The role of BRCA2 in replication-coupled DNA interstrand cross-link repair in vitro

Using a defined substrate DNA with a single psoralen interstrand cross-link (ICL), we studied the molecular mechanism of human ICL repair. In vitro ICL repair by human extracts is dependent on replication and is a largely error-free process. Extracts from a human BRCA2-defective mutant cell line, CAPAN-1, are severely compromised in ICL repair. Specifically, 'unhooked' but not fully repaired products accumulate in the reaction with CAPAN-1, and transient expression of BRCA2 in CAPAN-1 restores repair activity. Together, these results reveal that BRCA2 participates in repair of replication-mediated double-strand breaks generated when replication forks encounter ICLs. We also show that nucleotide excision repair is essential for the removal of the lesion left behind on one strand after unhooking. This study provides new mechanistic insights into the repair of ICLs in human cells.     

DNA interstrand crosslink repair during G1 involves nucleotide excision repair and DNA polymerase zeta

The repair mechanisms acting on DNA interstrand crosslinks (ICLs) in eukaryotes are poorly understood. Here, we provide evidence for a pathway of ICL processing that uses components from both nucleotide excision repair (NER) and translesion synthesis (TLS) and predominates during the G1 phase of the yeast cell cycle. Our results suggest that repair is initiated by the NER apparatus and is followed by a thwarted attempt at gap-filling by the replicative Polymerase delta, which likely stalls at the site of the remaining crosslinked oligonucleotide. This in turn leads to ubiquitination of PCNA and recruitment of the damage-tolerant Polymerase zeta that can perform TLS. The ICL repair factor Pso2 acts downstream of the incision step and is not required for Polymerase zeta activation. We show that this combination of NER and TLS is the only pathway of ICL repair available to the cell in G1 phase and is essential for viability in the presence of DNA crosslinks.     

BRCA1 functions independently of homologous recombination in DNA interstrand crosslink repair

Brca1 is required for DNA repair by homologous recombination (HR) and normal embryonic development. Here we report that deletion of the DNA damage response factor 53BP1 overcomes embryonic lethality in Brca1-nullizygous mice and rescues HR deficiency, as measured by hypersensitivity to polyADP-ribose polymerase (PARP) inhibition. However, Brca1,53BP1 double-deficient cells are hypersensitive to DNA interstrand crosslinks (ICLs), indicating that BRCA1 has an additional role in DNA crosslink repair that is distinct from HR. Disruption of the nonhomologous end-joining (NHEJ) factor, Ku, promotes DNA repair in Brca1-deficient cells; however deletion of either Ku or 53BP1 exacerbates genomic instability in cells lacking FANCD2, a mediator of the Fanconi anemia pathway for ICL repair. BRCA1 therefore has two separate roles in ICL repair that can be modulated by manipulating NHEJ, whereas FANCD2 provides a key activity that cannot be bypassed by ablation of 53BP1 or Ku.     

S. cerevisiae has three pathways for DNA interstrand crosslink repair.

Yeast mutants, snm1 (pso2-1), rev3 (pso1-1), and rad51, which display significant sensitivity to interstrand crosslinks (ICLs) have low relative sensitivity to other DNA damaging agents. SNM1, REV3, and RAD51 were disrupted in the same haploid strain, singly and in combination. The double mutants, snm1 Delta rev3 Delta, snm1 Delta rad51 Delta and rev3 Delta rad51 Delta were all more sensitive to ICLs than any of the single mutants, indicating that they are in separate epistasis groups for survival. A triple mutant displayed greater sensitivity to ICLs than any of the double mutants, with one ICL per genome being lethal. Therefore, Saccharomyces cerevisiae appears to have three separate ICL repair pathways, but no more. S-phase delay was not observed after ICL damage introduced by cisplatin (CDDP) or 8-methoxypsoralen (8-MOP) during the G1-phase, in any of the above mutants, or in an isogenic rad14 Delta mutant deficient in nucleotide excision repair. However, the psoralen analog angelicin (monoadduct damage) induced a significant S-phase delay in the rad14 Delta mutant. Thus, normal S-phase in the presence of ICLs does not seem to be due to rapid excision repair. The results also indicate that monoadduct formation by CDDP or 8-MOP at the doses used is not sufficient to delay S-phase in the rad14 Delta mutant. While the sensitivity of a rev3 Delta mutant indicates Pol zeta is needed for optimal ICL repair, isogenic cells deficient in Pol eta (rad30 Delta cells) were not significantly more sensitive to ICL agents than wild-type cells, and have no S-phase delay.     

FANCP/SLX4: A Swiss army knife of DNA interstrand crosslink repair

Fanconi anemia (FA) is a rare genetic disease characterized by congenital abnormalities, bone marrow failure and heightened cancer susceptibility. The FA proteins are known to function in the cellular defense against DNA interstrand crosslinks (ICLs), a process that remains poorly understood. A recent spate of discoveries has led to the identification of one new FA gene, FANCP/SLX4, and two strong candidate FA genes, FAN1 and RAD51C. In this perspective we describe the discovery of FANCP/SLX4 and discuss how these new findings collectively refine our understanding of DNA ICL repair.Key words: Fanconi anemia, DNA repair, ubiquitin, FANCP/SLX4, DNA interstrand crosslink repair     

Mechanism and regulation of incisions during DNA interstrand cross-link repair

A critical step in DNA interstrand cross-link repair is the programmed collapse of replication forks that have stalled at an ICL. This event is regulated by the Fanconi anemia pathway, which suppresses bone marrow failure and cancer. In this perspective, we focus on the structure of forks that have stalled at ICLs, how these structures might be incised by endonucleases, and how incision is regulated by the Fanconi anemia pathway.     

p21 promotes error-free replication-coupled DNA double-strand break repair

p21 is a well-established regulator of cell cycle progression. The role of p21 in DNA repair, however, remains poorly characterized. Here, we describe a critical role of p21 in a replication-coupled DNA double-strand break (DSB) repair that is mechanistically distinct from its cell cycle checkpoint function. We demonstrate that p21-deficient cells exhibit elevated chromatid-type aberrations, including gaps and breaks, dicentrics and radial formations, following exposure to several DSB-inducing agents. p21(-/-) cells also exhibit an increased DNA damage-inducible DNA-PK(CS) S2056 phosphorylation, indicative of elevated non-homologous DNA end joining. Concomitantly, p21(-/-) cells are defective in replication-coupled homologous recombination (HR), exhibiting decreased sister chromatid exchanges and HR-dependent repair as determined using a crosslinked GFP reporter assay. Importantly, we establish that the DSB hypersensitivity of p21(-/-) cells is associated with increased cyclin-dependent kinase (CDK)-dependent BRCA2 S3291 phosphorylation and MRE11 nuclear foci formation and can be rescued by inhibition of CDK or MRE11 nuclease activity. Collectively, our results uncover a novel mechanism by which p21 regulates the fidelity of replication-coupled DSB repair and the maintenance of chromosome stability distinct from its role in the G1-S phase checkpoint.     

DNA interstrand cross-link repair in Saccharomyces cerevisiae

DNA interstrand cross-links (ICL) present a formidable challenge to the cellular DNA repair apparatus. For Escherichia coli, a pathway which combines nucleotide excision repair (NER) and homologous recombination repair (HRR) to eliminate ICL has been characterized in detail, both genetically and biochemically. Mechanisms of ICL repair in eukaryotes have proved more difficult to define, primarily as a result of the fact that several pathways appear compete for ICL repair intermediates, and also because these competing activities are regulated in the cell cycle. The budding yeast Saccharomyces cerevisiae has proven a powerful tool for dissecting ICL repair. Important roles for NER, HRR and postreplication/translesion synthesis pathways have all been identified. Here we review, with reference to similarities and differences in higher eukaryotes, what has been discovered to date concerning ICL repair in this simple eukaryote.     

DNA interstrand crosslinks repair in mammalian cells

We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked plasmids and the ICL repair was monitored by following the restoration of the GFP expression. It was accompanied by gamma-H2AX foci formation suggesting that DSBs were formed during the process. However, the same amount of gamma-H2AX foci was observed when cells were transfected with native plasmid, which indicated that gamma-H2AX foci appearance could not be used to determine the amount of DSBs connected with the ICL repair in this system. For this reason we further monitored the DSB formation by determining the amount of linearized plasmids, since having one crosslink per plasmid on average, any ICL-driven DSB formation would lead to plasmid linearization. Native and crosslinked plasmids were incubated in repair-competent cell-free extracts from G1 and S phase HeLa cells. Although a considerable part of the ICLs was repaired, no linearization of the plasmids was observed in the extracts, which was interpreted that DSBs were not formed as intermediates in the process of ICL repair. In another set of experiments HR-proficient HeLa and HR-deficient irs3 cells were transfected with native and crosslinked plasmids, and 6 h and 12 h later the plasmid DNA was isolated and analyzed by electrophoresis. The same amount of linear plasmid molecules was observed in both cell lines, regardless of whether they were transfected with native or crosslinked pEGFP-N1, which further confirmed that DSB formation was not an obligatory step in the process of ICL repair by HR.     

Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1-/- mice

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1-/- mice was compared to that in young and old wild-type mice. Ercc1-/- mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1-/- mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa-/- mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging.     

Enhanced repair of DNA interstrand crosslinks in S phase

Hela cells synchronized in G1 and S phases of the cell cycle were transfected with pEGFP crosslinked with trioxsalen. Twelve hours later the number of fluorescent cells was determined by fluorescent microscopy. Cells in S phase have repaired 0.2-0.3 ICL/kb over the 12h period, while cells in G1 phase repaired interstrand crosslinks much more poorly. The crosslinked plasmids were efficiently recruited to the nuclear matrix both in G1 phase and S-phase, which showed that the poor repair of G1 cells was a result of a lack of DNA replication rather than of a lack of matrix attachment.     

DNA repair defects channel interstrand DNA cross-links into alternate recombinational and error-prone repair pathways

The repair of psoralen interstrand cross-links in the yeast Saccharomyces cerevisiae involves the DNA repair groups nucleotide excision repair (NER), homologous recombination (HR), and post-replication repair (PRR). In repair-proficient yeast cells cross-links induce double-strand breaks, in an NER-dependent process; the double-strand breaks are then repaired by HR. An alternate error-prone repair pathway generates mutations at cross-link sites. We have characterized the repair of plasmid molecules carrying a single psoralen cross-link, psoralen monoadduct, or double-strand break in yeast cells with deficiencies in NER, HR, or PRR genes, measuring the repair efficiencies and the levels of gene conversions, crossing over, and mutations. Strains with deficiencies in the NER genes RAD1, RAD3, RAD4, and RAD10 had low levels of cross-link-induced recombination but higher mutation frequencies than repair-proficient cells. Deletion of the HR genes RAD51, RAD52, RAD54, RAD55, and RAD57 also decreased induced recombination and increased mutation frequencies above those of NER-deficient yeast. Strains lacking the PRR genes RAD5, RAD6, and RAD18 did not have any cross-link-induced mutations but showed increased levels of recombination; rad5 and rad6 cells also had altered patterns of cross-link-induced gene conversion in comparison with repair-proficient yeast. Our observations suggest that psoralen cross-links can be repaired by three pathways: an error-free recombinational pathway requiring NER and HR and two PRR-dependent error-prone pathways, one NER-dependent and one NER-independent.     

Deubiquitination of FANCD2 is required for DNA crosslink repair

Monoubiquitination of FANCD2 and PCNA promotes DNA repair. It causes chromatin accumulation of FANCD2 and facilitates PCNA's recruitment of translesion polymerases to stalled replication. USP1, a protease that removes monoubiquitin from FANCD2 and PCNA, was thought to reverse the DNA damage response of these substrates. We disrupted USP1 in chicken cells to dissect its role in a stable genetic system. USP1 ablation increases FANCD2 and PCNA monoubiquitination but unexpectedly results in DNA crosslinker sensitivity. This defective DNA repair is associated with constitutively chromatin-bound, monoubiquitinated FANCD2. In contrast, persistent PCNA monoubiquitination has negligible impact on DNA repair or mutagenesis. USP1 was previously shown to autocleave after DNA damage. In DT40, USP1 autocleavage is not stimulated by DNA damage, and expressing a noncleavable mutant in the USP1 knockout strain partially rescues crosslinker sensitivity. We conclude that efficient DNA crosslink repair requires FANCD2 deubiquitination, whereas FANCD2 monoubiquitination is not dependent on USP1 autocleavage.     

Loss of homologous recombination or non-homologous end-joining leads to radial formation following DNA interstrand crosslink damage

High levels of interstrand cross-link damage in mammalian cells cause chromatid breaks and radial formations recognizable by cytogenetic examination. The mechanism of radial formation observed following DNA damage has yet to be determined. Due to recent findings linking homologous recombination and non-homologous end-joining to the action of the Fanconi anemia pathway, we speculated that radials might be the result of defects in either of the pathways of DNA repair. To test this hypothesis, we have investigated the role of homologous recombination proteins RAD51 and RAD52, non-homologous end-joining proteins Ku70 and LIG4, and protein MRE11 in radial formation and cell survival following interstrand crosslink damage with mitomycin C. For the studies we used small inhibitory RNA to deplete the proteins from cells, allowing for evaluation of radial formation and cell survival. In transformed normal human fibroblasts, depletion of these proteins increased interstrand crosslink sensitivity as manifested by decreased cell survival and increased radial formation. These results demonstrate that inactivation of proteins from either of the two separate DNA repair pathways increases cellular sensitivity to interstrand crosslinks, indicating each pathway plays a role in the normal response to interstrand crosslink damage. We can also conclude that homologous recombination or non-homologous end-joining are not required for radial formation, since radials occur with depletion of these pathways.     

DNA interstrand crosslink formation by mechlorethamine at a cytosine-cytosine mismatch pair: kinetics and sequence dependence

Expansion of the triplet repeat DNA sequence d[CGG]n.d[CCG]n is a characteristic of Fragile X syndrome, a human neurodegenerative disease. Stable intrastrand conformations formed by both d[CGG]n and d[CCG]n, and involving G-G and C-C mismatch pairs, respectively, are believed to be of importance in the development of the disease. We have shown previously that C-C mismatch pairs can be crosslinked covalently by mechlorethamine, a nitrogen mustard alkylating agent, and hence this reaction may be of value as a probe for conformers of d[CCG]n. To characterize the mechlorethamine C-C crosslink reaction further, here we report the kinetics and sequence dependence of formation of the crosslink species, using a series of model duplexes. The rate of reaction depends on the base sequence proximal to the C-C mismatch pair. Hence, in 19mer duplexes containing a central d[M4M3M2M1Cn1n2n3n4].d[N4N3N2N1Cm1m2m3m4] sequence, where M-m and N-n are complementary base pairs, the amount of crosslink increased with increasing G-C content of the eight base pairs neighboring the C-C mismatch and with the proximity of the G-C pairs to the C-C mismatch. Molecular dynamics simulations of the solvated duplexes provided an explanation of these data. Hence, for a C-C pair flanked by G-C base pairs the mismatched cytosine bases remain stacked within the duplex, but for a C-C pair flanked by A-T base pairs, the simulations suggested local opening of the duplex around the C-C pair, making it a less effective target for mechlorethamine.     

The Fanconi anemia pathway and the DNA interstrand cross-links repair

Fanconi anemia (FA) is a genetic cancer-predisposition syndrome characterized by bone marrow failure and cellular and chromosomal hypersensitivity to DNA cross-linking agents. Seven FA genes have been isolated and their products associate to form a pathway that interacts functionally or physically with several DNA-damage response proteins involved in cell cycle checkpoints and/or DNA repair. These proteins include BLM, ATM, BRCA1, XPF and the MRE11/RAD50/NBS1 complex. In spite of several recent striking progresses in the biochemistry and the molecular biology of the disorder, the precise function(s) of the FA proteins remain(s) poorly determined. However, several recent data indicate that the FA pathway could be involved in the coordination of both cell cycle checkpoints and DNA repair.     

Recruitment and positioning determine the specific role of the XPF‐ERCC1 endonuclease in interstrand crosslink repair

XPF‐ERCC1 is a structure‐specific endonuclease pivotal for several DNA repair pathways and, when mutated, can cause multiple diseases. Although the disease‐specific mutations are thought to affect different DNA repair pathways, the molecular basis for this is unknown. Here we examine the function of XPF‐ERCC1 in DNA interstrand crosslink (ICL) repair. We used Xenopus egg extracts to measure both ICL and nucleotide excision repair, and we identified mutations that are specifically defective in ICL repair. One of these separation‐of‐function mutations resides in the helicase‐like domain of XPF and disrupts binding to SLX4 and recruitment to the ICL. A small deletion in the same domain supports recruitment of XPF to the ICL, but inhibited the unhooking incisions most likely by disrupting a second, transient interaction with SLX4. Finally, mutation of residues in the nuclease domain did not affect localization of XPF‐ERCC1 to the ICL but did prevent incisions on the ICL substrate. Our data support a model in which the ICL repair‐specific function of XPF‐ERCC1 is dependent on recruitment, positioning and substrate recognition.