Psoralen Interstrand Crosslinks Research Articles

Single Molecule Analysis of Laser Localized Interstrand Crosslinks.

DNA interstrand crosslinks (ICLs) block unwinding of the double helix, and have always been regarded as major challenges to replication and transcription. Compounds that form these lesions are very toxic and are frequently used in cancer chemotherapy. We have developed two strategies, both based on immunofluorescence (IF), for studying cellular responses to ICLs. The basis of each is psoralen, a photoactive (by long wave ultraviolet light, UVA) DNA crosslinking agent, to which we have linked an antigen tag. In the one approach, we have taken advantage of DNA fiber and immuno-quantum dot technologies for visualizing the encounter of replication forks with ICLs induced by exposure to UVA lamps. In the other, psoralen ICLs are introduced into nuclei in live cells in regions of interest defined by a UVA laser. The antigen tag can be displayed by conventional IF, as can the recruitment and accumulation of DNA damage response proteins to the laser localized ICLs. However, substantial difference between the technologies creates considerable uncertainty as to whether conclusions from one approach are applicable to those of the other. In this report, we have employed the fiber/quantum dot methodology to determine lesion density and spacing on individual DNA molecules carrying laser localized ICLs. We have performed the same measurements on DNA fibers with ICLs induced by exposure of psoralen to UVA lamps. Remarkably, we find little difference in the adduct distribution on fibers prepared from cells exposed to the different treatment protocols. Furthermore, there is considerable similarity in patterns of replication in the vicinity of the ICLs introduced by the two techniques.

HMGB1 interacts with XPA to facilitate the processing of DNA interstrand crosslinks in human cells.

Many effective agents used in cancer chemotherapy cause DNA interstrand crosslinks (ICLs), which covalently link both strands of the double helix together resulting in cytotoxicity. ICLs are thought to be processed by proteins from a variety of DNA repair pathways; however, a clear understanding of ICL recognition and repair processing in human cells is lacking. Previously, we found that the high mobility group box 1 (HMGB1) protein bound to triplex-directed psoralen ICLs (TFO-ICLs) in vitro, cooperatively with NER damage recognition proteins, promoted removal of UVC-induced lesions and facilitated error-free repair of TFO-ICLs in mouse fibroblasts. Here, we demonstrate that HMGB1 recognizes TFO-ICLs in human cells, and its depletion increases ICL-induced mutagenesis in human cells without altering the mutation spectra. In contrast, HMGB1 depletion in XPA-deficient human cells significantly altered the ICL-induced mutation spectrum from predominantly T→A to T→G transversions. Moreover, the recruitment of XPA and HMGB1 to the ICLs is co-dependent. Finally, we show that HMGB1 specifically introduces negative supercoils in ICL-containing plasmids in HeLa cell extracts. Taken together, our data suggest that in human cells, HMGB1 functions in association with XPA on ICLs and facilitates the formation of a favorable architectural environment for ICL repair processing.

Incision‐independent Repair of a Psoralen‐interstrand Crosslink

DNA interstrand crosslinks (ICLs) are extremely cytotoxic because they prevent strand separation and thus interfere with DNA replication and transcription. ICLs are caused by endogenous metabolites, as well as chemotherapeutics such as cisplatin, nitrogen mustards, and psoralen. A major ICL repair pathway in metazoans is coupled with DNA replication and requires the Fanconi anemia pathway. Using Xenopus egg extracts, our lab previously described the mechanism of replication‐coupled cisplatin‐ICL repair. In this process, two DNA replication forks first converge at the ICL. Following CMG helicase dissociation, the ubiquitylated FANCI‐FANCD2 complex stimulates dual incisions on both sides of the ICL to unhook the lesion, creating a double‐strand break (DSB). Translesion DNA synthesis bypasses the lesion on one sister and the DSB in the other sister is repaired by inter‐sister recombination. Here, we investigated the repair of a psoralen‐ICL formed between two thymines. Strikingly, repair of this ICL does not require the FANCI‐FANCD2 complex and does not involve incision of the parental DNA surrounding the ICL. Instead, the ICL appears to be unhooked via cleavage of the glycosidic bond of one of the adducted thymines by an unknown glycosylase. This generates an abasic site on one parental strand while leaving a psoralen adduct on the other. No DSB is generated. Next, translesion DNA synthesis bypasses the lesions on both daughter molecules. As seen for cisplatin ICLs, the repair of psoralen ICLs requires replication fork convergence. Our data identify a novel pathway that uses DNA glycosylase activity to resolve an ICL in the absence of DSB formation. We are currently investigating which DNA glycosylase is involved in this reaction.

Abstract 39: FANCA regulates MUS81-EME1 mediated DNA incision in a damage-dependent manner

Abstract MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand crosslinks (ICL). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, interstrand crosslink repair protein, FANCA, greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner. Citation Format: Anaid Benitez, Fenghua Yuan, Satoshi Nakajima, Leizhen Wei, Liangyue Qian, Richard Myers, Jennifer J. Hu, Li Lan, Yanbin Zhang. FANCA regulates MUS81-EME1 mediated DNA incision in a damage-dependent manner. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr 39. doi:10.1158/1538-7445.CANSUSC14-39

Pyrene chromophores for the photoreversal of psoralen interstrand crosslinks.

Applying psoralen interstrand crosslinks for the photoactivation of nucleic acids is a new concept. To find chromophores that can efficiently stimulate crosslink repair we screened several pyrenes and appended them to peptide nucleic acids for their site-selective addressing. Even though pyrenes conjugated to uracil revealed desirable spectroscopic properties they were not effective in crosslink reversal. In contrast, bare pyrenes are well suitable for crosslink repair with 350 nm light showing an uncaging efficiency similar to classical photocaging groups.

Damage-dependent regulation of MUS81-EME1 by Fanconi anemia complementation group A protein

MUS81-EME1 is a DNA endonuclease involved in replication-coupled repair of DNA interstrand cross-links (ICLs). A prevalent hypothetical role of MUS81-EME1 in ICL repair is to unhook the damage by incising the leading strand at the 3′ side of an ICL lesion. In this study, we report that purified MUS81-EME1 incises DNA at the 5′ side of a psoralen ICL residing in fork structures. Intriguingly, ICL repair protein, Fanconi anemia complementation group A protein (FANCA), greatly enhances MUS81-EME1-mediated ICL incision. On the contrary, FANCA exhibits a two-phase incision regulation when DNA is undamaged or the damage affects only one DNA strand. Studies using truncated FANCA proteins indicate that both the N- and C-moieties of the protein are required for the incision regulation. Using laser-induced psoralen ICL formation in cells, we find that FANCA interacts with and recruits MUS81 to ICL lesions. This report clarifies the incision specificity of MUS81-EME1 on ICL damage and establishes that FANCA regulates the incision activity of MUS81-EME1 in a damage-dependent manner.

Replication-Fork Stalling and Processing at a Single Psoralen Interstrand Crosslink in Xenopus Egg Extracts

Interstrand crosslink (ICL)-inducing agents block the separation of the two DNA strands. They prevent transcription and replication and are used in clinics for the treatment of cancer and skin diseases. Here, we have introduced a single psoralen ICL at a specific site in plasmid DNA using a triplex-forming-oligonucleotide (TFO)-psoralen conjugate and studied its repair in Xenopus egg extracts that support nuclear assembly and replication of plasmid DNA. Replication forks arriving from either side stalled at the psoralen ICL. In contrast to previous observations with other ICL-inducing agents, the leading strands advanced up to the lesion without any prior pausing. Subsequently, incisions were introduced on one parental strand on both sides of the ICL. These incisions could be detected whether one or both forks reached the ICL. Using small molecule inhibitors, we found that the ATR-Chk1 pathway, but not the ATM-Chk2 pathway, stimulated both the incision step and the subsequent processing of the broken replication intermediates. Our results highlight both similarities and differences in fork stalling and repair induced by psoralen and by other ICL-forming agents.

MSH3 Mediates Sensitization of Colorectal Cancer Cells to Cisplatin, Oxaliplatin, and a Poly(ADP-ribose) Polymerase Inhibitor

The MSH3 gene is one of the DNA mismatch repair (MMR) genes that has undergone somatic mutation frequently in MMR-deficient cancers. MSH3, together with MSH2, forms the MutSβ heteroduplex, which interacts with interstrand cross-links (ICLs) induced by drugs such as cisplatin and psoralen. However, the precise role of MSH3 in mediating the cytotoxic effects of ICL-inducing agents remains poorly understood. In this study, we first examined the effects of MSH3 deficiency on cytotoxicity caused by cisplatin and oxaliplatin, another ICL-inducing platinum drug. Using isogenic HCT116-derived clones in which MSH3 expression is controlled by shRNA expression in a Tet-off system, we discovered that MSH3 deficiency sensitized cells to both cisplatin and oxaliplatin at clinically relevant doses. Interestingly, siRNA-induced down-regulation of the MLH1 protein did not affect MSH3-dependent toxicity of these drugs, indicating that this process does not require participation of the canonical MMR pathway. Furthermore, MSH3-deficient cells maintained higher levels of phosphorylated histone H2AX and 53BP1 after oxaliplatin treatment in comparison with MSH3-proficient cells, suggesting that MSH3 plays an important role in repairing DNA double strand breaks (DSBs). This role of MSH3 was further supported by our findings that MSH3-deficient cells were sensitive to olaparib, a poly(ADP-ribose) polymerase inhibitor. Moreover, the combination of oxaliplatin and olaparib exhibited a synergistic effect compared with either treatment individually. Collectively, our results provide novel evidence that MSH3 deficiency contributes to the cytotoxicity of platinum drugs through deficient DSB repair. These data lay the foundation for the development of effective prediction and treatments for cancers with MSH3 deficiency.

Targeted gene conversion induced by triplex-directed psoralen interstrand crosslinks in mammalian cells

Correction of a defective gene is a promising approach for both basic research and clinical gene therapy. However, the absence of site-specific targeting and the low efficiency of homologous recombination in human cells present barriers to successful gene targeting. In an effort to overcome these barriers, we utilized triplex-forming oligonucleotides (TFOs) conjugated to a DNA interstrand crosslinking (ICL) agent, psoralen (pTFO-ICLs), to improve the gene targeting efficiency at a specific site in DNA. Gene targeting events were monitored by the correction of a deletion on a recipient plasmid with the homologous sequence from a donor plasmid in human cells. The mechanism underlying this event is stimulation of homologous recombination by the pTFO-ICL. We found that pTFO-ICLs are efficient in inducing targeted gene conversion (GC) events in human cells. The deletion size in the recipient plasmid influenced both the recombination frequency and spectrum of recombinants; i.e. plasmids with smaller deletions had a higher frequency and proportion of GC events. The polarity of the pTFO-ICL also had a prominent effect on recombination. Our results suggest that pTFO-ICL induced intermolecular recombination provides an efficient method for targeted gene correction in mammalian cells.

Mismatch repair and nucleotide excision repair proteins cooperate in the recognition of DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) are among the most cytotoxic types of DNA damage, thus ICL-inducing agents such as psoralen, are clinically useful chemotherapeutics. Psoralen-modified triplex-forming oligonucleotides (TFOs) have been used to target ICLs to specific genomic sites to increase the selectivity of these agents. However, how TFO-directed psoralen ICLs (Tdp-ICLs) are recognized and processed in human cells is unclear. Previously, we reported that two essential nucleotide excision repair (NER) protein complexes, XPA–RPA and XPC–RAD23B, recognized ICLs in vitro, and that cells deficient in the DNA mismatch repair (MMR) complex MutSβ were sensitive to psoralen ICLs. To further investigate the role of MutSβ in ICL repair and the potential interaction between proteins from the MMR and NER pathways on these lesions, we performed electrophoretic mobility-shift assays and chromatin immunoprecipitation analysis of MutSβ and NER proteins with Tdp-ICLs. We found that MutSβ bound to Tdp-ICLs with high affinity and specificity in vitro and in vivo, and that MutSβ interacted with XPA–RPA or XPC–RAD23B in recognizing Tdp-ICLs. These data suggest that proteins from the MMR and NER pathways interact in the recognition of ICLs, and provide a mechanistic link by which proteins from multiple repair pathways contribute to ICL repair.

Human HMGB1 directly facilitates interactions between nucleotide excision repair proteins on triplex-directed psoralen interstrand crosslinks

Psoralen is a chemotherapeutic agent that acts by producing DNA interstrand crosslinks (ICLs), which are especially cytotoxic and mutagenic because their complex chemical nature makes them difficult to repair. Proteins from multiple repair pathways, including nucleotide excision repair (NER), are involved in their removal in mammalian cells, but the exact nature of their repair is poorly understood. We have shown previously that HMGB1, a protein involved in chromatin structure, transcriptional regulation, and inflammation, can bind cooperatively to triplex-directed psoralen ICLs with RPA, and that mammalian cells lacking HMGB1 are hypersensitive to psoralen ICLs. However, whether this effect is mediated by a role for HMGB1 in DNA damage recognition is still unknown. Given HMGB1's ability to bind to damaged DNA and its interaction with the RPA protein, we hypothesized that HMGB1 works together with the NER damage recognition proteins to aid in the removal of ICLs. We show here that HMGB1 is capable of binding to triplex-directed psoralen ICLs with the dedicated NER damage recognition complex XPC-RAD23B, as well as XPA-RPA, and that they form a higher-order complex on these lesions. In addition, we demonstrate that HMGB1 interacts with XPC-RAD23B and XPA in the absence of DNA. These findings directly demonstrate interactions between HMGB1 and the NER damage recognition proteins, and suggest that HMGB1 may affect ICL repair by enhancing the interactions between NER damage recognition factors.

Psoralen-Induced DNA Interstrand Cross-Links Block Transcription and Induce p53 in an Ataxia-Telangiectasia and Rad3-Related-Dependent Manner

Psoralen plus UVA light (PUVA) is commonly used to treat psoriasis, a common skin disorder associated with rapid proliferation of cells. PUVA exerts its antiproliferative activity through formation of DNA monoadducts and interstrand cross-links (ICLs). However, this treatment may lead to skin malignancies as a direct result of inducing carcinogenic DNA damage. Inactivation of the p53 tumor suppressor gene is an important event in the development of skin cancer. p53 is rapidly phosphorylated and stabilized in response to DNA damage, and the induction of apoptosis by p53 is an important mechanism by which p53 exerts its tumor-suppressive activity. To better understand the mechanism by which PUVA treatment induces p53, we exposed human skin fibroblasts with PUVA under conditions that differentially produce monoadducts and ICLs and found that psoralen-induced ICLs induced phosphorylation of the Ser-15 site of p53 and apoptosis much more effectively than psoralen-induced monoadducts. The induction of p53 phosphorylation by psoralen ICLs did not require factors believed to be involved in the repair of psoralen ICLs [xeroderma pigmentosum (XP)-A, XP-C, XP-F, Cockayne's syndrome-B, Fanconi anemia] but did require the ataxia-telangiectasia and Rad3-related but not the ataxia-telangiectasia mutated kinase. Psoralen-induced ICLs blocked transcription and replication more efficiently than monoadducts, and induction of p53 and apoptosis correlated with doses causing interference with transcription rather than DNA replication. Our finding that cells underwent apoptosis preferentially during S-phase suggests that the combined blockade of transcription and DNA replication by psoralen ICLs during S-phase elicits a strong apoptotic response.

Efficient processing of TFO-directed psoralen DNA interstrand crosslinks by the UvrABC nuclease

Photoreactive psoralens can form interstrand crosslinks (ICLs) in double-stranded DNA. In eubacteria, the endonuclease UvrABC plays a key role in processing psoralen ICLs. Psoralen-modified triplex-forming oligonucleotides (TFOs) can be used to direct ICLs to specific genomic sites. Previous studies of pyrimidine-rich methoxypsoralen–modified TFOs indicated that the TFO inhibits cleavage by UvrABC. Because different chemistries may alter the processing of TFO-directed ICLs, we investigated the effect of another type of triplex formed by purine-rich TFOs on the processing of 4′-(hydroxymethyl)-4,5′,8-trimethylpsoralen (HMT) ICLs by the UvrABC nuclease. Using an HMT-modified TFO to direct ICLs to a specific site, we found that UvrABC made incisions on the purine-rich strand of the duplex ∼3 bases from the 3′-side and ∼9 bases from the 5′-side of the ICL, within the TFO-binding region. In contrast to previous reports, the UvrABC nuclease cleaved the TFO-directed psoralen ICL with a greater efficiency than that of the psoralen ICL alone. Furthermore, the TFO was dissociated from its duplex binding site by UvrA and UvrB. As mutagenesis by TFO-directed ICLs requires nucleotide excision repair, the efficient processing of these lesions supports the use of triplex technology to direct DNA damage for genome modification.

Human MLH1 Protein Participates in Genomic Damage Checkpoint Signaling in Response to DNA Interstrand Crosslinks, while MSH2 Functions in DNA Repair

DNA interstrand crosslinks (ICLs) are among the most toxic types of damage to a cell. For this reason, many ICL-inducing agents are effective therapeutic agents. For example, cisplatin and nitrogen mustards are used for treating cancer and psoralen plus UVA (PUVA) is useful for treating psoriasis. However, repair mechanisms for ICLs in the human genome are not clearly defined. Previously, we have shown that MSH2, the common subunit of the human MutSα and MutSβ mismatch recognition complexes, plays a role in the error-free repair of psoralen ICLs. We hypothesized that MLH1, the common subunit of human MutL complexes, is also involved in the cellular response to psoralen ICLs. Surprisingly, we instead found that MLH1-deficient human cells are more resistant to psoralen ICLs, in contrast to the sensitivity to these lesions displayed by MSH2-deficient cells. Apoptosis was not as efficiently induced by psoralen ICLs in MLH1-deficient cells as in MLH1-proficient cells as determined by caspase-3/7 activity and binding of annexin V. Strikingly, CHK2 phosphorylation was undetectable in MLH1-deficient cells, and phosphorylation of CHK1 was reduced after PUVA treatment, indicating that MLH1 is involved in signaling psoralen ICL-induced checkpoint activation. Psoralen ICLs can result in mutations near the crosslinked sites; however, MLH1 function was not required for the mutagenic repair of these lesions, and so its signaling function appears to have a role in maintaining genomic stability following exposure to ICL-induced DNA damage. Distinguishing the genetic status of MMR-deficient tumors as MSH2-deficient or MLH1-deficient is thus potentially important in predicting the efficacy of treatment with psoralen and perhaps with other ICL-inducing agents.

3-Methyladenine DNA glycosylase is important for cellular resistance to psoralen interstrand cross-links

DNA interstrand cross-links (ICLs), widely used in chemotherapy, are cytotoxic lesions because they block replication and transcription. Repair of ICLs involves proteins from different repair pathways however the precise mechanism is still not completely understood. Here, we report that the 3-methyladenine DNA glycosylase (Aag), an enzyme that initiates base excision repair at a variety of alkylated bases, is also involved in the repair of ICLs. Aag−/− mouse embryonic stem cells were shown to be more sensitive to the cross-linking agent 4,5′,8-trimethylpsoralen than wild-type cells, but no more sensitive than wild-type to the psoralen derivative Angelicin that forms only monoadducts. We show that γ-H2AX foci formation, a marker for double strand breaks that are formed during ICL repair, is impaired in psoralen treated Aag−/− cells in both quantity and kinetics. However, in our in vitro system, purified human AAG can neither bind to the ICL nor cleave it. Taken together, our results suggest that Aag is important for the resistance of mouse ES cells to psoralen-induced ICLs.

DNA Polymerase I-Mediated Translesion Synthesis in RecA-Independent DNA Interstrand Cross-Link Repair in E. coli

DNA interstrand cross-links (ICLs) are mainly repaired by the combined action of nucleotide excision repair and homologous recombination in E. coli. Genetic data also suggest the existence of a nucleotide excision repair-dependent, homologous recombination-independent ICL repair pathway. The involvement of translesion synthesis in this pathway has been postulated; however, the molecular mechanism of this pathway is not understood. To examine the role of translesion synthesis in ICL repair, we generated a defined substrate with a single psoralen ICL that mimics a postincision structure generated by nucleotide excision repair. We demonstrated that the Klenow fragment (DNA polymerase I) performs translesion synthesis on this model substrate. This in vitro translesion synthesis assay will help in understanding the basic mechanism of a postincision translesion synthesis process in ICL repair.

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

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 ICL-specific 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.

Deficiency in Incisions Produced by XPF at the Site of a DNA Interstrand Cross-Link in Fanconi Anemia Cells

Repair of DNA interstrand cross-links is a multistep process, critical to which is production of incisions at the site of the lesion resulting in the unhooking of the cross-link from DNA. We have previously shown that XPF is involved in production of incisions at the site of a psoralen interstrand cross-link and that in Fanconi anemia, complementation group A (FA-A) cells, there is a deficiency in these incisions. We now demonstrate that in FA complementation group B, C, D2, F, and G cells there is also a deficiency in production of these incisions. Involvement of FA proteins in this process is demonstrated by the ability of FA cells, corrected with the appropriate FANC cDNAs, to produce these incisions and by inhibition of these incisions by antibodies against these proteins. This incision deficiency correlates with reduced levels of DNA repair synthesis in these cells and is not due to reduced levels of XPF. FA proteins could be influencing this incision process by interacting either with proteins involved in the unhooking step or with damaged DNA, acting as a damage sensor. The results also demonstrate that FA cells are undergoing apoptosis by 12 h after interstrand cross-link damage. It is thus proposed that the single-strand breaks known to be created in DNA during apoptosis could mask the deficiency in ability of FA cells to incise cross-linked DNA and could account for the reported discrepancy as to whether FA cells are deficient in the incision step of the repair process.

DNA polymerase η reduces the γ-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 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 coincident 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.

Double-strand breaks induce homologous recombinational repair of interstrand cross-links via cooperation of MSH2, ERCC1-XPF, REV3, and the Fanconi anemia pathway

DNA interstrand cross-linking agents have been widely used in chemotherapeutic treatment of cancer. The majority of interstrand cross-links (ICLs) in mammalian cells are removed via a complex process that involves the formation of double-strand breaks at replication forks, incision of the ICL, and subsequent error-free repair by homologous recombination. How double-strand breaks effect the removal of ICLs and the downstream homologous recombination process is not clear. Here, we describe a plasmid-based recombination assay in which one copy of the CFP gene is inactivated by a site-specific psoralen ICL and can be repaired by gene conversion with a mutated homologous donor sequence. We found that the homology-dependent recombination (HDR) is inhibited by the ICL. However, when we introduced a double-strand break adjacent to the site of the ICL, the removal of the ICL was enhanced and the substrate was funneled into a HDR repair pathway. This process was not dependent on the nucleotide excision repair pathway, but did require the ERCC1-XPF endonuclease and REV3. In addition, both the Fanconi anemia pathway and the mismatch repair protein MSH2 were required for the recombinational repair processing of the ICL. These results suggest that the juxtaposition of an ICL and a DSB stimulates repair of ICLs through a process requiring components of mismatch repair, ERCC1-XPF, REV3, Fanconi anemia proteins, and homologous recombination repair factors.