Alternative end joining (Alt-EJ) chromosomal break repair involves bypassing classical non-homologous end joining (c-NHEJ), and such repair causes mutations often with microhomology at the repair junction. Since the mediators of Alt-EJ are not well understood, we have sought to identify DNA damage response (DDR) factors important for this repair event. Using chromosomal break reporter assays, we surveyed an RNAi library targeting known DDR factors for siRNAs that cause a specific decrease in Alt-EJ, relative to an EJ event that is a composite of Alt-EJ and c-NHEJ (Distal-EJ between two tandem breaks). From this analysis, we identified several DDR factors that are specifically important for Alt-EJ relative to Distal-EJ. While these factors are from diverse pathways, we also found that most of them also promote homologous recombination (HR), including factors important for DNA crosslink repair, such as the Fanconi Anemia factor, FANCA. Since bypass of c-NHEJ is likely important for both Alt-EJ and HR, we disrupted the c-NHEJ factor Ku70 in Fanca-deficient mouse cells and found that Ku70 loss significantly diminishes the influence of Fanca on Alt-EJ. In contrast, an inhibitor of poly ADP-ribose polymerase (PARP) causes a decrease in Alt-EJ that is enhanced by Ku70 loss. Additionally, the helicase/nuclease DNA2 appears to have distinct effects from FANCA and PARP on both Alt-EJ, as well as end resection. Finally, we found that the proteasome inhibitor Bortezomib, a cancer therapeutic that has been shown to disrupt FANC signaling, causes a significant reduction in both Alt-EJ and HR, relative to Distal-EJ, as well as a substantial loss of end resection. We suggest that several distinct DDR functions are important for Alt-EJ, which include promoting bypass of c-NHEJ and end resection.
Alternative EJ (Alt-EJ) is a chromosomal double strand break (DSB) repair pathway that often uses short stretches of homology (microhomology) to bridge the break during repair. Alt-EJ involves bypass of the classical non-homologous end joining (c-NHEJ) pathway, and hence may be important for DSBs that are not readily repaired by c-NHEJ, such as DSBs requiring extensive end processing prior to ligation. Since the factors that mediate Alt-EJ are unclear, we identified DNA damage response factors that differentially promote Alt-EJ relative to an EJ event that is a composite of c-NHEJ and Alt-EJ. Several of these factors promote other repair events that are enhanced by loss of c-NHEJ, namely homologous recombination (HR), including DNA crosslink repair factors, such as FANCA. We then investigated distinctions among individual factors. For instance, we found that loss of c-NHEJ appears to diminish the influence of FANCA on Alt-EJ, but enhances the effect of PARP inhibition. Furthermore, we find that FANCA and DNA2 differentially affect another aspect of the DNA damage response, namely end resection. Based on these findings, we suggest that several aspects of the DNA damage response are important for Alt-EJ.
DNA end resection; DNA recombination; DNA repair; DNA2; nuclease
The E3 ubiquitin ligase RNF168 is a DNA damage response (DDR) factor that promotes monoubiquitination of H2A/H2AX at K13/15, facilitates recruitment of other DDR factors (e.g. 53BP1) to DNA damage, and inhibits homologous recombination (HR) in cells deficient in the tumor suppressor BRCA1. We have examined the domains of RNF168 important for these DDR events, including chromosomal HR that is induced by several nucleases (I-SceI, CAS9-WT and CAS9-D10A), since the inducing nuclease affects the relative frequency of distinct repair outcomes. We found that an N-terminal fragment of RNF168 (1-220/N221*) efficiently inhibits HR induced by each of these nucleases in BRCA1 depleted cells, and promotes recruitment of 53BP1 to DNA damage and H2AX monoubiquitination at K13/15. Each of these DDR events requires a charged residue in RNF168 (R57). Notably, RNF168-N221* fails to self-accumulate into ionizing radiation induced foci (IRIF). Furthermore, expression of RNF168 WT and N221* can significantly bypass the role of another E3 ubiquitin ligase, RNF8, for inhibition of HR in BRCA1 depleted cells, and for promotion of 53BP1 IRIF. We suggest that the ability for RNF168 to promote H2A/H2AX monoubiquitination and 53BP1 IRIF, but not RNF168 self-accumulation into IRIF, is important for inhibition of HR in BRCA1 deficient cells.
Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was significantly expressed in a broad range of cancer cells and tumor tissues, but not in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interconnector domain of PCNA that is known to be a major binding site for many of PCNA's interacting proteins. We hypothesized that therapeutic agents targeting protein-protein interactions mediated through this region may confer differential toxicity to normal and malignant cells. To test this hypothesis, we designed a cell permeable peptide containing the PCNA L126-Y133 sequence. Here, we report that this peptide selectively kills human neuroblastoma cells, especially those with MYCN gene amplification, with much less toxicity to non-malignant human cells. Mechanistically, the peptide is able to block PCNA interactions in cancer cells. It interferes with DNA synthesis and homologous recombination-mediated double-stranded DNA break repair, resulting in S-phase arrest, accumulation of DNA damage, and enhanced sensitivity to cisplatin. These results demonstrate conceptually the utility of this peptide for treating neuroblastomas, particularly, the unfavorable MYCN-amplified tumors.
In mammalian cells, the phospho-dependent interaction between BRCA1 and CtIP is not required for homology-directed DNA repair or tumor suppression.
The CtIP protein facilitates homology-directed repair (HDR) of double-strand DNA breaks (DSBs) by initiating DNA resection, a process in which DSB ends are converted into 3′-ssDNA overhangs. The BRCA1 tumor suppressor, which interacts with CtIP in a phospho-dependent manner, has also been implicated in DSB repair through the HDR pathway. It was recently reported that the BRCA1–CtIP interaction is essential for HDR in chicken DT40 cells. To examine the role of this interaction in mammalian cells, we generated cells and mice that express Ctip polypeptides (Ctip-S326A) that fail to bind BRCA1. Surprisingly, isogenic lines of Ctip-S326A mutant and wild-type cells displayed comparable levels of HDR function and chromosomal stability. Although Ctip-S326A mutant cells were modestly sensitive to topoisomerase inhibitors, mice expressing Ctip-S326A polypeptides developed normally and did not exhibit a predisposition to cancer. Thus, in mammals, the phospho-dependent BRCA1–CtIP interaction is not essential for HDR-mediated DSB repair or for tumor suppression.
BCR-ABL transforms bone marrow progenitor cells and promotes genome instability, leading to development of chronic myelogenous leukemia (CML). The tyrosine kinase inhibitor imatinib effectively treats CML, but acquired resistance can develop due to BCR-ABL mutations. Mechanisms for acquisition of BCR-ABL mutations are not fully understood. Using a novel culture model of CML acquired resistance, we show that inhibition of SIRT1 deacetylase by small molecule inhibitors or gene knockdown blocks acquisition of BCR-ABL mutations and relapse of CML cells on tyrosine kinase inhibitors. SIRT1 knockdown also suppresses de novo genetic mutations of HPRT (hypoxanthine phosphoribosyl transferase) gene in CML and non-CML cells upon treatment with DNA damaging agent camptothecin. Although SIRT1 can enhance cellular DNA damage response, it alters functions of DNA repair machineries in CML cells and stimulates activity of error-prone DNA damage repair, in association with acquisition of genetic mutations. These results reveal a previously unrecognized role of SIRT1 for promoting mutation acquisition in cancer, and have implication for targeting SIRT1 to overcome CML drug resistance.
Autophagy defects have been recently associated with chromosomal instability (CIN), a hallmark of human cancer. However, the functional specificity and mechanism of action of autophagy-related factors in genome stability remain elusive. Here we report that UVRAG, an autophagic tumor suppressor, plays a dual role in chromosomal stability, surprisingly independent of autophagy. We establish that UVRAG promotes DNA double-strand-breaks repair by directly binding and activating DNA-PK in non-homologous end-joining. Disruption of UVRAG increases genetic instability and sensitivity of cells to irradiation. Furthermore, UVRAG was found also localized at centrosomes and physically associated with CEP63, an integral component of centrosomes. Disruption of the association of UVRAG with centrosomes causes centrosome instability and aneuploidy. UVRAG thus represents an autophagy-related molecular factor that also has a convergent role in patrolling both the structural integrity and proper segregation of chromosomes, which may confer autophagy-independent tumor suppressor activity.
UVRAG; genomic integrity; DNA damage repair; centrosome; DNA-PK
The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the γH2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G2/M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.
Production of concatemeric DNA is an essential step during HSV infection, as the packaging machinery must recognize longer-than-unit-length concatemers; however, the mechanism by which they are formed is poorly understood. Although it has been proposed that the viral genome circularizes and rolling circle replication leads to the formation of concatemers, several lines of evidence suggest that HSV DNA replication involves recombination-dependent replication reminiscent of bacteriophages λ and T4. Similar to λ, HSV-1 encodes a 5′-to-3′ exonuclease (UL12) and a single strand annealing protein [SSAP (ICP8)] that interact with each other and can perform strand exchange in vitro. By analogy with λ phage, HSV may utilize viral and/or cellular recombination proteins during DNA replication. At least four double strand break repair pathways are present in eukaryotic cells, and HSV-1 is known to manipulate several components of these pathways. Chromosomally integrated reporter assays were used to measure the repair of double strand breaks in HSV-infected cells. Single strand annealing (SSA) was increased in HSV-infected cells, while homologous recombination (HR), non-homologous end joining (NHEJ) and alternative non-homologous end joining (A-NHEJ) were decreased. The increase in SSA was abolished when cells were infected with a viral mutant lacking UL12. Moreover, expression of UL12 alone caused an increase in SSA, which was completely eliminated when a UL12 mutant lacking exonuclease activity was expressed. UL12-mediated stimulation of SSA was decreased in cells lacking the cellular SSAP, Rad52, and could be restored by coexpressing the viral SSAP, ICP8, indicating that an SSAP is also required. These results demonstrate that UL12 can specifically stimulate SSA and that either ICP8 or Rad52 can function as an SSAP. We suggest that SSA is the homology-mediated repair pathway utilized during HSV infection.
The repair of DNA damage is essential to maintain genomic stability. Cells have at least four distinct DNA repair pathways, and defects in any of them can lead to tumor formation and cancer progression. Herpes Simplex Virus-1 (HSV-1) manipulates components of the host DNA repair pathways. In this paper we showed that DNA repair by the single strand annealing (SSA) pathway was increased during HSV infection and that other pathways were inhibited. We also show that a viral nuclease in conjunction with either a viral or cellular single strand annealing protein can stimulate the SSA pathway. We suggest that viral DNA synthesis occurs via an SSAdependent mechanism that is reminiscent of that used by bacterial viruses such as λ. Interestingly, λ has evolved an SSA-mediated repair mechanism to exchange genetic information that has also been used to enhance gene targeting in bacteria. It is thus possible that HSV proteins could be similarly used as tools to stimulate gene targeting in human cells leading to more effective strategies for gene therapy. Furthermore, the diversity of HSV reported in human populations, combined with the high rate of genetic exchange during infection, suggests that SSA may play a role in viral evolution and pathogenesis.
Homologous recombination serves multiple roles in DNA repair that are essential for maintaining genomic stability. We here describe RI-1, a small molecule that inhibits the central recombination protein RAD51. RI-1 specifically reduces gene conversion in human cells while stimulating single strand annealing. RI-1 binds covalently to the surface of RAD51 protein at cysteine 319 that likely destabilizes an interface used by RAD51 monomers to oligomerize into filaments on DNA. Correspondingly, the molecule inhibits the formation of subnuclear RAD51 foci in cells following DNA damage, while leaving replication protein A focus formation unaffected. Finally, it potentiates the lethal effects of a DNA cross-linking drug in human cells. Given that this inhibitory activity is seen in multiple human tumor cell lines, RI-1 holds promise as an oncologic drug. Furthermore, RI-1 represents a unique tool to dissect the network of reaction pathways that contribute to DNA repair in cells.
We have previously demonstrated that double-strand breaks (DSBs) in regions near telomeres are much more likely to result in large deletions, gross chromosome rearrangements, and chromosome instability than DSBs at interstitial sites within chromosomes. In the present study, we investigated whether this response of subtelomeric regions to DSBs is a result of a deficiency in DSB repair by comparing the frequency of homologous recombination repair (HRR) and nonhomologous end joining (NHEJ) at interstitial and telomeric sites following the introduction of DSBs by I-SceI endonuclease. We also monitored the frequency of small deletions, which have been shown to be the most common mutation at I-SceI-induced DSBs at interstitial sites. We observed no difference in the frequency of small deletions or HRR at interstitial and subtelomeric DSBs. However, the frequency of NHEJ was significantly lower at DSBs near telomeres compared to interstitial sites. The frequency of NHEJ was also lower at DSBs occurring at interstitial sites containing telomeric repeat sequences. We propose that regions near telomeres are deficient in classical NHEJ as a result of the presence of cis-acting telomere-binding proteins that cause DSBs to be processed as though they were telomeres, resulting in excessive resection, telomere loss, and eventual chromosome rearrangements by alternative NHEJ.
Chromosome instability; double-strand break; nonhomologous end joining; telomere
DNA double-strand breaks (DSBs) are among the most lethal lesions associated with genome stability, which, when destabilized, predisposes organs to cancers. DSBs are primarily fixed either with little fidelity by non-homologous end joining (NHEJ) repair or with high fidelity by homology-directed repair (HDR). The phosphorylated form of H2AX on serine 139 (γ-H2AX) is a marker of DSBs. In this study, we explored if the protein phosphatase PP6 is involved in DSB repair by depletion of its expression in human cancer cell lines, and determined PP6 expression in human breast cancer tissues by immunohistochemistry staining. We found that bacterially produced PP6c (the catalytic subunit of PP6)-containing heterotrimeric combinations exhibit phosphatase activity against γ-H2AX in the in vitro phosphatase assays. Depletion of PP6c or PP6R2 led to persistent high levels of γ-H2AX after DNA damage and a defective HDR. Chromatin immunoprecipitation assays demonstrated that PP6c was recruited to the region adjacent to the DSB sites. Expression of PP6c, PP6R2 and PP6R3 in human breast tumors was significantly lower than those in benign breast diseases. Taken together, our results suggest that γ-H2AX is a physiological substrate of PP6 and PP6 is required for HDR and its expression may harbor a protective role during the development of breast cancer.
protein phosphatase; PP6; γ-H2AX; DNA double-strand break; homology-directed repair
We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1f/f), and the other with a homozygous disruption (Fbh1−/−). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1−/− cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1f/f and Fbh1−/− cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.
Fbh1; Rad51; decatenation; bisdioxopiperazine; topoisomerase; mitosis
Defective DNA repair by homologous recombination (HR) is thought to be a major contributor to tumorigenesis in individuals carrying Brca1 mutations. Here we show that DNA breaks in Brca1-deficient cells are aberrantly joined into complex chromosome rearrangements by a process dependent on the non-homologous end joining (NHEJ) factors, 53BP1 and DNA Ligase 4. Loss of 53BP1 alleviates hypersensitivity of Brca1 mutant cells to PARP inhibition and restores error-free repair by HR. Mechanistically, 53BP1 deletion promotes ATM-dependent processing of broken DNA ends to produce recombinogenic single-stranded DNA competent for HR. In contrast, Lig4 deficiency does not rescue the HR defect in Brca1 mutant cells, but prevents the joining of chromatid breaks into chromosome rearrangements. Our results illustrate that HR and NHEJ compete to process DNA breaks that arise during DNA replication, and that shifting the balance between these pathways can be exploited to selectively protect or kill cells harboring Brca1 mutations.
Homology-directed repair (HDR) is essential to limit mutagenesis, chromosomal instability (CIN) and tumorigenesis. We have characterized the consequences of HDR deficiency on anaphase, using markers for incomplete chromosome separation: DAPI-bridges and Ultra-fine bridges (UFBs). We show that multiple HDR factors (Rad51, Brca2 and Brca1) are critical for complete chromosome separation during anaphase, while another chromosome break repair pathway, non-homologous end joining, does not affect chromosome segregation. We then examined the consequences of mild versus severe HDR disruption, using two different dominant-negative alleles of the strand exchange factor, Rad51. We show that mild HDR disruption is viable, but causes incomplete chromosome separation, as detected by DAPI-bridges and UFBs, while severe HDR disruption additionally results in multipolar anaphases and loss of clonogenic survival. We suggest that mild HDR disruption favors the proliferation of cells that are prone to CIN due to defective chromosome separation during anaphase, whereas, severe HDR deficiency leads to multipolar divisions that are prohibitive for cell proliferation.
Chromosome rearrangements can form when incorrect ends are matched during end joining (EJ) repair of multiple chromosomal double-strand breaks (DSBs). We tested whether the ATM kinase limits chromosome rearrangements via suppressing incorrect end utilization during EJ repair of multiple DSBs. For this, we developed a system for monitoring EJ of two tandem DSBs that can be repaired using correct ends (Proximal-EJ) or incorrect ends (Distal-EJ, which causes loss of the DNA between the DSBs). In this system, two DSBs are induced in a chromosomal reporter by the meganuclease I-SceI. These DSBs are processed into non-cohesive ends by the exonuclease Trex2, which leads to the formation of I-SceI–resistant EJ products during both Proximal-EJ and Distal-EJ. Using this method, we find that genetic or chemical disruption of ATM causes a substantial increase in Distal-EJ, but not Proximal-EJ. We also find that the increase in Distal-EJ caused by ATM disruption is dependent on classical non-homologous end joining (c-NHEJ) factors, specifically DNA-PKcs, Xrcc4, and XLF. We present evidence that Nbs1-deficiency also causes elevated Distal-EJ, but not Proximal-EJ, to a similar degree as ATM-deficiency. In addition, to evaluate the roles of these factors on end processing, we examined Distal-EJ repair junctions. We found that ATM and Xrcc4 limit the length of deletions, whereas Nbs1 and DNA-PKcs promote short deletions. Thus, the regulation of end processing appears distinct from that of end utilization. In summary, we suggest that ATM is important to limit incorrect end utilization during c-NHEJ.
When a chromosome is fragmented by multiple double-strand breaks (DSBs), each set of DSB ends needs to be matched correctly during repair to avoid chromosomal rearrangements. Considering the case of two tandem DSBs, if the ends of different breaks (incorrect ends) are used for repair, loss of the intervening DNA can occur. Alternatively, when the ends of a single DSB (correct ends) are used for repair, the original order of the chromosome is restored. Deficiencies in the factors ATM and Nbs1, as seen in patients with Ataxia Telangiectasia and Nijmegen Breakage Syndrome, respectively, have been associated with elevated chromosome rearrangements and cancer predisposition. Hence, we examined the possibility that these factors may be important for the usage of correct ends during repair of multiple DSBs. For this, we developed a reporter system to examine end usage during repair of two tandem DSBs in mammalian chromosomes and found that disruption of ATM or Nbs1 leads to elevated usage of incorrect ends. Furthermore, we found that the role of ATM during end usage depends on a repair pathway called classical non-homologous end joining (c-NHEJ). We suggest that ATM suppresses genome rearrangements via limiting incorrect end utilization during c-NHEJ.
Homologous recombination (HR) provides an efficient mechanism for error-free repair of DNA double-strand breaks (DSBs). However, HR can be also harmful as inappropriate or untimely HR events can give rise to lethal recombination intermediates and chromosome rearrangements. A critical step of HR is the formation of a RAD51 filament on single-stranded (ss)DNA, which mediates the invasion of a homologous DNA molecule. In mammalian cells, several DNA helicases have been implicated in the regulation of this process. RECQ5, a member of the RecQ family of DNA helicases, interacts physically with the RAD51 recombinase and disrupts RAD51 presynaptic filaments in a reaction dependent on ATP hydrolysis. Here, we have precisely mapped the RAD51-interacting domain of RECQ5 and generated mutants that fail to interact with RAD51. We show that although these mutants retain normal ATPase activity, they are impaired in their ability to displace RAD51 from ssDNA. Moreover, we show that ablation of RECQ5-RAD51 complex formation by a point mutation alleviates the inhibitory effect of RECQ5 on HR-mediated DSB repair. These findings provide support for the proposal that interaction with RAD51 is critical for the anti-recombinase attribute of RECQ5.
Chromosomes; DNA Damage; DNA Helicase; DNA Recombination; DNA Repair; DNA Double-Strand Break; Genomic Instability; RAD51; RecQ
To characterize the repair pathways of chromosome double-strand breaks (DSBs), one approach involves monitoring the repair of site-specific DSBs generated by rare-cutting endonucleases, such as I-SceI. Using this method, we first describe the roles of Ercc1, Msh2, Nbs1, Xrcc4, and Brca1 in a set of distinct repair events. Subsequently, we considered that the outcome of such assays could be influenced by the persistent nature of I-SceI-induced DSBs, in that end-joining (EJ) products that restore the I-SceI site are prone to repeated cutting. To address this aspect of repair, we modified I-SceI-induced DSBs by co-expressing I-SceI with a non-processive 3′ exonuclease, Trex2, which we predicted would cause partial degradation of I-SceI 3′ overhangs. We find that Trex2 expression facilitates the formation of I-SceI-resistant EJ products, which reduces the potential for repeated cutting by I-SceI and, hence, limits the persistence of I-SceI-induced DSBs. Using this approach, we find that Trex2 expression causes a significant reduction in the frequency of repair pathways that result in substantial deletion mutations: EJ between distal ends of two tandem DSBs, single-strand annealing, and alternative-NHEJ. In contrast, Trex2 expression does not inhibit homology-directed repair. These results indicate that limiting the persistence of a DSB causes a reduction in the frequency of repair pathways that lead to significant genetic loss. Furthermore, we find that individual genetic factors play distinct roles during repair of non-cohesive DSB ends that are generated via co-expression of I-SceI with Trex2.
A deleterious lesion in DNA is a break of both strands, or a chromosome double-strand break (DSB). DSBs can arise during normal cellular metabolism, but are also a consequence of many forms of cancer therapy. If DSBs are not repaired prior to cell division, entire segments of a chromosome can be lost. Several pathways ensure that DSBs are repaired, though some pathways are prone to causing mutations and/or chromosomal rearrangements, each of which can contribute to cancer development. In the first part of this study, we describe the roles of individual genetic factors in distinct repair pathways of DSBs generated by the I-SceI endonuclease. From these studies, we find that some factors can function in multiple repair pathways. In the second part of this study, we present a method for partially degrading the cohesive DSB overhangs that are generated by I-SceI, which we find facilitates repair products that are not prone to being re-cut by the endonuclease. As a consequence, we have limited the persistence of such breaks, which we find causes a reduction in repair pathways that lead to significant genetic loss. As well, we use this method to characterize the role of individual genetic factors during the repair of non-cohesive DSB ends.
Characterizing the functional overlap and mutagenic potential of different pathways of chromosomal double-strand break (DSB) repair is important to understand how mutations arise during cancer development and treatment. To this end, we have compared the role of individual factors in three different pathways of mammalian DSB repair: alternative-nonhomologous end joining (alt-NHEJ), single-strand annealing (SSA), and homology directed repair (HDR/GC). Considering early steps of repair, we found that the DSB end-processing factors KU and CtIP affect all three pathways similarly, in that repair is suppressed by KU and promoted by CtIP. In contrast, both KU and CtIP appear dispensable for the absolute level of total-NHEJ between two tandem I-SceI–induced DSBs. During later steps of repair, we find that while the annealing and processing factors RAD52 and ERCC1 are important to promote SSA, both HDR/GC and alt-NHEJ are significantly less dependent upon these factors. As well, while disruption of RAD51 causes a decrease in HDR/GC and an increase in SSA, inhibition of this factor did not affect alt-NHEJ. These results suggest that the regulation of DSB end-processing via KU/CtIP is a common step during alt-NHEJ, SSA, and HDR/GC. However, at later steps of repair, alt-NHEJ is a mechanistically distinct pathway of DSB repair, and thus may play a unique role in mutagenesis during cancer development and therapy.
Changes to the sequence of DNA, or mutations, can disrupt cellular growth control genes, which can lead to cancer development. Such mutations likely arise from damage to DNA that is repaired in a way that fails to restore the original sequence. One type of DNA damage is a chromosomal double-strand break. We have developed assays to measure how these breaks are repaired, and also how such repair can lead to mutations. In particular, we present an assay to measure a pathway of repair that results in deletion mutations, often with evidence of short homologous sequences at the repair junctions (alt-NHEJ). We have compared the genetic requirements of this repair pathway in relation to other pathways of repair that use extensive homology. We find that factors KU and CtIP appear to affect the initial stages of repair of each of these pathways, regardless of the length of homology. However, these pathways appear to diverge at later steps, as relates to the role of the repair factors RAD52, ERCC1, and RAD51. Given that mutations observed in some cancer cells are consistent with alt-NHEJ repair, these mechanistic descriptions provide models for how such mutations could arise in cancer.
Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52−/− mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.
Loss of heterozygosity (LOH) is a common genetic alteration in tumors and often extends several megabases to encompass multiple genetic loci or even whole chromosome arms. Based on marker and karyotype analysis of tumor samples, a significant fraction of LOH events appears to arise from mitotic recombination between homologous chromosomes, reminiscent of recombination during meiosis. As DNA double-strand breaks (DSBs) initiate meiotic recombination, a potential mechanism leading to LOH in mitotically dividing cells is DSB repair involving homologous chromosomes. We therefore sought to characterize the extent of LOH arising from DSB-induced recombination between homologous chromosomes in mammalian cells. To this end, a recombination reporter was introduced into a mouse embryonic stem cell line that has nonisogenic maternal and paternal chromosomes, as is the case in human populations, and then a DSB was introduced into one of the chromosomes. Recombinants involving alleles on homologous chromosomes were readily obtained at a frequency of 4.6 × 10−5; however, this frequency was substantially lower than that of DSB repair by nonhomologous end joining or the inferred frequency of homologous repair involving sister chromatids. Strikingly, the majority of recombinants had LOH restricted to the site of the DSB, with a minor class of recombinants having LOH that extended to markers 6 kb from the DSB. Furthermore, we found no evidence of LOH extending to markers 1 centimorgan or more from the DSB. In addition, crossing over, which can lead to LOH of a whole chromosome arm, was not observed, implying that there are key differences between mitotic and meiotic recombination mechanisms. These results indicate that extensive LOH is normally suppressed during DSB-induced allelic recombination in dividing mammalian cells.