DNA Interstrand Cross-Links Induce Futile Repair Synthesis in Mammalian Cell Extracts

Mu, David; Bessho, Tadayoshi; Nechev, Lubomir V.; Chen, David J.; Harris, Thomas M.; Hearst, John E.; Sancar, Aziz

doi:10.1128/mcb.20.7.2446-2454.2000

Cited by 124 publications

(134 citation statements)

References 36 publications

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“…Instead, DSBs form in S phase when cross-links or perhaps cross-link repair intermediates encounter and stall a replication fork. The mammalian NER factors act on psoralen interstrand cross-links in vitro to produce two incisions, both 5Ј to the cross-link, but leave the cross-link in place (41). Cross-links can be unhooked by the exonuclease activity of XPF-ERCC1 (41), the NER factor homologous to S. cerevisiae Rad1⅐Rad10.…”

Section: Discussionmentioning

confidence: 99%

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

Saffran

Ahmed

Bellevue

et al. 2004

Journal of Biological Chemistry

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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-...

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Section: Discussionmentioning

confidence: 99%

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

Saffran

Ahmed

Bellevue

et al. 2004

Journal of Biological Chemistry

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“…The human XPF-ERCC1 complex and the yeast homolog, RAD1-RAD10 complex, contain a 3Ј 5Ј-exonuclease activity (13,22). An ICL-specific 3Ј-incision by the XPF-ERCC1 complex might generate a substrate for its own 3Ј 5Ј-exonuclease activity, which would unhook the ICL (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast to NER-defective mutants in E. coli and yeast, the NER-defective mammalian cells (except for ERCC-1 and ERCC-4 cells) are only moderately sensitive to DNA cross-linking agents such as mitomycin C (MMC) (8 -11). Moreover, biochemical studies demonstrated that the dual incisions by mammalian NER do not "unhook" ICLs (12,13). Therefore, mammalian cells evidently have a unique mechanism(s) to initiate ICL repair.…”

Section: Dna Interstrand Cross-links (Icls)mentioning

confidence: 99%

Processing of a Psoralen DNA Interstrand Cross-link by XPF-ERCC1 Complex in Vitro

Fisher

Bessho

2008

Journal of Biological Chemistry

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The processing of stalled forks caused by DNA interstrand cross-links (ICLs) has been proposed to be an important step in initiating mammalian ICL repair. To investigate a role of the XPF-ERCC1 complex in this process, we designed a model substrate DNA with a single psoralen ICL at a three-way junction (Y-shaped DNA), which mimics a stalled fork structure. We found that the XPF-ERCC1 complex makes an incision 5 to a psoralen lesion on Y-shaped DNA in a damage-dependent manner. Furthermore, the XPF-ERCC1 complex generates an ICLspecific incision on the 3-side of an ICL. The ICL-specific 3-incision, along with the 5-incision, on the cross-linked Y-shaped DNA resulted in the separation of the two cross-linked strands (the unhooking of the ICL) and the induction of a double strand break near the cross-linked site. These results implicate the XPF-ERCC1 complex in initiating ICL repair by unhooking the ICL, which simultaneously induces a double strand break at a stalled fork. DNA interstrand cross-links (ICLs)2 are formed as a result of DNA damage to each strand of the duplex. These lesions are located one or two nucleotides apart on the opposite strands and covalently link them, blocking DNA replication and transcription (1-3). Therefore, ICLs are considered to be the most cytotoxic DNA lesion (2-4).Several models for mammalian ICL repair have been proposed (2, 5-7). The first critical step for repairing ICLs in any given model is to release the ICL from one of the strands by making two incisions that bracket an ICL (unhooking the ICL). The unhooking of ICLs permits strand separation. In Escherichia coli and yeast, nucleotide excision repair (NER) makes dual incisions bracketing the ICL on one of the cross-linked strands to unhook ICLs (2, 3). Interestingly, NER is dispensable in mammalian ICL repair (5,8). In contrast to NER-defective mutants in E. coli and yeast, the NER-defective mammalian cells (except for ERCC-1 and ERCC-4 cells) are only moderately sensitive to DNA cross-linking agents such as mitomycin C (MMC) (8 -11). Moreover, biochemical studies demonstrated that the dual incisions by mammalian NER do not "unhook" ICLs (12, 13). Therefore, mammalian cells evidently have a unique mechanism(s) to initiate ICL repair.Notably, double strand break (DSB) repair-defective mutant cells, including those deficient in Rad51 paralogs or BRCA2, are hypersensitive to DNA cross-linking agents such as MMC (10). It has also been reported that ICLs in the mammalian genome are removed during S-phase and the DNA replication-mediated formation of a DSB is the key intermediate in ICL repair in mammalian cells (1,8,14,15). These data indicate that mammalian ICL repair goes through three critical processes: the formation of DNA replication-mediated DSBs, unhooking of the ICLs (separation of the two linked strands), and repair of the DSBs (restoration of the collapsed replication fork). It is unknown whether the formation of DSBs precedes the unhooking reaction. When the DNA replication complex encounters an ICL, the strand separat...

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“…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].…”

Section: Introductionmentioning

confidence: 99%

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

Mogi

Butcher

2008

Experimental Cell Research

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DNA interstrand crosslinks are processed by multiple mechanisms whose relationships to each other are unclear. Xeroderma pigmentosum-variant (XP-V) cells lacking DNA polymerase η are sensitive to psoralen photoadducts created under conditions favoring crosslink formation, suggesting a role for translesion synthesis in crosslink repair. Because crosslinks can lead to double strand breaks, we monitored phosphorylated H2AX (γ-H2AX), which is typically generated near double-strand breaks but also in response to single-stranded DNA, following psoralen photoadduct formation in XP-V fibroblasts to assess whether polymerase η is involved in processing crosslinks. In contrast to conditions favoring monoadducts, conditions favoring psoralen crosslinks induced γ-H2AX levels in both XP-V and nucleotide excision repair-deficient XP-A cells relative to control repair-proficient cells; ectopic expression of polymerase η in XP-V cells normalized the γ-H2AX response. In response to psoralen crosslinking, γ-H2AX as well as 53BP1 formed co-incident foci that were more numerous and intense in XP-V and XP-A cells than in controls. Psoralen photoadducts induced γ-H2AX throughout the cell cycle in XP-V cells. These results indicate that polymerase η is important in responding to psoralen crosslinks, and are consistent with a model in which nucleotide excision repair and polymerase η are involved in processing crosslinks and avoiding γ-H2AX associated with double strand breaks and single-stranded DNA in human cells.

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DNA Interstrand Cross-Links Induce Futile Repair Synthesis in Mammalian Cell Extracts

Cited by 124 publications

References 36 publications

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

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

Processing of a Psoralen DNA Interstrand Cross-link by XPF-ERCC1 Complex in Vitro

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

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