Partial reconstitution of human interstrand cross-link repair in vitro: characterization of the roles of RPA and PCNA

The repair of DNA interstrand cross-links (ICLs) remains largely ill-defined in higher eukaryotic cells. Previously, we have developed assays that can be used to monitor the early stages of processing of ICLs in vitro. Here, we have used P11 phosphocellulose chromatography to fractionate HeLa nuclear extracts and have subsequently reconstituted these assays with the resulting fractions. RPA and PCNA were found in a single fraction, and were the only factors in this fraction required for the reconstitution of these assays. The roles of RPA and PCNA in the formation of incisions at ICLs and in the subsequent DNA synthesis step were assessed. RPA was found to be essential for both stages of ICL processing indicating that it is required for lesion recognition and/or for the subsequent endonucleolytic processing. PCNA is required for the DNA synthesis stage and although it is not critical for the incision stage of the reaction it does enhance this step presumably by a stimulation of lesion recognition by MutSbeta. These findings define novel roles for RPA and PCNA in the processing of ICLs in mammalian cells.     

A role for the base excision repair enzyme NEIL3 in replication-dependent repair of interstrand DNA cross-links derived from psoralen and abasic sites

Interstrand DNA–DNA cross-links are highly toxic lesions that are important in medicinal chemistry, toxicology, and endogenous biology. In current models of replication-dependent repair, stalling of a replication fork activates the Fanconi anemia pathway and cross-links are “unhooked” by the action of structure-specific endonucleases such as XPF-ERCC1 that make incisions flanking the cross-link. This process generates a double-strand break, which must be subsequently repaired by homologous recombination. Recent work provided evidence for a new, incision-independent unhooking mechanism involving intrusion of a base excision repair (BER) enzyme, NEIL3, into the world of cross-link repair. The evidence suggests that the glycosylase action of NEIL3 unhooks interstrand cross-links derived from an abasic site or the psoralen derivative trioxsalen. If the incision-independent NEIL3 pathway is blocked, repair reverts to the incision-dependent route. In light of the new model invoking participation of NEIL3 in cross-link repair, we consider the possibility that various BER glycosylases or other DNA-processing enzymes might participate in the unhooking of chemically diverse interstrand DNA cross-links.     

A versatile new tool to quantify abasic sites in DNA and inhibit base excision repair

A number of endogenous and exogenous agents, and cellular processes create abasic (AP) sites in DNA. If unrepaired, AP sites cause mutations, strand breaks and cell death. Aldehyde-reactive agent methoxyamine reacts with AP sites and blocks their repair. Another alkoxyamine, ARP, tags AP sites with a biotin and is used to quantify these sites. We have combined both these abilities into one alkoxyamine, AA3, which reacts with AP sites with a better pH profile and reactivity than ARP. Additionally, AA3 contains an alkyne functionality for bioorthogonal click chemistry that can be used to link a wide variety of biochemical tags to AP sites. We used click chemistry to tag AP sites with biotin and a fluorescent molecule without the use of proteins or enzymes. AA3 has a better reactivity profile than ARP and gives much higher product yields at physiological pH than ARP. It is simpler to use than ARP and its use results in lower background and greater sensitivity for AP site detection. We also show that AA3 inhibits the first enzyme in the repair of abasic sites, APE-1, to about the same extent as methoxyamine. Furthermore, AA3 enhances the ability of an alkylating agent, methylmethane sulfonate, to kill human cells and is more effective in such combination chemotherapy than methoxyamine.     

Identification of the first small-molecule inhibitor of the REV7 DNA repair protein interaction

DNA interstrand crosslink (ICL) repair (ICLR) has been implicated in the resistance of cancer cells to ICL-inducing chemotherapeutic agents. Despite the clinical significance of ICL-inducing chemotherapy, few studies have focused on developing small-molecule inhibitors for ICLR. The mammalian DNA polymerase ζ, which comprises the catalytic subunit REV3L and the non-catalytic subunit REV7, is essential for ICLR. To identify small-molecule compounds that are mechanistically capable of inhibiting ICLR by targeting REV7, high-throughput screening and structure–activity relationship (SAR) analysis were performed. Compound 1 was identified as an inhibitor of the interaction of REV7 with the REV7-binding sequence of REV3L. Compound 7 (an optimized analog of compound 1) bound directly to REV7 in nuclear magnetic resonance analyses, and inhibited the reactivation of a reporter plasmid containing an ICL in between the promoter and reporter regions. The normalized clonogenic survival of HeLa cells treated with cisplatin and compound 7 was lower than that for cells treated with cisplatin only. These findings indicate that a small-molecule inhibitor of the REV7/REV3L interaction can chemosensitize cells by inhibiting ICLR.     

Repair of psoralen interstrand cross-links in Xenopus laevis egg extracts is highly mutagenic

The recognition and removal of interstrand cross-links is perhaps the least understood of all repair pathways in eukaryotic cells. We have shown previously that uncoupling of cross-links occurs in mammalian cell extracts and have identified a number of factors that mediate this process. However, we have not observed complete repair of the substrate in this system. Here, we show that uncoupling of interstrand cross-links also occurs in Xenopus laevis egg extracts, and that the initial products of this reaction are identical to the products observed in mammalian cell extracts suggesting a common mechanism. However in contrast to mammalian cell extracts, we observe repair of the cross-linked substrate in the Xenopus extracts presumably by a translesion bypass mechanism that allows replication past the uncoupled monoadduct, and its likely subsequent removal by nucleotide excision repair. This repair process is shown to be highly mutagenic consistent with bypass synthesis.     

Chemosensitivity of primary human fibroblasts with defective unhooking of DNA interstrand cross-links

Xeroderma pigmentosum (XP) is characterised by defects in nucleotide excision repair, ultraviolet (UV) radiation sensitivity and increased skin carcinoma. Compared to other complementation groups, XP-F patients show relatively mild cutaneous symptoms. DNA interstrand cross-linking agents are a highly cytotoxic class of DNA damage induced by common cancer chemotherapeutics such as cisplatin and nitrogen mustards. Although the XPF-ERCC1 structure-specific endonuclease is required for the repair of ICLs cellular sensitivity of primary human XP-F cells has not been established. In clonogenic survival assays, primary fibroblasts from XP-F patients were moderately sensitive to both UVC and HN2 compared to normal cells (2- to 3-fold and 3- to 5-fold, respectively). XP-A fibroblasts were considerably more sensitive to UVC (10- to 12-fold) but not sensitive to HN2. The sensitivity of XP-F fibroblasts to HN2 correlated with the defective incision or 'unhooking' step of ICL repair. Using the comet assay, XP-F cells exhibited only 20% residual unhooking activity over 24 h. Over the same time, normal and XP-A cells unhooked greater than 95% and 62% of ICLs, respectively. After HN2 treatment, ICL-associated DNA double-strand breaks (DSBs) are detected by pulse field gel electrophoresis in dividing cells. Induction and repair of DNA DSBs was normal in XP-F fibroblasts. These findings demonstrate that in primary human fibroblasts, XPF is required for the unhooking of ICLs and not for the induction or repair of ICL-associated DNA DSBs induced by HN2. In terms of cancer chemotherapy, people with mild DNA repair defects affecting ICL repair may be more prevalent in the general population than expected. Since cellular sensitivity of primary human fibroblasts usually reflects clinical sensitivity such patients with cancer would be at risk of increased toxicity.     

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.     

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.     

DNA polymerase eta reduces the gamma-H2AX response to psoralen interstrand crosslinks in human cells

DNA interstrand crosslinks are processed by multiple mechanisms whose relationships to each other are unclear. Xeroderma pigmentosum-variant (XP-V) cells lacking DNA polymerase eta 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 (gamma-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 eta is involved in processing crosslinks. In contrast to conditions favoring monoadducts, conditions favoring psoralen crosslinks induced gamma-H2AX levels in both XP-V and nucleotide excision repair-deficient XP-A cells relative to control repair-proficient cells; ectopic expression of polymerase eta in XP-V cells normalized the gamma-H2AX response. In response to psoralen crosslinking, gamma-H2AX as well as 53BP1 formed coincident foci that were more numerous and intense in XP-V and XP-A cells than in controls. Psoralen photoadducts induced gamma-H2AX throughout the cell cycle in XP-V cells. These results indicate that polymerase eta is important in responding to psoralen crosslinks, and are consistent with a model in which nucleotide excision repair and polymerase eta are involved in processing crosslinks and avoiding gamma-H2AX associated with double-strand breaks and single-stranded DNA in human cells.     

Induction of direct repeat recombination by psoralen–DNA adducts in Saccharomyces cerevisiae: Defects in DNA repair increase gene copy number variation

Psoralen photoreaction produces covalent monoadducts and interstrand crosslinks in DNA. The interstrand DNA crosslinks are complex double strand lesions that require the involvement of multiple pathways for repair. Homologous recombination, which can carry out error-free repair, is a major pathway for crosslink repair; however, some recombination pathways can also produce DNA rearrangements. Psoralen photoreaction-induced recombination in yeast was measured using direct repeat substrates that can detect gene conversions, a form of conservative recombination, as well as deletions and triplications, which generate gene copy number changes. In repair-proficient cells the major products of recombination were gene conversions, along with substantial fractions of deletions. Deficiencies in DNA repair pathways increased non-conservative recombination products. Homologous recombination-deficient rad51, rad54, and rad57 strains had low levels of crosslink-induced recombination, and most products were deletions produced by single strand annealing. Nucleotide excision repair-deficient rad1 and rad2 yeast had increased levels of triplications, and rad1 cells had lower crosslink-induced recombination. Deficiencies in post-replication repair increased crosslink-induced recombination and gene copy number changes. Loss of REV3 function, in the error-prone branch, and of RAD5 and UBC13, in the error-free branch, produced moderate increases in deletions and triplications; rad18 cells, deficient in both post-replication repair sub-pathways, exhibited hyperrecombination, with primarily non-conservative products. Proper functioning of all the DNA repair pathways tested was required to maintain genomic stability and avoid gene copy number variation in response to interstrand crosslinks.     

Mammalian INO80 chromatin remodeler cooperates with FANCM to mediate DNA interstrand crosslink-induced checkpoint activation and repair

Chromatin regulators play crucial roles in the DNA damage response. While the chromatin changes needed for double-strand break repair and nucleotide excision repair have been intensely studied, the chromatin requirements of interstrand crosslink (ICL) repair have remained largely unexplored. Here, we studied the effect of silencing the INO80 chromatin remodeler subunits on the cellular response to ICLs. Cells depleted of Ino80 ATPase were more sensitive to mitomycin C (MMC) and defective in FANCD2 chromatin recruitment. Ino80-deficient cells displayed strongly reduced Chk1 phosphorylation after MMC treatment indicating impaired ATR-dependent DNA damage signaling, likely due to the significantly slower RPA foci formation which we observed in these cells. MMC treatment of cells silenced for FANCM - a protein required for ICL-induced checkpoint signaling, Ino80 or both genes simultaneously led to similar decreases in RPA phosphorylation suggesting that the two proteins were involved in the same checkpoint pathway. Co-immunoprecipitation data indicated that Ino80 and FANCM interact physically. Taken together our data demonstrate for the first time that the INO80 chromatin remodeler cooperates with FANCM to mediate ICL-induced checkpoint activation by promoting accumulation of RPA at the lesion sites. This constitutes a novel mechanism by which the INO80 chromatin remodeler participates in the repair of ICLs and genome integrity maintenance.     

Differential repair and recombination of psoralen damaged plasmid DNA in Saccharomyces cerevisiae.

Summary Psoralen photoreaction with DNA produces interstrand crosslinks, which require the activity of excision and recombinational pathways for repair. Yeast replicating plasmids, carrying the HIS3, TRP1, and URA3 genes, were photoreacted with psoralen in vitro and transfected into Saccharomyces cerevisiae cells. Repair was assayed as the relative transformation efficiency. A recombination-deficient rad52 strain was the least efficient in the repair of psoralen-damaged plasmids; excision repair-deficient rad1 and rad3 strains had repair efficiencies intermediate between those of rad52 and RAD cells. The level of repair also depended on the conditions of transformant selection; repair was more efficient in medium lacking tryptophan than in medium from which either histidine or uracil was omitted. The plasmid repair differential between these selective media was greatest in rad1 cells, and depended on RAD52. Plasmid-chromosome recombination was stimulated by psoralen damage, and required RAD52 function. Chromosome to plasmid gene conversion was seen most frequently at the HIS3 locus. In RAD and rad3 cells, the majority of the conversions were associated with plasmid integration, while in rad1 cells most were non-crossover events. Plasmid to chromosome gene conversion was observed most frequently at the TRP1 locus, and was accompanied by plasmid loss.     

The Fanconi anemia pathway and DNA interstrand cross-link repair

Fanconi anemia (FA) is an autosomal or X-linked recessive disorder characterized by chromosomal instability, bone marrow failure, cancer susceptibility, and a profound sensitivity to agents that produce DNA interstrand cross-link (ICL). To date, 15 genes have been identified that, when mutated, result in FA or an FA-like syndrome. It is believed that cellular resistance to DNA interstrand cross-linking agents requires all 15 FA or FA-like proteins. Here, we review our current understanding of how these FA proteins participate in ICL repair and discuss the molecular mechanisms that regulate the FA pathway to maintain genome stability.     

Active DNA demethylation and DNA repair

DNA methylation on cytosine is an epigenetic modification and is essential for gene regulation and genome stability in vertebrates. Traditionally DNA methylation was considered as the most stable of all heritable epigenetic marks. However, it has become clear that DNA methylation is reversible by enzymatic “active” DNA demethylation, with examples in plant cells, animal development and immune cells. It emerges that “pruning” of methylated cytosines by active DNA demethylation is an important determinant for the DNA methylation signature of a cell. Work in plants and animals shows that demethylation occurs by base excision and nucleotide excision repair. Far from merely protecting genomic integrity from environmental insult, DNA repair is therefore at the heart of an epigenetic activation process.     

FANCI functions as a repair/apoptosis switch in response to DNA crosslinks

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SCAI promotes error‐free repair of DNA interstrand crosslinks via the Fanconi anemia pathway

DNA interstrand crosslinks (ICLs) are cytotoxic lesions that threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI‐FANCD2 (ID2) marking the activation step that triggers incisions on DNA to unhook the ICL. Restoration of intact DNA requires the coordinated actions of polymerase ζ (Polζ)‐mediated translesion synthesis (TLS) and homologous recombination (HR). While the proteins mediating FA pathway activation have been well characterized, the effectors regulating repair pathway choice to promote error‐free ICL resolution remain poorly defined. Here, we uncover an indispensable role of SCAI in ensuring error‐free ICL repair upon activation of the FA pathway. We show that SCAI forms a complex with Polζ and localizes to ICLs during DNA replication. SCAI‐deficient cells are exquisitely sensitive to ICL‐inducing drugs and display major hallmarks of FA gene inactivation. In the absence of SCAI, HR‐mediated ICL repair is defective, and breaks are instead re‐ligated by polymerase θ‐dependent microhomology‐mediated end‐joining, generating deletions spanning the ICL site and radial chromosomes. Our work establishes SCAI as an integral FA pathway component, acting at the interface between TLS and HR to promote error‐free ICL repair.     

DNA2 and EXO1 in replication-coupled,homology-directed repair and in the interplay between HDR and the FA/BRCA network

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.     

DNA2 and EXO1 in replication-coupled,homology-directed repair and in the interplay between HDR and the FA/BRCA network

During DNA replication, stalled replication forks and DSBs arise when the replication fork encounters ICLs (interstrand crosslinks), covalent protein/DNA intermediates or other discontinuities in the template. Recently, homologous recombination proteins have been shown to function in replication-coupled repair of ICLs in conjunction with the Fanconi anemia (FA) regulatory factors FANCD2-FANCI, and, conversely, the FA gene products have been shown to play roles in stalled replication fork rescue even in the absence of ICLs, suggesting a broader role for the FA network than previously appreciated. Here we show that DNA2 helicase/nuclease participates in resection during replication-coupled repair of ICLs and other replication fork stresses. DNA2 knockdowns are deficient in HDR (homology-directed repair) and the S phase checkpoint and exhibit genome instability and sensitivity to agents that cause replication stress. DNA2 is partially redundant with EXO1 in these roles. DNA2 interacts with FANCD2, and cisplatin induces FANCD2 ubiquitylation even in the absence of DNA2. DNA2 and EXO1 deficiency leads to ICL sensitivity but does not increase ICL sensitivity in the absence of FANCD2. This is the first demonstration of the redundancy of human resection nucleases in the HDR step in replication-coupled repair, and suggests that DNA2 may represent a new mediator of the interplay between HDR and the FA/BRCA pathway.