Fanconi anemia FANCG protein in mitigating radiation- and enzyme-induced DNA double-strand breaks by homologous recombination in vertebrate cells

The rare hereditary disorder Fanconi anemia (FA) is characterized by progressive bone marrow failure, congenital skeletal abnormality, elevated susceptibility to cancer, and cellular hypersensitivity to DNA cross-linking chemicals and sometimes other DNA-damaging agents. Molecular cloning identified six causative genes (FANCA, -C, -D2, -E, -F, and -G) encoding a multiprotein complex whose precise biochemical function remains elusive. Recent studies implicate this complex in DNA damage responses that are linked to the breast cancer susceptibility proteins BRCA1 and BRCA2. Mutations in BRCA2, which participates in homologous recombination (HR), are the underlying cause in some FA patients. To elucidate the roles of FA genes in HR, we disrupted the FANCG/XRCC9 locus in the chicken B-cell line DT40. FANCG-deficient DT40 cells resemble mammalian fancg mutants in that they are sensitive to killing by cisplatin and mitomycin C (MMC) and exhibit increased MMC and radiation-induced chromosome breakage. We find that the repair of I-SceI-induced chromosomal double-strand breaks (DSBs) by HR is decreased approximately 9-fold in fancg cells compared with the parental and FANCG-complemented cells. In addition, the efficiency of gene targeting is mildly decreased in FANCG-deficient cells, but depends on the specific locus. We conclude that FANCG is required for efficient HR-mediated repair of at least some types of DSBs.     

Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination

Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs.     

The controlling role of ATM in homologous recombinational repair of DNA damage

The human genetic disorder ataxia telangiectasia (A-T), caused by mutation in the ATM gene, is characterized by chromosomal instability, radiosensitivity and defective cell cycle checkpoint activation. DNA double-strand breaks (dsbs) persist in A-T cells after irradiation, but the underlying defect is unclear. To investigate ATM's interactions with dsb repair pathways, we disrupted ATM along with other genes involved in the principal, complementary dsb repair pathways of homologous recombination (HR) or non-homologous end-joining (NHEJ) in chicken DT40 cells. ATM(-/-) cells show altered kinetics of radiation-induced Rad51 and Rad54 focus formation. Ku70-deficient (NHEJ(-)) ATM(-/-) chicken DT40 cells show radiosensitivity and high radiation-induced chromosomal aberration frequencies, while Rad54-defective (HR(-)) ATM(-/-) cells show only slightly elevated aberration levels after irradiation, placing ATM and HR on the same pathway. These results reveal that ATM defects impair HR-mediated dsb repair and may link cell cycle checkpoints to HR activation.     

c-Abl tyrosine kinase is not essential for ataxia telangiectasia mutated functions in chromosomal maintenance

c-Abl is activated by DNA damage in an ataxia telangiectasia mutated (ATM)-dependent manner and plays important roles in growth arrest and apoptosis induced by DNA damage. c-Abl also interacts physically and functionally with Rad51, a key molecule in homologous recombinational (HR) DNA repair. To study further the roles of c-Abl in HR DNA repair, we generated c-Abl(-/-) and ATM(-/-)/c-Abl(-/-) mutant cell lines from a chicken B lymphocyte DT40 cell line, comparing the phenotypes of these mutants to those of ATM(-/-) DT40 cells that we had created previously. We found that the time course of radiation-induced Rad51 focus formation is abnormal in ATM(-/-) DT40 cells, consistent with the observation that ATM(-/-) DT40 cells display hypersensitivity to ionizing radiation and highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In contrast, c-Abl(-/-) cells did not show these ATM-related defects in their cellular response to radiation, nor did the disruption of c-Abl in ATM(-/-) DT40 cells exacerbate these ATM-related defects. However, c-Abl(-/-) DT40 cells, but not ATM(-/-) DT40 cells, were resistant to radiation-induced apoptosis, indicating an important role for c-Abl in this cellular response to ionizing radiation. These results therefore indicate that, although ATM plays an important role in genome maintenance, c-Abl is not essential for this ATM function. These findings suggest that c-Abl and ATM play important roles in the maintenance of the cell homeostasis in response to DNA damage that are, at least in part, independent.     

Defective nucleotide excision repair with normal centrosome structures and functions in the absence of all vertebrate centrins

The principal microtubule-organizing center in animal cells, the centrosome, contains centrin, a small, conserved calcium-binding protein unique to eukaryotes. Several centrin isoforms exist and have been implicated in various cellular processes including nuclear export and deoxyribonucleic acid (DNA) repair. Although centrins are required for centriole/basal body duplication in lower eukaryotes, centrin functions in vertebrate centrosome duplication are less clear. To define these roles, we used gene targeting in the hyperrecombinogenic chicken DT40 cell line to delete all three centrin genes in individual clones. Unexpectedly, centrin-deficient cells underwent normal cellular division with no detectable cell cycle defects. Light and electron microscopy analyses revealed no significant difference in centrosome composition or ultrastructure. However, centrin deficiency made DT40 cells highly sensitive to ultraviolet (UV) irradiation, with Cetn3 deficiency exacerbating the sensitivity of Cetn4/Cetn2 double mutants. DNA damage checkpoints were intact, but repair of UV-induced DNA damage was delayed in centrin nulls. These data demonstrate a role for vertebrate centrin in nucleotide excision repair.     

Evaluation of recombination repair pathways in thermal radiosensitization

Thermal radiosensitization has been shown to cause inhibition of repair of sublethal and potentially lethal damage and DNA DSBs. In this study we assessed thermal radiosensitization in mutants deficient in homologous recombinational (HR) repair and nonhomologous end joining repair (NHEJ). Using cells of the mouse wild-type embryo fibroblast cell line MEF and its Ku80(-/-) derivative that is deficient in NHEJ, we showed that thermal radiosensitization is the same in both cell lines. Further studies with cells of the wild-type CHO-AA8 cell line and its derivative IRS(ISF), which is deficient in HR, also showed comparable thermal radiosensitization in both cell lines. Further experiments using cells of chicken DT40 cell lines also showed comparable thermal radiosensitization between the wild-type HR mutant Rad54, the NHEJ mutant Ku70, and the double mutant Rad 54-Ku70. These results indicate that the HR and NHEJ pathways may not be targets for thermal radiosensitization.     

The Fanconi anaemia components UBE2T and FANCM are functionally linked to nucleotide excision repair

The many proteins that function in the Fanconi anaemia (FA) monoubiquitylation pathway initiate replicative DNA crosslink repair. However, it is not clear whether individual FA genes participate in DNA repair pathways other than homologous recombination and translesion bypass. Here we show that avian DT40 cell knockouts of two integral FA genes--UBE2T and FANCM are unexpectedly sensitive to UV-induced DNA damage. Comprehensive genetic dissection experiments indicate that both of these FA genes collaborate to promote nucleotide excision repair rather than translesion bypass to protect cells form UV genotoxicity. Furthermore, UBE2T deficiency impacts on the efficient removal of the UV-induced photolesion cyclobutane pyrimidine dimer. Therefore, this work reveals that the FA pathway shares two components with nucleotide excision repair, intimating not only crosstalk between the two major repair pathways, but also potentially identifying a UBE2T-mediated ubiquitin-signalling response pathway that contributes to nucleotide excision repair.     

Recombination repair pathway in the maintenance of chromosomal integrity against DNA interstrand crosslinks

DNA interstrand crosslinks (ICL) present a major threat to cell viability and genome integrity. In eukaryotic cells, the ICLs have been suggested to be repaired by a complex process involving Xpf/Ercc1-mediated endonucleolytic incision and homologous recombination (HR). However, the entire feature of the ICL tolerating mechanism is still poorly understood. Here we studied chromosome aberrations (CA) and sister chromatid exchanges (SCE) by the use of the crosslinking agent mitomycin C (MMC), in chicken DT40 cells with the HR genes disrupted by targeted replacement. The disruption of the Rad54, Rad51B, Rad51C, Rad51D, Xrcc2 and Xrcc3 genes resulted in a dramatic reduction of spontaneous and MMC-induced SCEs. Interestingly, while HR-deficient cells were hypersensitive to cell killing by MMC, MMC-induced CAs were also suppressed in the HR-deficient cells except for Rad51D-, Xrcc2- and Xrcc3-deficient cells. These observations indicate that DNA double strand breaks (DSB) at stalled replication forks and those arising as repair intermediates present strong signals to cell death but can be tolerated by the HR repair pathway, where Rad54, Rad51B and Rad51C have an initiative role and repair can be completed by their paralogs Rad51D, Xrcc2 and Xrcc3. The impairment of the HR pathway, which otherwise leads to cell death, may be somewhat substituted by an alternative mechanism such as the Mre11/Rad50/Nbs1 pathway, resulting in reduced frequencies of SCEs and CAs.     

Involvement of vertebrate polkappa in Rad18-independent postreplication repair of UV damage

DNA damage, which is left unrepaired by excision repair pathways, often blocks replication, leading to lesions such as breaks and gaps on the sister chromatids. These lesions may be processed by either homologous recombination (HR) repair or translesion DNA synthesis (TLS). Vertebrate Polkappa belongs to the DNA polymerase Y family, as do most TLS polymerases. However, the role for Polkappa in vertebrate cells is unclear because of the lack of reverse genetic studies. Here, we generated cells deficient in Polkappa (polkappa cells) from the chicken B lymphocyte line DT40. Although purified Polkappa is unable to bypass ultraviolet (UV) damage, polkappa cells exhibited increased UV sensitivity, and the phenotype was suppressed by expression of human and chicken Polkappa, suggesting that Polkappa is involved in TLS of UV photoproduct. Defects in both Polkappa and Rad18, which regulates TLS in yeast, in DT40 showed an additive effect on UV sensitivity. Interestingly, the level of sister chromatid exchange, which reflects HR-mediated repair, was elevated in normally cycling polkappa cells. This implies functional redundancy between HR and Polkappa in maintaining chromosomal DNA. In conclusion, vertebrate Polkappa is involved in Rad18-independent TLS of UV damage and plays a role in maintaining genomic stability.     

Post-replication repair in DT40 cells: translesion polymerases versus recombinases

Replication forks inevitably stall at damaged DNA in every cell cycle. The ability to overcome DNA lesions is an essential feature of the replication machinery. A variety of specialized polymerases have recently been discovered, which enable cells to replicate past various forms of damage by a process termed translesion synthesis. Alternatively, homologous recombination can be used to restart DNA replication across the lesion. Genetic and biochemical studies have shed light on the impact of these two post-replication repair pathways in bacteria and yeast. In vertebrates, however, a genetic approach to study post-replication repair has been compromised because many of the genes involved appear to be essential for embryonic development. We have taken advantage of the chicken cell line DT40 to perform a genetic analysis of translesion synthesis and homologous recombination and to characterize genetic interactions between these two pathways in vertebrates. In this article, we aim to summarize our current understanding of post-replication repair in DT40 in the perspective of bacterial, yeast and mammalian genetics.     

Genetic dissection of vertebrate 53BP1: a major role in non-homologous end joining of DNA double strand breaks

53BP1 (p53 binding protein) is a BRCT domain-containing protein that is rapidly recruited to DNA double strand breaks (DSBs). To investigate the role of 53BP1 in the DNA damage response, we generated 53BP1(-/-) cells from the chicken DT40 cell line. As in mammalian cells, mutation of 53BP1 increased cellular sensitivity to ionizing radiation. Although depletion of 53BP1 resulted in checkpoint defects in mammalian cells, DT40 53BP1(-/-) cells had normal intra S phase and G2/M checkpoints. G1 specific radiosensitivity and a higher sensitivity to topoisomerase II suggested defective non-homologous end joining (NHEJ) defects in DT40 53BP1(-/-) cells. Genetic analyses confirm this suggestion as we have demonstrated an epistatic relationship between 53BP1 and the NHEJ genes, Ku70 and Artemis, but not with Rad54, a gene essential for repair of DSBs by homologous recombination. We conclude that the major role of 53BP1 in supporting survival of DT40 cells that have suffered DNA DSBs is in facilitating repair by NHEJ.     

Ubc13 dosage is critical for immunoglobulin gene conversion and gene targeting in vertebrate cells

In contrast to lower eukaryotes, most vertebrate cells are characterized by a moderate efficiency of homologous recombination (HR) and limited feasibility of targeted genetic modifications. As a notable exception, the chicken DT40 B cell line is distinguished by efficient homology-mediated repair of DNA lesions during Ig gene conversion, and also shows exceptionally high gene-targeting efficiencies. The molecular basis of these phenomena is elusive. Here we show that the activity levels of Ubc13, the E2 enzyme responsible for non-canonical K63-linked polyubiquitination, are critical for high efficiency of Ig gene conversion and gene targeting in DT40. Ubc13^+/− cells show substantially lower homology-mediated repair, yet do not display changes in somatic hypermutation, overall DNA repair or cell proliferation. Our results suggest that modulation of the activity of K63-linked polyubiquitination may be used to customize HR efficiencies in vertebrate cells.     

Fanconi's anemia: what have we learned from the genes so far?

Fanconi's anemia (FA) is a rare genetic disorder affecting children at an early age; patients suffer from progressive bone marrow failure and, in many cases, from congenital malformations. As cells from FA patients have an increased sensitivity to DNA-crosslinking agents, FA has been included among the group of DNA repair disorders. However, identification of a specific DNA repair defect in FA has not been firmly established. None the less, this cellular phenotype has allowed the classification of FA patients into eight complementation groups defining eight possible FA genes. Two of these genes have now been cloned and, although they have raised more questions than they have answered, are facilitating the identification of cellular processes implicated in the pathophysiology of FA, and the design of new therapies.     

Functional relationships of FANCC to homologous recombination, translesion synthesis, and BLM

Some of the restarting events of stalled replication forks lead to sister chromatid exchange (SCE) as a result of homologous recombination (HR) repair with crossing over. The rate of SCE is elevated by the loss of BLM helicase or by a defect in translesion synthesis (TLS). We found that spontaneous SCE levels were elevated approximately 2-fold in chicken DT40 cells deficient in Fanconi anemia (FA) gene FANCC. To investigate the mechanism of the elevated SCE, we deleted FANCC in cells lacking Rad51 paralog XRCC3, TLS factor RAD18, or BLM. The increased SCE in fancc cells required Xrcc3, whereas the fancc/rad18 double mutant exhibited higher SCE than either single mutant. Unexpectedly, SCE in the fancc/blm mutant was similar to that in blm cells, indicating functional linkage between FANCC and BLM. Furthermore, MMC-induced formation of GFP-BLM nuclear foci was severely compromised in both human and chicken fancc or fancd2 cells. Our cell survival data suggest that the FA proteins serve to facilitate HR, but not global TLS, during crosslink repair.     

Interplay between Fanconi anemia and homologous recombination pathways in genome integrity

The Fanconi anemia (FA) pathway plays a central role in the repair of DNA interstrand crosslinks (ICLs) and regulates cellular responses to replication stress. Homologous recombination (HR), the error‐free pathway for double‐strand break (DSB) repair, is required during physiological cell cycle progression for the repair of replication‐associated DNA damage and protection of stalled replication forks. Substantial crosstalk between the two pathways has recently been unravelled, in that key HR proteins such as the RAD51 recombinase and the tumour suppressors BRCA1 and BRCA2 also play important roles in ICL repair. Consistent with this, rare patient mutations in these HR genes cause FA pathologies and have been assigned FA complementation groups. Here, we focus on the clinical and mechanistic implications of the connection between these two cancer susceptibility syndromes and on how these two molecular pathways of DNA replication and repair interact functionally to prevent genomic instability.     

The Walker B motif in avian FANCM is required to limit sister chromatid exchanges but is dispensable for DNA crosslink repair

FANCM, the most highly conserved component of the Fanconi Anaemia (FA) pathway can resolve recombination intermediates and remodel synthetic replication forks. However, it is not known if these activities are relevant to how this conserved protein activates the FA pathway and promotes DNA crosslink repair. Here we use chicken DT40 cells to systematically dissect the function of the helicase and nuclease domains of FANCM. Our studies reveal that these domains contribute distinct roles in the tolerance of crosslinker, UV light and camptothecin-induced DNA damage. Although the complete helicase domain is critical for crosslink repair, a predicted inactivating mutation of the Walker B box domain has no impact on FA pathway associated functions. However, this mutation does result in elevated sister chromatid exchanges (SCE). Furthermore, our genetic dissection indicates that FANCM functions with the Blm helicase to suppress spontaneous SCE events. Overall our results lead us to reappraise the role of helicase domain associated activities of FANCM with respect to the activation of the FA pathway, crosslink repair and in the resolution of recombination intermediates.     

Mcm8 and Mcm9 Form a Complex that Functions in Homologous Recombination Repair Induced by DNA Interstrand Crosslinks

DNA interstrand crosslinks (ICLs) are highly toxic lesions that stall the replication fork to initiate the repair process during the S phase of vertebrates. Proteins involved in Fanconi anemia (FA), nucleotide excision repair (NER), and translesion synthesis (TS) collaboratively lead to homologous recombination (HR) repair. However, it is not understood how ICL-induced HR repair is carried out and completed. Here, we showed that the replicative helicase-related Mcm family of proteins, Mcm8 and Mcm9, forms?a complex required for HR repair induced by ICLs. Chicken DT40 cells lacking MCM8 or MCM9 are viable but highly sensitive to ICL-inducing agents, and exhibit more chromosome aberrations in the presence of mitomycin C compared with wild-type cells. During ICL repair, Mcm8 and Mcm9 form nuclear foci that partly colocalize with Rad51. Mcm8-9 works downstream of the FA and BRCA2/Rad51 pathways, and is required for HR that promotes sister chromatid exchanges, probably as a hexameric ATPase/helicase.     

Parp-1 protects homologous recombination from interference by Ku and Ligase IV in vertebrate cells

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1(-/-) DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB-binding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1(-/-) cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.     

Fen-1 facilitates homologous recombination by removing divergent sequences at DNA break ends

Homologous recombination (HR) requires nuclease activities at multiple steps, but the contribution of individual nucleases to the processing of double-strand DNA ends at different stages of HR has not been clearly defined. We used chicken DT40 cells to investigate the role of flap endonuclease 1 (Fen-1) in HR. FEN-1-deficient cells exhibited a significant decrease in the efficiency of immunoglobulin gene conversion while being proficient in recombination between sister chromatids, suggesting that Fen-1 may play a role in HR between sequences of considerable divergence. To clarify whether sequence divergence at DNA ends is truly the reason for the observed HR defect in FEN-1(-/-) cells we inserted a unique I-SceI restriction site in the genome and tested various donor and recipient HR substrates. We found that the efficiency of HR-mediated DNA repair was indeed greatly diminished when divergent sequences were present at the DNA break site. We conclude that Fen-1 eliminates heterologous sequences at DNA damage site and facilitates DNA repair by HR.     

Reverse genetic studies of homologous DNA recombination using the chicken B-lymphocyte line, DT40

DT40 is an avian leucosis virus-transformed chicken B-lymphocyte line which exhibits high ratios of targeted to random integration of transfected DNA constructs. This efficient targeted integration may be related to the ongoing diversification of the variable segment of the immunoglobulin gene through homologous DNA recombination-controlled gene conversion. DT40s are a convenient model system for making gene-targeted mutants. Another advantage is the relative tractability of these cells, which makes it possible to disrupt multiple genes in a single cell and to generate conditionally gene-targeted mutants including temperature-sensitive mutants. There are strong phenotypic similarities between murine and DT40 mutants of various genes involved in DNA recombination. These similarities confirm that the DT40 cell line is a reasonable model for the analysis of vertebrate DNA recombination, despite obvious concerns associated with the use of a transformed cell line, which may have certain cell-line-specific characteristics. Here we describe our studies of homologous DNA recombination in vertebrate somatic cells using reverse genetics in DT40 cells.