Transcription-Coupled Repair of Psoralen Cross-Links but Not Monoadducts in Chinese Hamster Ovary Cells

Islas, Ángel L.; Baker, Felix J.; Hanawalt, Philip C.

doi:10.1021/bi00201a029

Cited by 37 publications

(21 citation statements)

References 35 publications

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“…In our study, both the CSA and the CSB mutant were significantly defective in the repair of MMC ICLs located in the actively transcribed region of the reporter substrate. These observations are consistent with the report that TCR is involved in the repair of psoralen cross-links in hamster cells (20). In our study, a profound strand bias toward the transcribed strand was found, whereas the loss of transcription completely abolished this effect.…”

Section: Discussionsupporting

confidence: 94%

Nucleotide Excision Repair- and Polymerase η-Mediated Error-Prone Removal of Mitomycin C Interstrand Cross-Links

Zheng

Wang

Warren

et al. 2003

Molecular and Cellular Biology

137

180

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Interstrand cross-links (ICLs) make up a unique class of DNA lesions in which both strands of the double helix are covalently joined, precluding strand opening during replication and transcription. The repair of DNA ICLs has become a focus of study since ICLs are recognized as the main cytotoxic lesion inflicted by an array of alkylating compounds used in cancer treatment. As is the case for double-strand breaks, a damage-free homologous copy is essential for the removal of ICLs in an error-free manner. However, recombinationindependent mechanisms may exist to remove ICLs in an error-prone fashion. We have developed an in vivo reactivation assay that can be used to examine the removal of site-specific mitomycin C-mediated ICLs in mammalian cells. We found that the removal of the ICL from the reporter substrate could take place in the absence of undamaged homologous sequences in repair-proficient cells, suggesting a cross-link repair mechanism that is independent of homologous recombination. Systematic analysis of nucleotide excision repair mutants demonstrated the involvement of transcription-coupled nucleotide excision repair and a partial requirement for the lesion bypass DNA polymerase encoded by the human POLH gene. From these observations, we propose the existence of a recombination-independent and mutagenic repair pathway for the removal of ICLs in mammalian cells.A DNA interstrand cross-link (ICL) is formed when both strands of the double helix are covalently joined by a single molecule. Since ICLs effectively prevent strand separation, essential metabolic functions of DNA such as transcription, replication, and recombination are severely blocked by these lesions. The formation of DNA ICLs appears to be an essential prerequisite for the potent cytotoxicity and antitumor activity of a large array of chemotherapeutic compounds used in cancer treatment (41).In Escherichia coli and lower eukaryotes, the repair of ICLs is carried out primarily by a combination of the nucleotide excision repair (NER) and homologous recombination pathways. In a model proposed by Cole et al. (9, 10) based on genetic evidence, the NER mechanism introduces incisions flanking the site of the cross-link on the same strand. The resulting gap is then repaired by using a lesion-free homologous chromosome as a donor via the recA-dependent homologous recombination pathway. Subsequent biochemical analyses fully confirmed that the removal of ICLs in E. coli is mediated by both NER and homologous recombination (39,44,45). Similarly, with Saccharomyces cerevisiae, a group of RAD3 mutants (deficient in NER) and a group of RAD52 mutants (deficient in homologous recombination) are hypersensitive to the killing of bifunctional alkylating agents, suggesting that both pathways are essential for the repair of ICLs (21,28,30,38). These observations also indicated the presence of a combination of NER and homologous-recombination mechanisms in ICL repair. More recently, direct evidence of psoralen ICL-induced homologous recombination in budding yeast h...

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Section: Discussionsupporting

confidence: 94%

Nucleotide Excision Repair- and Polymerase η-Mediated Error-Prone Removal of Mitomycin C Interstrand Cross-Links

Zheng

Wang

Warren

et al. 2003

Molecular and Cellular Biology

137

180

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“…DNA adducts are preferentially removed from active genes by transcription-coupled repair (55). NER excises adducts more quickly from the transcribed strand than the non-transcribed strand (56), leading to more mutations on the non-transcribed strand.…”

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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“…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]. For example, the contribution of NER proteins to ICL repair is unclear.…”

Section: Introductionmentioning

confidence: 99%

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

Mogi

2006

DNA Repair

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To further define the molecular mechanisms involved in processing interstrand crosslinks, we monitored the formation of phosphorylated histone H2AX (γ-H2AX), which is generated in chromatin near double strand break sites, following DNA damage in normal and repair-deficient human cells. Following treatment with a psoralen derivative and ultraviolet A radiation doses that produce significant numbers of crosslinks, γ-H2AX levels in nucleotide excision repair-deficient XP-A fibroblasts (XP12RO-SV) increased to levels that were twice those observed in normal control GM637 fibroblasts. A partial XPA revertant cell line (XP129) that is proficient in crosslink removal, exhibited reduced γ-H2AX levels that were intermediate between those of GM637 and XP-A cells. XP-F fibroblasts (XP2YO-SV and XP3YO) that are also repair-deficient exhibited γ-H2AX levels below even control fibroblasts following treatment with psoralen and ultraviolet A radiation. Similarly, another crosslinking agent, mitomycin C, did not induce γ-H2AX in XP-F cells, although it did induce equivalent levels of γ-H2AX in XPA and control GM637 cells. Ectopic expression of XPF in XP-F fibroblasts restored γ-H2AX induction following treatment with crosslinking agents. Angelicin, a furocoumarin which forms only monoadducts and not crosslinks following ultraviolet A radiation, as well as ultraviolet C radiation, resulted only in weak induction of γ-H2AX in all cells, suggesting that the double strand breaks observed with psoralen and ultraviolet A treatment result preferentially following crosslink formation. These results indicate that XPF is required to form γ-H2AX and likely double strand breaks in response to interstrand crosslinks in human cells. Furthermore, XPA may be important to allow psoralen interstrand crosslinks to be processed without forming a double strand break intermediate.

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Transcription-Coupled Repair of Psoralen Cross-Links but Not Monoadducts in Chinese Hamster Ovary Cells

Cited by 37 publications

References 35 publications

Nucleotide Excision Repair- and Polymerase η-Mediated Error-Prone Removal of Mitomycin C Interstrand Cross-Links

Nucleotide Excision Repair- and Polymerase η-Mediated Error-Prone Removal of Mitomycin C Interstrand Cross-Links

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

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

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