DNA Repair Defects Channel Interstrand DNA Cross-links into Alternate Recombinational and Error-prone Repair Pathways

Abstract

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 crosslink-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 errorprone pathways, one NER-dependent and one NERindependent.DNA interstrand cross-links are complex lesions, highly toxic to cells, and difficult to repair because of the involvement of both strands of the DNA duplex. A single unrepaired interstrand cross-link is sufficient to kill a cell, and multiple DNA repair pathways may be required to complete cross-link removal (1-7).The yeast Saccharomyces cerevisiae has three major DNA repair epistasis groups, all of which are involved in cross-link repair; they are nucleotide excision repair, recombinational repair, and post-replication repair (8). Nucleotide excision repair (NER) 1 is a general system that recognizes bulky and helix-distorting lesions (9, 10). Endonucleases nick the affected DNA strand on both the 5Ј and 3Ј sides of the lesion; after removal of the damaged oligonucleotide, the resulting singlestrand gap is filled in by polymerase activity using the undamaged strand as a template. This mechanism produces error-free repair of single-strand damage. Recombinational repair is the major pathway for repair of double-strand damage, such as double-strand breaks (DSBs) or gaps in yeast (11,12). In homologous recombination (HR) the broken DNA molecule is repaired, and missing genetic information is restored through interactions with intact homologous sequences. This pathway is also non-mutagenic but can result in DNA rearrangements. Post-replication repair (PRR) acts on damage in the context of DNA replication, allowing for bypass of replication fork-...

γ-H2AX formation in response to interstrand crosslinks requires XPF in human cells

“…Cisplatin forms a small number of ICL and fails to induce DSB in rodent cells [48]. NER processing of psoralen ICL that depends on XPA and other NER proteins to uncouple one strand of the ICL [21,22], followed by translesion synthesis with a bypass polymerase may be one scenario that could account for reduced DSB formation in GM637 cells relative to XP-A cells [11,20,50,51].…”

“…The processing of ICL has been extensively studied in prokaryotes and yeast and appears to involve the nucleotide excision repair (NER) and recombination pathways, as well as lesion bypass and post-replication repair [1,10,11]. Processing of ICL in mammalian cells appears to be mediated by similarly diverse mechanisms as well as transcription [12,13] and certain mismatch repair proteins [14], although the relative importance of differing pathways and the detailed sequence of events remain to be elucidated [1].…”

REV3 and REV1 Play Major Roles in Recombination-independent Repair of DNA Interstrand Cross-links Mediated by Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA)

“…This error-prone mechanism may account for the mutagenic impact of ICLs. A similar mechanism has been demonstrated in budding yeast, suggesting that recombination-independent ICL repair may be a highly conserved mechanism in eukaryotes (5,6).…”

DNA polymerase η reduces the γ-H2AX response to psoralen interstrand crosslinks in human cells

“…In yeast and mammalian cells, nucleotide excision repair (NER), recombination, and translesion synthesis, as well as certain mismatch repair proteins, have been reported to participate in ICL repair [2,[5][6][7][8][9][10]. Recombinational repair is in part thought to be a response to formation of double-strand breaks (DSBs) at ICL that appear to be unique to eukaryotes and that arise either as a result of collapsed replication forks blocked at ICL, or as true repair intermediates [8,9,[11][12][13].…”