. The final concentration of Mn2+ was 1 mM. Buffers and ddH2O were used to adjust the salt concentration to approximately 250 mM. Buffer E was used to supplement the system volume to 100 μL. The reaction was incubated at 37°C for 30 min. For the detection of samples with low ssDNA content, 50 μL of the sample was added to the system, maintaining the same concentrations of the enzyme, substrate and manganese ions as mentioned above, and buffer E was finally used to make up the detection volume to 100 μL.

Statistical analysis and reproducibility

All the experiment results have been successfully repeated for at least three times. Results of FRET assays were determined using GraphPad Prism 8. The results represented the means and standard errors (SE) of three independent experiments. One‐way ANOVA method followed by Tukey's post‐hoc test was performed to compare the significant differences.

RESULTS AND DISCUSSION

Construction and activity test of the substrate

The eYFP and eCFP tags were fused to the N‐terminus and C‐terminus of DdrO (DR_2574 from Deinococcus radiodurans) by flexible linker peptide GGGGSGGGGS respectively (Figure 1A, Figure S1A), which was named YDC for convenience. After induction and multi‐step purification, we obtained protein samples that met the experimental purity requirements (Figure S1B), followed by fluorescence excitation test ensuring the correct folding of the domains and proper fluorescence resonance energy transfer (FRET) between the two fluorophores (Figure 1B). When DdrO is intact and eYFP is close enough to eCFP (less than 5 nm), indicated as in Figure 1C, the light emitted from eCFP after being excitated can excitate eYFP to emit a longer‐wavelength light. The FRET between the two fluorophores would be eliminated when DdrO is hydrolysed by PprI and eYFP on the N‐terminus departs from eCFP on the C‐terminus. From the emission spectrum, we can see that YDC has two distinct emission peaks (Figure 1B), one located around 480 nm and the other around 530 nm, corresponding to the emission peaks of eCFP and eYFP (Figure 1B), respectively. Compared to the mixed solution of eYFP and eCFP, YDC exhibits significantly stronger emission at 530 nm than at 480 nm (Figure 1B), indicating that FRET indeed occurs in YDC. To conveniently describe the efficiency of FRET, we used the intensity ratio of 530 nm emission light to 480 nm emission light (R530/480) to reflect the efficiency of FRET.

FIGURE 1 Construction and function verification. (A) Schematic diagram of YDC composition. The yellow part is the eYFP protein, the cyan part is the eCFP protein, and the grey part is the DdrO protein. (B) Emission spectra of eYFP, eCFP, eYFP and eCFP mixed solution (Y + C), and YDC with 440 nm and 480 nm light used as the excitation sources, respectively. The final concentration of the proteins is 3 μM. (C) Crystal structure of DdrO protein (PDB: 6JQ1). The two monomers of the DdrO dimer are coloured yellow and pink, respectively. The N‐terminal is marked in green and the C‐terminal is marked in red. The distance between the N‐terminal and the C‐terminal is indicated by the dashed line. (D) Size exclusion chromatography of YDC. The molecular weight corresponding to the peak is 136.3 kD, while the molecular weight of the YDC monomer is 72.27 kD, indicating that YDC forms a dimer like natural DdrO. (E) SDS‐PAGE results after reaction. ‘+’ represents the addition of PprI, while ‘‐’ indicates its absence. The bands corresponding to the cleaved substrate protein fragments are indicated by black arrows. (F) The emission spectrum of YDC and its emission spectra at different reaction times. (G) The R530/480 of YDC and its R530/480 at different reaction times.

YDC protein was confirmed as a dimer in solution by size exclusion chromatography (Figure 1D), consistent with the dimerization of wild‐type DdrO (Lu et al., 2019), which further ensures the correct folding and following recognition and cleavage by PprI (Figure 1C). To test whether the two fluorescent groups in YDC protein would block the enzyme cleavage site on DdrO, we conducted cleavage assay demonstrating that the cleavage reaction could occur normally: PprI can hydrolyse YDC protein as well as DdrO, and produce two fragments (Figure 1E). As the reaction time increased, the emission spectrum of YDC after being cleaved changed significantly and the peak of 530 nm emission light was no longer prominent (Figure 1F,G). This indicates that FRET efficiency can indeed reflect the degree of cleavage, which is in line with our design philosophy for the detection system.

Detection system optimization

Since the intrinsic enzymatic activity of PprI would reduce the contrast between the absence and presence of ssDNA (Figure S2A), we tried to construct a mutation that only affect the protease activity without ssDNA. The alignment between DR_0167 (AlphaFold Protein Structure Database: Q9RXY7) and Dgeo_0395 complex with ssDNA (PDB: 8SLN) (Figure 2A) revealed several conserved amino acid residues (D91, R111 and N153) locating between the DNA‐binding domain and N‐terminal β‐sheets (Figure 2A and Figure S2B), which seems to stabilize the conformation of the N‐terminal protease domain. We mutated these residues to alanine and performed cleavage assays with the mutations, which showed that D91A and N153A weaken PprI's cleavage activity in the absence of ssDNA, while R111A does not significantly affect cleavage activity (Figure 2B).

FIGURE 2 Adjustment and optimization of the detection system. (A) Comparison of the structures of DR_0167 and Dgeo_0395, coloured in light blue and wheat, respectively. The structure of DR_0167 is from AlphaFold Protein Structure Database (ID: Q9RXY7), while that of Dgeo_0395 is a complex structure coordinating with ssDNA (PDB: 8SLN). The root mean square deviation (RMSD) for the structural alignment is 0.732. The selected mutation sites are coloured red and indicated with black arrows. (B) The cleavage results of PprI and its mutants on DdrO. (C, D) The SDS‐PAGE and FRET results of YDC cleavage by PprI and D91A. The final concentrations of ssDNA were 0 μM, 0.05 μM and 0.5 μM, respectively. NS: p ≥ 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. (E) The emission spectra of YDC protein under different excitation. Excessive wavelength can cause significant errors in the emission light near 460 nm. (F) R530/480 of YDC protein under different excitation. Longer wavelength excitation light can increase the R530/480 value. (G) R530/480 of YDC protein under different salt concentrations. It can be clearly seen that with the increase of salt concentration, R530/480 has a very obvious downward trend.

However, the decreased protease activity of D91A mutant could be restored after the addition of ssDNA, while N153A shows some activation but less pronounced than D91A (Figure 2B). Additionally, N153A tends to form inclusion bodies during induction expression (Figure S1C), which is unfavourable for protein purification. Furthermore, we conducted cleavage experiments with YDC, showing that D91A significantly reduced cleavage activity in the absence of ssDNA and gradually restored activated activity with increasing ssDNA concentration, demonstrating it a suitable mutant that meets our requirements (Figure 2C,D, Figure S2C,D).

Moreover, we tested the emission spectra of YDC protein under excitation light of different wavelengths (Figure 2E,F). It can be seen that using longer wavelength excitation light can increase the value of R530/480 (Figure 2F), but too long a wavelength can cause significant errors in the emitted light near 460 nm (Figure 2E). Therefore, we used light at 440 nm as the excitation light and scanned the changes in the emission light of YDC protein before and after the cleavage.

While testing the effect of salt concentration on the fluorescence excitation, we found that increasing salt concentration would lead to a decrease in the intensity of 530 nm emission light and an increase in the intensity of 480 nm emission light for YDC protein (Figure 2G, Figure S2E). Besides, it can be seen from Figure 2G that with the increase of salt concentration, R530/480 of YDC decreased significantly, which would lead to misjudgement of the FRET efficiency. We finally chose 250 mM NaCl as the salt concentration of the test solution for subsequent experiments, which is suitable for protein activity and meets the condition for fluorescence excitation and FRET efficiency.

According to existing research, ssDNA is the activation factor for PprI enzymatic cleavage. We also designed different types of DNA to explore the activation effect, and the results showed that ssDNA was the main factor for activating enzymatic cleavage (Figure S3A–D). Although blunt‐end dsDNA had some activation effect, we hypothesized that this might be due to the presence of partially dissociated ssDNA. Therefore, we believed that the FRET efficiency of the sample primarily reflects the amount of ssDNA present in the sample. Meanwhile, we also tested the same DNA samples with the Qubit ssDNA assay kit, and the results showed that the Qubit kit cannot distinguish ssDNA from dsDNA; its fluorescence values are positively correlated with the total amount of DNA (Figure S3E). This indicates that distinguishing between ssDNA and dsDNA is an important feature of our method.

Construction of standard curve and protein storage

After determining the wavelength and salt concentration of the system, as well as the introduction of D91A mutation into PprI, we started to prepare a series of ssDNA gradients and draw the standard curve by curve fitting. In order to obtain better enzymatic cleavage results, we adjusted the final concentration of PprI‐D91A in the reaction system to 0.1 μM and the final concentration of YDC to 3 μM. We prepared ssDNA samples with different concentrations ranging from 5 μM–1 nM by diluting them with water according to a gradient. The total volume of the reaction system was 100 μL, which contained 5 μL of ssDNA samples and 1 mM of manganese ions. Water and buffers were added to adjust the salt concentration of the system to 250 mM. The reaction system was incubated at 37°C for 30 min.

The SDS‐PAGE electrophoresis results showed that the cleavage of YDC protein corresponded to the concentration gradient of ssDNA (Figure 3A). Although the amount of PprI‐D91A was very low and not visible on the gel, its activation effect by ssDNA was still evident (Figure 3A). Likewise, R530/480 also had shown a good correlation with the ssDNA concentration (Figure 3B).

FIGURE 3 Enzyme activity reaction results and drawing of standard curves. (A) SDS‐PAGE electrophoresis results after different concentrations of ssDNA were added to the enzyme activity system. The enzymatic cleavage results are consistent with the ssDNA concentration gradient trend. (B) The R530/480 after reaction of ssDNA samples with different concentrations. The trend of R530/480 with the change of ssDNA concentration is very obvious. (C) The rR530/480 and standard curve after reaction of ssDNA samples with different concentrations. The standard curve fits the data points very well. (D) The detection results of the Qubit ssDNA assay kit for ssDNA samples at different concentrations. Linear fitting was performed on the data points.

To better describe the FRET efficiency of the samples, we decided to take the ratio between R530/480 of each sample to R530/480 of a blank control without ssDNA added as the relative R530/480 (rR530/480). Based on the rR530/480 of each sample, we used an improved logistic equation to fit the data points, and obtained an equation in this form: y=a1+ebxc+1−a (Figure 3C). The values, standard deviations and significance levels of each fitting parameter are listed in Table 1. The R‐squared value of the fitting result is 0.98426, which is very close to 1, indicating a good fit for our model.

TABLE 1 The results of parameter fitting for the detection system.

	Value	Standard error	t‐Value	Prob > |t|	Dependency	
a	0.4715	0.03929	12.00124	7.69313e‐7	0.87258	
b	0.98204	0.37381	2.62708	0.02749	0.92912	
c	0.90257	0.13855	6.51424	1.09621e‐4	0.81049	

According to the fitting results, our detection system has a detection range between approximately 0.01 μM and 5 μM, and a linear range from about 0.05 μM to 1 μM (Figure 3C). With this standard curve, we can estimate the concentration of ssDNA in a given sample based on its rR530/480 value, and thus evaluate the degree of DNA damage of the samples.

In addition, we also tested these gradient ssDNA samples with the Qubit ssDNA assay kit (Figure 3D). Since the Qubit assay typically uses linear fitting, we have also adopted linear fitting here (y=A+Bx, R 2 = 0.99905), and the fitting results are shown in Table 2. From the fitting results, it can be seen that when the concentration of the ssDNA sample is below 0.05 μM, the data points deviate from the linear fitting (Figure 3D). This is consistent with our detection method. Moreover, both detection methods are unable to significantly distinguish ssDNA samples with concentrations below 0.01 μM (Figure 3C,D), indicating that the detection limits of the two methods are close.

TABLE 2 The results of parameter fitting for the Qubit assay.

	Value	Standard error	t‐Value	Prob > |t|	Dependency	
A	−9.3898	166.9669	−0.05624	0.95626	0.21724	
B	10794.2971	105.06982	102.73452	1.8709e‐16	0.21724	

In an attempt to increase the sensitivity of the detection method, we adjusted the concentrations of YDC and enzyme and extended the reaction time (Figure S4A). However, even after these adjustments, the detection method still could not effectively distinguish between the 1 nM ssDNA sample and the blank sample, which implies that the detection limit of our method is approximately 10 nM.

We also conducted stability tests on YDC and enzyme, storing both proteins at 4°C, −20°C, and −80°C for 15 days before testing (Figure S4B–G). The results indicate that short‐term storage does not significantly impact the enzymes and YDC, and it does not greatly affect the quantitative trend. Based on our experimental experience, we recommended that proteins should not be stored at 4°C for more than 3 weeks, at −20°C for more than 6 months, and for longer storage, proteins should be kept at −80°C.

The application of genome damage detection

To demonstrate the practical application of the detection system, we extracted the genome of E. coli and divided it into four parts, which were treated as follows: (I) stored at 4°C for 10 days, (II) incubated at 37°C for 5 days and then stored at 4°C for 5 days, (III) incubated at 37°C for 10 days, (IV) incubated at 70°C for 10 days. After treatment, we checked the degradation of the genome by agarose gel electrophoresis and found that sample IV was almost completely degraded (Figure 4A).

FIGURE 4 The application of genome damage detection. I: Stored at 4°C for 10 days, II: Incubated at 37°C for 5 days and then stored at 4°C for 5 days, III: Incubated at 37°C for 10 days, IV: Incubated at 70°C for 10 days. (A) The result of genome electrophoresis on 1% agarose gel. It can be seen that sample IV has been almost completely degraded. (B) The SDS‐PAGE gel image of the samples after completing cleavage incubation. Similarly, sample IV shows the most obvious cleavage effect. (C) The rR530/480 of each sample. The result is very consistent with the two electrophoresis images.

After that, 5 μL of each sample was added to the detection system for detection as described above. The results of the reaction are shown in Figure 4B,C. According to the standard curve and the rR530/480 of each sample, we can calculate the concentration of ssDNA in the samples: (I) 0.051 ± 0.011 μM, (II) 0.029 ± 0.012 μM, (III) 0.044 ± 0.013 μM and (IV) 1.762 ± 0.132 μM. From the results, only the ssDNA concentration of sample IV fell within the linear range. This also indicates that the extracted genome samples will not degrade significantly if stored briefly at 37°C for a few days, while higher temperatures will lead to significant degradation of DNA.

Detection in different solutions

To explore the applicability of our detection method, we further tested its capability for ssDNA in different solutions. We selected four commonly used solutions in the laboratory for testing, namely PBS, LB liquid medium, FBS and DMEM (containing 10% FBS). Using these four solutions, we prepared ssDNA samples at concentrations of 0 μM, 0.1 μM and 1 μM, respectively, and performed ssDNA detection as described above. The results showed that the cleavage reaction occurred normally in all four solutions, and the R530/480 reflected the ssDNA concentration gradient in these solutions well (Figure 5A,B), indicating the feasibility of the detection method for ssDNA in various solutions. However, in LB, samples without additional ssDNA also caused a significant enzymatic cleavage reaction, likely due to ssDNA presence in the components used in LB, such as yeast extract. Although this does not affect the detection system's ability to distinguish ssDNA gradients, it is advisable to avoid ssDNA contamination in the blank control during actual use, as it may affect the accuracy of detection.

FIGURE 5 Detection for ssDNA in different solutions. (A, B) The detection results of the system for ssDNA in PBS, LB liquid medium, FBS and DMEM (containing 10% FBS). (C) The detection results of ssDNA in the PBS incubated with E. coli. The red dashed line represents the R530/480 of the blank control, and the pink area indicates the standard error range of the blank control. (D, E) The detection results of ssDNA in the DMEM incubated with HeLa cells. The red dashed line represents the R530/480 of the blank control, and the pink area indicates the standard error range of the blank control. The sample volume used for the detection system are 5 μL (D) and 50 μL (E), respectively.

Additionally, to demonstrate the detection of ssDNA in extracellular solutions to reflect intracellular damage, we prepared extracellular solution samples using E. coli and HeLa cells that were either UV‐irradiated or left untreated. The experimental data indicated that E. coli continuously releases ssDNA outward, and UV irradiation increases the liberation (Figure 5C, Figure S5A). However, similar phenomena were not observed in the medium incubating HeLa cells (Figure 5D, Figure S5B), possibly due to the limited sensitivity of the detection system. We increased the sample volume in the 100 μL detection system from 5 to 50 μL to increase the ssDNA concentration. The results indicated that HeLa cells also release ssDNA outward, and UV damage increases this release. (Figure 5E, Figure S5C).

It is evident that our detection system can successfully detect ssDNA in various solutions and reflect the state of cells by detecting ssDNA in extracellular solutions, indicating the potential application for cfDNA detection. Reports indicate that the concentration of cfDNA in healthy individuals is generally between 1 and 10 ng/mL (most are ssDNA), while the average concentration of cfDNA of cancer patients can reach tens of ng/mL, and sometimes even hundreds of ng/mL (Bettegowda et al., 2014; Bryzgunova et al., 2021; Cohen et al., 2018; Diehl et al., 2008; Huang et al., 2020; Leon et al., 1977). Based on our standard curve and the ssDNA used (oligonucleotide fp‐pet‐f), with 10 nM as the detection limit of the system, the lower limit is approximately 100 ng/mL. The limit can be even improved by increasing the addition of sample, meeting the ssDNA concentration levels in cfDNA of cancer patients. Therefore, our detection system can effectively distinguish the ssDNA levels between healthy individuals and cancer patients, aiding in cancer diagnosis.

CONCLUSION

In this article, we introduced a quantitative DNA damage detection method based on ssDNA activation of PprI, which is capable of detecting ssDNA content in various samples. We constructed a designed protein (YDC) by fusing eYFP, eCFP and DdrO, which can produce FRET and be cleaved by PprI. In addition, we mutated three amino acid residues of PprI and chose D91A as the enzyme in our detection system after testing enzyme activity. We further optimized the detection method and determined the appropriate detection steps. Based on the optimized method, we drew a standard curve for the ssDNA concentration gradient to evaluate the degradation of purified genome samples, and tested ssDNA samples in different solutions.

Comparing the detection limit of the detection system with reported cfDNA concentrations, we believe this method has the potential utility in distinguishing between the extracellular ssDNA levels of healthy individuals and cancer patients, thereby aiding in cancer diagnosis.

AUTHOR CONTRIBUTIONS

Shitong Zhong: Writing – original draft; investigation; formal analysis; data curation; conceptualization. Shuang Song: Methodology; investigation; software; data curation. Linjia Wang: Visualization; formal analysis. Yufeng Liu: Methodology. Hong Xu: Methodology. Liangyan Wang: Writing – review and editing. Huizhi Lu: Writing – review and editing; supervision; funding acquisition; conceptualization. Yuejin Hua: Writing – review and editing; supervision; project administration; funding acquisition; conceptualization.

FUNDING INFORMATION

This work was supported by the National Natural Science Foundation of China (32370028 and 32200016), and the Zhejiang Provincial Natural Science Foundation of China (LQ23C010002).

CONFLICT OF INTEREST STATEMENT

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Supporting information

Data S1.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.
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REFERENCES

Battista, J.R. , Earl, A.M. & Park, M.J. (1999) Why is Deinococcus radiodurans so resistant to ionizing radiation? Trends in Microbiology, 7 , 362–365.10470044
Bettegowda, C. , Sausen, M. , Leary, R.J. , Kinde, I. , Wang, Y. , Agrawal, N. et al. (2014) Detection of circulating tumor DNA in early‐ and late‐stage human malignancies. Science Translational Medicine, 6 , 224r.
Blanchard, L. , Guerin, P. , Roche, D. , Cruveiller, S. , Pignol, D. , Vallenet, D. et al. (2017) Conservation and diversity of the IrrE/DdrO‐controlled radiation response in radiation‐resistant Deinococcus bacteria. Microbiology, 6 , e00477.
Bryzgunova, O.E. , Konoshenko, M.Y. & Laktionov, P.P. (2021) Concentration of cell‐free DNA in different tumor types. Expert Review of Molecular Diagnostics, 21 , 63–75.33270495
Butt, A.N. & Swaminathan, R. (2008) Overview of circulating nucleic acids in plasma/serum. Annals of the New York Academy of Sciences, 1137 , 236–242.18837954
Casadio, V. , Calistri, D. , Salvi, S. , Gunelli, R. , Carretta, E. , Amadori, D. et al. (2013) Urine cell‐free DNA integrity as a marker for early prostate cancer diagnosis: a pilot study. BioMed Research International, 2013 , 270457.23509700
Chowdhury, D. , Xu, X. , Zhong, X. , Ahmed, F. , Zhong, J. , Liao, J. et al. (2008) A PP4‐phosphatase complex dephosphorylates gamma‐H2AX generated during DNA replication. Molecular Cell, 31 , 33–46.18614045
Cohen, J.D. , Li, L. , Wang, Y. , Thoburn, C. , Afsari, B. , Danilova, L. et al. (2018) Detection and localization of surgically resectable cancers with a multi‐analyte blood test. Science, 359 , 926–930.29348365
Cortes‐Gutierrez, E.I. , Davila‐Rodriguez, M.I. , Fernandez, J.L. , Lopez‐Fernandez, C. & Gosalvez, J. (2012) DNA breakage detection‐fluorescence in situ hybridization (DBD‐FISH) in buccal cells. European Journal of Histochemistry, 56 , e49.23361245
Diehl, F. , Schmidt, K. , Choti, M.A. , Romans, K. , Goodman, S. , Li, M. et al. (2008) Circulating mutant DNA to assess tumor dynamics. Nature Medicine, 14 , 985–990.
Hua, Y. , Narumi, I. , Gao, G. , Tian, B. , Satoh, K. , Kitayama, S. et al. (2003) PprI: a general switch responsible for extreme radioresistance of Deinococcus radiodurans . Biochemical and Biophysical Research Communications, 306 , 354–360.12804570
Huang, X. , Zhao, Q. , An, X. , Pan, J. , Zhao, L. , Shen, L. et al. (2020) The ratio of ssDNA to dsDNA in circulating cell‐free DNA extract is a stable indicator for diagnosis of gastric cancer. Pathology Oncology Research, 26 , 2621–2632.32632900
Ismail, I.H. , Wadhra, T.I. & Hammarsten, O. (2007) An optimized method for detecting gamma‐H2AX in blood cells reveals a significant interindividual variation in the gamma‐H2AX response among humans. Nucleic Acids Research, 35 , e36.17284459
Krisko, A. & Radman, M. (2013) Biology of extreme radiation resistance: the way of Deinococcus radiodurans . Cold Spring Harbor Perspectives in Biology, 5 , a012765.23818498
Leon, S.A. , Shapiro, B. , Sklaroff, D.M. & Yaros, M.J. (1977) Free DNA in the serum of cancer patients and the effect of therapy. Cancer Research, 37 , 646–650.837366
Liu, Y.Q. , Zhou, J.Z. , Omelchenko, M.V. , Beliaev, A.S. , Venkateswaran, A. , Stair, J. et al. (2003) Transcriptome dynamics of Deinococcus radiodurans recovering from ionizing radiation. Proceedings of the National Academy of Sciences of the United States of America, 100 , 4191–4196.12651953
Lou, Z. & Chen, J. (2006) Cellular senescence and DNA repair. Experimental Cell Research, 312 , 2641–2646.16893723
Lu, H. , Chen, H. , Xu, G. , Shah, A.M. & Hua, Y. (2012) DNA binding is essential for PprI function in response to radiation damage in Deinococcus radiodurans . DNA Repair, 11 , 139–145.22051194
Lu, H. , Chen, Z. , Xie, T. , Zhong, S. , Suo, S. , Song, S. et al. (2024) The Deinococcus protease PprI senses DNA damage by directly interacting with single‐stranded DNA. Nature Communications, 15 , 1892.
Lu, H. , Wang, L. , Li, S. , Pan, C. , Cheng, K. , Luo, Y. et al. (2019) Structure and DNA damage‐dependent derepression mechanism for the XRE family member DG‐DdrO. Nucleic Acids Research, 47 , 9925–9933.31410466
Ludanyi, M. , Blanchard, L. , Dulermo, R. , Brandelet, G. , Bellanger, L. , Pignol, D. et al. (2014) Radiation response in Deinococcus deserti: IrrE is a metalloprotease that cleaves repressor protein DdrO. Molecular Microbiology, 94 , 434–449.25170972
Mardis, E. & McCombie, W.R. (2017) Library quantification: fluorometric quantitation of double‐stranded or single‐stranded DNA samples using the Qubit system. Cold Spring Harbor Perspectives in Biology, 2017 , pdb.prot094730.
Moeglin, E. , Desplancq, D. , Conic, S. , Oulad‐Abdelghani, M. , Stoessel, A. , Chiper, M. et al. (2019) Uniform widespread nuclear phosphorylation of histone H2AX is an indicator of lethal DNA replication stress. Cancers, 11 , 355.30871194
Ostling, O. & Johanson, K.J. (1984) Microelectrophoretic study of radiation‐induced DNA damages in individual mammalian cells. Biochemical and Biophysical Research Communications, 123 , 291–298.6477583
Ponti, G. , Maccaferri, M. , Manfredini, M. , Kaleci, S. , Mandrioli, M. , Pellacani, G. et al. (2018) The value of fluorimetry (Qubit) and spectrophotometry (NanoDrop) in the quantification of cell‐free DNA (cfDNA) in malignant melanoma and prostate cancer patients. Clinica Chimica Acta, 479 , 14–19.
Singh, N.P. , McCoy, M.T. , Tice, R.R. & Schneider, E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research, 175 , 184–191.3345800
Slade, D. & Radman, M. (2011) Oxidative stress resistance in Deinococcus radiodurans . Microbiology and Molecular Biology Reviews, 75 , 133–191.21372322
Wang, Y. , Xu, Q. , Lu, H. , Lin, L. , Wang, L. , Xu, H. et al. (2015) Protease activity of PprI facilitates DNA damage response: Mn2+‐dependence and substrate sequence‐specificity of the proteolytic reaction. PLoS One, 10 , e0122071.25811789
