Rad52 has a role in the repair of sodium selenite-induced DNA damage in Saccharomyces cerevisiae

Selenium (Se) is a chemo-preventive agent that has been shown to have a protective role against cancer. The inorganic form of Se, sodium selenite (Na2SeO3), has frequently been included in various chemo-prevention studies, and this commercially available form of Se is used as dietary supplement by the public. Because high doses of this Se compound can be toxic, the underlying molecular mechanisms of sodium selenite toxicity need to be elucidated. Recently, we have reported that sodium selenite is acting as an oxidizing agent in the budding yeast Saccharomyces cerevisiae, producing oxidative damage to DNA. This pro-oxidative activity of sodium selenite likely accounted for the observed DNA double-strand breaks (DSB) and yeast cell death. In this study we determine the genetic factors that are responsible for repair of sodium selenite-induced DSB. We report that the Rad52 protein is indispensable for repairing sodium selenite-induced DSB, suggesting a fundamental role of homologous recombination (HR) in this repair process. These results provide the first evidence that HR may have a fundamental role in the repair of sodium selenite-induced toxic DNA lesions.     

SUMOylation of Rad52-Rad59 synergistically change the outcome of mitotic recombination

Homologous recombination (HR) is essential for maintenance of genome stability through double-strand break (DSB) repair, but at the same time HR can lead to loss of heterozygosity and uncontrolled recombination can be genotoxic. The post-translational modification by SUMO (small ubiquitin-like modifier) has been shown to modulate recombination, but the exact mechanism of this regulation remains unclear. Here we show that SUMOylation stabilizes the interaction between the recombination mediator Rad52 and its paralogue Rad59 in Saccharomyces cerevisiae. Although Rad59 SUMOylation is not required for survival after genotoxic stress, it affects the outcome of recombination to promote conservative DNA repair. In some genetic assays, Rad52 and Rad59 SUMOylation act synergistically. Collectively, our data indicate that the described SUMO modifications affect the balance between conservative and non-conservative mechanisms of HR.     

Telomere-related functions of yeast KU in the repair of bleomycin-induced DNA damage

Bleomycins are small glycopeptide cancer chemotherapeutics that give rise to 3'-modified DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, DSBs are predominantly repaired by RAD52-dependent homologous recombination (HR) with some support by Yku70/Yku80 (KU)-dependent pathways. The main DSB repair function of KU is believed to be as part of the non-homologous end-joining (NHEJ) pathway, but KU also functions in a "chromosome healing" pathway that seals DSBs by de novo telomere addition. We report here that rad52Deltayku70Delta double mutants are considerably more bleomycin hypersensitive than rad52Deltalig4Delta cells that lack the NHEJ-specific DNA ligase 4. Moreover, the telomere-specific KU mutation yku80-135i also dramatically increases rad52Delta bleomycin hypersensitivity, almost to the level of rad52Deltayku80Delta. The results indicate that telomere-specific functions of KU play a more prominent role in the repair of bleomycin-induced damage than its NHEJ functions, which could have important clinical implications for bleomycin-based combination chemotherapies.     

Effect of DNA sequence and nucleotide cofactors on hRad51 binding to ssDNA: role of hRad52 in recruitment

hRad51 binding to ssDNA is significantly lowered in the presence of a nucleotide cofactor ATP/ADP/ATPgammaS. In these conditions, presence of trace amounts of hRad52 protein restores hRad51 binding to DNA. In the absence of any nucleotide cofactor where intrinsic binding of hRad51 to ssDNA is higher, hRad52 brings about no improved binding. hRad51 binding to ssDNA is strongly influenced by the DNA sequence. The protein binding to repeat sequences is poor compared to that of mixed DNA sequence. Interestingly, presence of hRad52 restores the ability of hRad51 binding to such DNA targets as well. Moreover, all the cooperative effects of hRad52 on hRad51 binding are highly specific to the latter's binding to ssDNA and not to dsDNA. These results help us to model important mechanistic steps of hRad51 presynapsis on ssDNA templates.     

Non‐recombinogenic roles for Rad52 in translesion synthesis during DNA damage tolerance

DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error‐prone, HR uses the sister chromatid and is mostly error‐free. We report that the HR protein Rad52—but not Rad51 and Rad57—acts in concert with the TLS machinery (Rad6/Rad18‐mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light‐induced ssDNA gaps through a non‐recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS‐ and UV‐induced HR); accordingly, Rad52 is required for efficient DNA damage‐induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage‐induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57‐dependent and ‐independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.     

Nuclear localization of Rad52 is pre-requisite for its sumoylation

In Saccharomyces cerevisiae, Rad52 plays major roles in several types of homologous recombination. Here, we found that rad52-K200R mutation greatly reduced sumoylation of Rad52. The rad52-K200R mutant exhibited defects in various types of recombination, such as intrachromosomal recombination and mating-type switching. The K200 residue of Rad52 is part of the nuclear localization signal (NLS), which is important for transport into the nucleus. Indeed, the addition of a SV40 NLS to Rad52-K200R suppressed the sumoylation defect of Rad52-K200R. These findings indicate that nuclear localization of Rad52 is pre-requisite for its sumoylation.     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

 We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome. Received: 9 February 1996/Accepted: 7 July 1996     

Plasmid multimerization is dependent on RAD52 activity in Saccharomyces cerevisiae

Summary A mutant plasmid, pX, derived from the 1453 base pair small plasmid, YARp1 (or TRP1 RI circle), consists of 849 base pairs of DNA bearing the TRP1 gene and the ARS1 sequence of Saccharomyces cerevisiae and, unlike YARp1 and other commonly used yeast plasmids, highly multimerizes in a S. cerevisiae host. The multimerization of pX was dependent on RAD52, which is known to be necessary for homologous recombination in S. cerevisiae. Based upon this observation, a regulated system of multimerization of pX with GAL1 promoter-driven RAD52 has been developed. We conclude that the regulated multimerization of pX could provide a useful model system to study genetic recombination in the eukaryotic cell, in particular to investigate recombination intermediates and the effects of various trans-acting mutations on the multimerization and recombination of plasmids.     

A physical assay for detection of early meiotic recombination intermediates in Saccharomyces cerevisiae

In most eukaryotic organisms, recombination events leading to exchanges between homologous chromosomes link the homologs in a manner that allows their proper attachment to the meiotic spindle. In the yeast Saccharomyces cerevisiae these exchanges are initiated in early prophase as double-strand breaks in the DNA. These breaks are processed through a series of intermediates to yield mature crossovers late in prophase. The following experiments were designed to monitor the appearance of the earliest recombinant DNA strands formed in this process. A polymerase chain reaction assay was devised that allows the detection of recombinant strands at a known initiation site for meiotic recombination. The time and rate of appearance of recombinant strands was found to coincide with commitment to recombination, demonstrating that DNA strands bearing sequences from both parental chromosomes are rapidly formed after the initiation of meiotic recombination. Received: 22 July 1997 / Accepted: 25 February 1998     

DNA double-strand break repair signalling: the case of RAD51 post-translational regulation

DNA double-strand breaks (DSBs) are the major lethal lesion induced by ionizing radiation or by replication block. However, cells can take advantage of DSB-induced recombination in order to generate genetic diversity in physiological processes such as meiosis and V(D)J recombination. Two main alternative pathways compete for DSB repair: homologous recombination (HR) and non-homologous end-joining (NHEJ). This review will briefly present the mechanisms and the enzymatic complex for HR and NHEJ. The signalling of the DSB through the ATM pathway will be presented. Then, we will focus on the case of the RAD51 protein, which plays a pivotal role in HR and is conserved from bacteria to humans. Post-translational regulation of RAD51 is presented. Two contrasting situations are discussed: one with up-regulation (expression of the oncogene BCR/ABL) and one with a down-regulation (expression of the oncogene BCL-2) of RAD51, associated with apoptosis inhibition and tumour predisposition.     

Chromatin PTEN is involved in DNA damage response partly through regulating Rad52 sumoylation

A pool of PTEN localizes to the nucleus. However, the exact mechanism of action of nuclear PTEN remains poorly understood. We have investigated PTEN’s role during DNA damage response. Here we report that PTEN undergoes chromatin translocation after DNA damage, and that its translocation is closely associated with its phosphorylation on S366/T370 but not on S380. Deletional analysis reveals that the C2 domain of PTEN is responsible for its nuclear translocation after exposure to genotoxin. Both casein kinase 2 and GSK3β are involved in the phosphorylation of the S366/T370 epitope, as well as PTEN’s association with chromatin after DNA damage. Significantly, PTEN specifically interacts with Rad52 and colocalizes with Rad52, as well as γH2AX, after genotoxic stress. Moreover, PTEN is involved in regulating Rad52 sumoylation. Combined, our studies strongly suggest that nuclear/chromatin PTEN mediates DNA damage repair through interacting with and modulating the activity of Rad52.     

Ionizing radiation-induced growth in soft agar is associated with miR-21 upregulation in wild-type and DNA double strand break repair deficient cells

DNA double strand breaks (DSBs) are a severe threat to genome integrity and a potential cause of tumorigenesis, which is a multi-stage process and involves many factors including the mutation of oncogenes and tumor suppressors, some of which are transcribed microRNAs (miRNAs). Among more than 2000 known miRNAs, miR-21 is a unique onco-miRNA that is highly expressed in almost all types of human tumors and is associated with tumorigenesis through its multiple targets. However, it remains unclear whether there is any functional link between DSBs and miR-21 expression and, if so, does the link contribute to DSB-induced genomic instability/tumorigenesis. To address this question, we used DNA-PKcs-/- (deficient in non-homologous end-joining (NHEJ)) and Rad54-/- (deficient in homologous recombination repair (HRR)) mouse embryonic fibroblasts (MEFs) since NHEJ and HRR are the major pathways for DSB repair in mammalian cells. Our results indicate that levels of miR-21 are elevated in these DSB repair (DSBR) deficient cells, and ionizing radiation (IR) further increases these levels in both wild-type (WT) and DSBR-deficient cells. Interestingly, IR stimulated growth in soft agar and this effect was greatly reduced by blocking miR-21 expression in both WT and DSBR-deficient cells. Taken together, our results suggest that either IR or DSBR-deficient can lead to an upregulation of miR-21 levels and that miR-21 is associated with IR-induced cell growth in soft agar. These results may help our understanding of DSB-induced tumorigenesis and provide information that could facilitate the development of new strategies to prevent DSB-induced carcinogenesis.     

Differential effect of the overexpression of Rad2/XPG family endonucleases on genome integrity in yeast and human cells

Eukaryotic cells possess several DNA endonucleases that are necessary to complete different steps in DNA metabolism. Rad2/XPG and Rad27/FEN1 belong to a group of evolutionary conserved proteins that constitute the Rad2 family. Given the important roles carried out by these nucleases in DNA repair and their capacity to create DNA breaks, we have investigated the effect that in vivo imbalance of these nucleases and others of the family have on genome integrity and cell proliferation. We show that overexpression of these nucleases causes genetic instability in both yeast and human cells. Interestingly, the type of recombination event and DNA damage induced suggest specific modes and timing of action of each nuclease that are beyond their known DNA repair function and are critical to preserve genome integrity. In addition to identifying new sources of genome instability, a hallmark of cancer cells, this study provides new genetic tools for studies of genome dynamics.     

Enhancing CRISPR/Cas9-mediated homology-directed repair in mammalian cells by expressing Saccharomyces cerevisiae Rad52

Precise genome editing with desired point mutations can be generated by CRISPR/Cas9-mediated homology-directed repair (HDR) and is of great significance for gene function study, gene therapy and animal breeding. However, HDR efficiency is inherently low and improvements are necessitated. Herein, we determined that the HDR efficiency could be enhanced by expressing Rad52, a gene that is involved in the homologous recombination process. Both the Rad52 co-expression and Rad52-Cas9 fusion strategies yielded approximately 3-fold increase in HDR during the surrogate reporter assays in human HEK293T cells, as well as in the genome editing assays. Moreover, the enhancement effects of the Rad52-Cas9 fusion on HDR mediated by different (plasmid, PCR and ssDNA) donor templates were confirmed. We found that the HDR efficiency could be significantly improved to about 40% by the combined usage of Rad52 and Scr7. In addition, we also applied the fusion strategy for modifying the IGF2 gene of porcine PK15 cells, which further demonstrated a 2.2-fold increase in HDR frequency. In conclusion, our data suggests that Rad52-Cas9 fusion is a good option for enhancing CRISPR/Cas9-mediated HDR, which may be of use in future studies involving precise genome editing.     

Differential effects of the mismatch repair genes MSH2 and MSH3 on homeologous recombination in Saccharomyces cerevisiae

The products of the yeast mismatch repair genes MSH2 and MSH3 participate in the inhibition of genetic recombination between homeologous (divergent) DNA sequences. In strains deficient for these genes, homeologous recombination rates between repeated elements are elevated due to the loss of this inhibition. In this study, the effects of these mutations were further analyzed by quantitation of mitotic homeologous recombinants as crossovers, gene conversions or exceptional events in wild-type, msh2, msh3 and msh2 msh3 mutant strains. When homeologous sequences were present as a direct repeat in one orientation, crossovers and gene conversions were elevated in msh2, msh3 and msh2 msh3 strains. The increases were greater in the msh2 msh3 double mutant than in either single mutant. When the order of the homeologous sequences was reversed, the msh2 mutation again yielded increased rates of crossovers and gene conversions. However, in an msh3 strain, gene conversions occurred at higher levels but interchromosomal crossovers were not increased and intrachromosomal crossovers were reduced relative to wild type. The msh2 msh3 double mutant behaved like the msh2 single mutant in this orientation. Control strains harboring homologous duplications were largely but not entirely unaffected in mutant strains, suggesting specificity for the mismatched intermediates of homeologous recombination. In all strains, very few (<10%) recombinants could be attributed to exceptional events. These results suggest that MSH2 and MSH3 can function differentially to control homeologous exchanges. Received: 24 December 1996 / Accepted: 24 July 1997     

Evidence for a role of vertebrate Rad52 in the repair of topoisomerase II-mediated DNA damage

DNA topoisomerase II (Top2) inhibitors are useful as anticancer agents, mostly by virtue of their ability to induce DNA double-strand breaks (DSBs). These DSBs are repaired almost exclusively by Rad52-dependent homologous recombination (HR) in yeast. However, we have recently shown that in vertebrate cells such lesions are primarily repaired by nonhomologous end-joining, but not HR. This finding, taken together with previous observations that disruption of RAD52 does not severely affect HR in vertebrate cells, makes it highly unlikely that Rad52 contributes to the repair of Top2-mediated DNA damage. However, in this paper we show that chicken cells lacking Rad52 do exhibit increased sensitivity to the Top2 inhibitor VP-16. Remarkably, the level of hypersensitivity of RAD52-null cells was comparable to that of RAD54-null cells, albeit only at high doses. Our data thus provide the first demonstration of a major repair defect associated with loss of Rad52 in vertebrate cells.     

Impairment of the non-homologous end joining and homologous recombination pathways of DNA double strand break repair: Impact on spontaneous and radiation-induced mammary and intestinal tumour risk in Apcmin/+ mice

Female Apc^min/+ mice carrying the BALB/c variant of Prkdc or heterozygous knockout for Xrcc2, were sham- or 2 Gy X-irradiated as adults to compare the effect of mild impairments of double–strand break (DSB) repair pathways, non-homologous end joining (NHEJ) and homologous recombination (HR) respectively on spontaneous and radiation–induced mammary and intestinal tumorigenesis. Mice with impaired NHEJ showed no difference in incidence of spontaneous mammary tumours, compared with matched controls, (2.46 fold, P = 0.121) and significantly less following irradiation (radiation–induced excess; 0.35 fold, P = 0.008). In contrast mice with impaired HR presented with significantly less spontaneous mammary tumours than matched controls (0.33 fold, P = 0.027) and significantly more following irradiation (radiation-induced excess; 3.3 fold, P = 0.016). Spontaneous and radiation-induced intestinal adenoma multiplicity in the same groups were significantly greater than matched controls for mice with impaired NHEJ (sham; 1.29 fold, P < 0.001, radiation–induced excess; 2.55 fold, P < 0.001) and mice with impaired HR showed no significant differences (sham; 0.92 fold, P = 0.166, radiation-induced excess; 1.16, P = 0.274). Genetic insertion events were common in spontaneous tumours from NHEJ impaired mice compared with matched controls. γH2AX foci analysis suggests a significantly faster rate of DSB repair (MANOVA P < 0.001) in intestinal than mammary tissue; apoptosis was also higher in irradiated intestine.To conclude, results suggest that pathway of choice for repair of spontaneous and radiation-induced DSBs is influenced by tissue type. NHEJ appears to play a greater role in DSB repair in intestinal tissue since impairment by functional change of Prkdc significantly increases the rate of mis-repair in intestinal but not mammary tissue. HR appears to play a greater role in DSB repair in adult mammary tissue since impaired HR results in significant changes in mammary but not in the intestinal tumorigenesis. This indicates that early DNA damage response and repair is important for cancer susceptibility and plays a role in determining tissue specificity of cancer risk.     

基因组编辑对酿酒酵母DNA的损伤作用及修复响应

【目的】为了研究基因组编辑工具CRISPR/Cas9和CRISPR/Cpf1所产生的DNA双链断裂(DNA doublestrandbreak,DSB)对酿酒酵母DNA的损伤作用及修复响应情况,对比化学物质甲基磺酸甲酯(methyl methanesulfonate,MMS)对酿酒酵母基因组DNA的损伤和修复,阐明编辑细胞在细胞水平和转录水平上的变化。【方法】起始细胞分为两种情况,包括未进行细胞周期同步化和被α-因子同步化细胞周期至G0/G1期。检测CRISPR/Cas9和CRISPR/Cpf1处理后编辑细胞的生长情况。利用流式细胞术检测编辑细胞的细胞周期延滞的情况。利用荧光定量PCR检测编辑细胞和MMS处理细胞后DNA损伤响应关键基因转录表达水平的变化情况。【结果】起始细胞无论是未同步化还是同步化,其生长均受到基因组编辑抑制,细胞存活率降低,细胞周期被滞留在G2/M期,而MMS处理导致细胞周期S期的滞留。此外,随编辑时间的延长,突变率增加,细胞存活率降低。CRISPR/Cpf1编辑细胞的突变率和存活率均低于CRISPR/Cas9,由此可见,CRISPR/Cpf1对细胞的损伤强度高于CRISPR/Cas9。两种编辑均诱导酵母DNA损伤响应关键基因RNR3及HUG1转录水平显著上调,并且CRISPR/Cpf1介导的上调幅度大于CRISPR/Cas9,但两者均低于MMS的处理。【结论】本研究解析了CRISPR/Cas9和CRISPR/Cpf1介导的基因组编辑在细胞水平和转录水平上对DNA损伤作用及修复响应,初步揭示了酿酒酵母应对不同类型的DSB损伤时响应程度的差异,为提高基因组编辑工具的编辑能力和评估基因编辑安全性提供了重要依据。