Mechanism of replication-coupled DNA interstrand crosslink repair

DNA interstrand crosslinks (ICLs) are toxic DNA lesions whose repair occurs in the S phase of metazoans via an unknown mechanism. Here, we describe a cell-free system based on Xenopus egg extracts that supports ICL repair. During DNA replication of a plasmid containing a site-specific ICL, two replication forks converge on the crosslink. Subsequent lesion bypass involves advance of a nascent leading strand to within one nucleotide of the ICL, followed by incisions, translesion DNA synthesis, and extension of the nascent strand beyond the lesion. Immunodepletion experiments suggest that extension requires DNA polymerase zeta. Ultimately, a significant portion of the input DNA is fully repaired, but not if DNA replication is blocked. Our experiments establish a mechanism for ICL repair that reveals how this process is coupled to DNA replication.     

DNA interstrand crosslinks induce a potent replication block followed by formation and repair of double strand breaks in intact mammalian cells

DNA interstrand crosslinks (ICLs) are highly toxic lesions that covalently link both strands of DNA and distort the DNA helix. Crosslinking agents have been shown to stall DNA replication and failure to repair ICL lesions before encountered by replication forks may induce severe DNA damage. Most knowledge of the ICL repair process has been revealed from studies in bacteria and cell extracts. However, for mammalian cells the process of ICL repair is still unclear and conflicting data exist. In this study we have explored the fate of psoralen-induced ICLs during replication, by employing intact mammalian cells and novel techniques. By comparative studies distinguishing between effects by monoadducts versus ICLs, we have been able to link the block of replication to the ICLs induction. We found that the replication fork was equally blocked by ICLs in wild-type cells as in cells deficient in ERCC1/XPF and XRCC3. The formation of ICL induced double strand breaks (DSBs), detected by formation of 53PB1 foci, was equally induced in the three cell lines suggesting that these proteins are involved at a later step of the repair process. Furthermore, we found that forks blocked by ICLs were neither bypassed, restarted nor restored for several hours. We propose that this process is different from that taking place following monoadduct induction by UV-light treatment where replication bypass is taking place as an early step. Altogether our findings suggest that restoration of an ICL blocked replication fork, likely initiated by a DSB occurs relatively rapidly at a stalled fork, is followed by restoration, which seems to be a rather slow process in intact mammalian cells.     

Structure-dependent bypass of DNA interstrand crosslinks by translesion synthesis polymerases

DNA interstrand crosslinks (ICLs), inhibit DNA metabolism by covalently linking two strands of DNA and are formed by antitumor agents such as cisplatin and nitrogen mustards. Multiple complex repair pathways of ICLs exist in humans that share translesion synthesis (TLS) past a partially processed ICL as a common step. We have generated site-specific major groove ICLs and studied the ability of Y-family polymerases and Pol ζ to bypass ICLs that induce different degrees of distortion in DNA. Two main factors influenced the efficiency of ICL bypass: the length of the dsDNA flanking the ICL and the length of the crosslink bridging two bases. Our study shows that ICLs can readily be bypassed by TLS polymerases if they are appropriately processed and that the structure of the ICL influences which polymerases are able to read through it.     

The structure and duplex context of DNA interstrand crosslinks affects the activity of DNA polymerase η

Several important anti-tumor agents form DNA interstrand crosslinks (ICLs), but their clinical efficiency is counteracted by multiple complex DNA repair pathways. All of these pathways require unhooking of the ICL from one strand of a DNA duplex by nucleases, followed by bypass of the unhooked ICL by translesion synthesis (TLS) polymerases. The structures of the unhooked ICLs remain unknown, yet the position of incisions and processing of the unhooked ICLs significantly influence the efficiency and fidelity of bypass by TLS polymerases. We have synthesized a panel of model unhooked nitrogen mustard ICLs to systematically investigate how the state of an unhooked ICL affects pol η activity. We find that duplex distortion induced by a crosslink plays a crucial role in translesion synthesis, and length of the duplex surrounding an unhooked ICL critically affects polymerase efficiency. We report the synthesis of a putative ICL repair intermediate that mimics the complete processing of an unhooked ICL to a single crosslinked nucleotide, and find that it provides only a minimal obstacle for DNA polymerases. Our results raise the possibility that, depending on the structure and extent of processing of an ICL, its bypass may not absolutely require TLS polymerases.     

Role for DNA polymerase kappa in the processing of N2-N2-guanine interstrand cross-links

Although there exists compelling genetic evidence for a homologous recombination-independent pathway for repair of interstrand cross-links (ICLs) involving translesion synthesis (TLS), biochemical support for this model is lacking. To identify DNA polymerases that may function in TLS past ICLs, oligodeoxynucleotides were synthesized containing site-specific ICLs in which the linkage was between N(2)-guanines, similar to cross-links formed by mitomycin C and enals. Here, data are presented that mammalian cell replication of DNAs containing these lesions was approximately 97% accurate. Using a series of oligodeoxynucleotides that mimic potential intermediates in ICL repair, we demonstrate that human polymerase (pol) kappa not only catalyzed accurate incorporation opposite the cross-linked guanine but also replicated beyond the lesion, thus providing the first biochemical evidence for TLS past an ICL. The efficiency of TLS was greatly enhanced by truncation of both the 5 ' and 3 ' ends of the nontemplating strand. Further analyses showed that although yeast Rev1 could incorporate a dCTP opposite the cross-linked guanine, no evidence was found for TLS by pol zeta or a pol zeta/Rev1 combination. Because pol kappa was able to bypass these ICLs, biological evidence for a role for pol kappa in tolerating the N(2)-N(2)-guanine ICLs was sought; both cell survival and chromosomal stability were adversely affected in pol kappa-depleted cells following mitomycin C exposure. Thus, biochemical data and cellular studies both suggest a role for pol kappa in the processing of N(2)-N(2)-guanine ICLs.     

Unique dynamic properties of DNA duplexes containing interstrand cross-links

Bifunctional DNA alkylating agents form a diverse assortment of covalent DNA interstrand cross-linked (ICL) structures that are potent cytotoxins. Because it is implausible that cells could possess distinct DNA repair systems for each individual ICL, it is believed that common structural and dynamic features of ICL damage are recognized, rather than specific structural characteristics of each cross-linking agent. Investigation of the structural and dynamic properties of ICLs that might be important for recognition has been complicated by heterogeneous incorporation of these lesions into DNA. To address this problem, we have synthesized and characterized several homogeneous ICL DNAs containing site-specific staggered N4-cytosine-ethyl-N4-cytosine cross-links. Staggered cross-links were introduced in two ways, in a manner that preserves the overall structure of B-form duplex DNA and in a manner that highly distorts the DNA structure, with the goal of understanding how structural and dynamic properties of diverse ICL duplexes might flag these sites for repair. Measurements of base pair opening dynamics in the B-form ICL duplex by (1)H NMR line width or imino proton solvent exchange showed that the guanine base opposite the cross-linked cytosine opened at least 1 order of magnitude more slowly than when in a control matched normal duplex. To a lesser degree, the B-form ICL also induced a decrease in base pair opening dynamics that extended from the site of the cross-link to adjacent base pairs. In contrast, the non-B-form ICL showed extensive conformational dynamics at the site of the cross-link, which extended over the entire DNA sequence. Because DNA duplexes containing the B-form and non-B-form ICL cross-links have both been shown to be incised when incubated in mammalian whole cell extracts, while a matched normal duplex is not, we conclude that intrinsic DNA dynamics is not a requirement for specific damage incision of these ICLs. Instead, we propose a general model in which destabilized ICL duplexes serve to energetically facilitate binding of DNA repair factors that must induce bubbles or other distortions in the duplex. However, the essential requirement for incision is an immobile Y-junction where the repair factors are stably bound at the site of the ICL, and the two DNA strands are unpaired.     

DNA interstrand crosslink repair during G1 involves nucleotide excision repair and DNA polymerase zeta

The repair mechanisms acting on DNA interstrand crosslinks (ICLs) in eukaryotes are poorly understood. Here, we provide evidence for a pathway of ICL processing that uses components from both nucleotide excision repair (NER) and translesion synthesis (TLS) and predominates during the G1 phase of the yeast cell cycle. Our results suggest that repair is initiated by the NER apparatus and is followed by a thwarted attempt at gap-filling by the replicative Polymerase delta, which likely stalls at the site of the remaining crosslinked oligonucleotide. This in turn leads to ubiquitination of PCNA and recruitment of the damage-tolerant Polymerase zeta that can perform TLS. The ICL repair factor Pso2 acts downstream of the incision step and is not required for Polymerase zeta activation. We show that this combination of NER and TLS is the only pathway of ICL repair available to the cell in G1 phase and is essential for viability in the presence of DNA crosslinks.     

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.     

Chromosomal Integrity after UV Irradiation Requires FANCD2-Mediated Repair of Double Strand Breaks

Fanconi Anemia (FA) is a rare autosomal recessive disorder characterized by hypersensitivity to inter-strand crosslinks (ICLs). FANCD2, a central factor of the FA pathway, is essential for the repair of double strand breaks (DSBs) generated during fork collapse at ICLs. While lesions different from ICLs can also trigger fork collapse, the contribution of FANCD2 to the resolution of replication-coupled DSBs generated independently from ICLs is unknown. Intriguingly, FANCD2 is readily activated after UV irradiation, a DNA-damaging agent that generates predominantly intra-strand crosslinks but not ICLs. Hence, UV irradiation is an ideal tool to explore the contribution of FANCD2 to the DNA damage response triggered by DNA lesions other than ICL repair. Here we show that, in contrast to ICL-causing agents, UV radiation compromises cell survival independently from FANCD2. In agreement, FANCD2 depletion does not increase the amount of DSBs generated during the replication of UV-damaged DNA and is dispensable for UV-induced checkpoint activation. Remarkably however, FANCD2 protects UV-dependent, replication-coupled DSBs from aberrant processing by non-homologous end joining, preventing the accumulation of micronuclei and chromatid aberrations including non-homologous chromatid exchanges. Hence, while dispensable for cell survival, FANCD2 selectively safeguards chromosomal stability after UV-triggered replication stress.     

REV3 and REV1 play major roles in recombination-independent repair of DNA interstrand cross-links mediated by monoubiquitinated proliferating cell nuclear antigen (PCNA)

DNA interstrand cross-links (ICLs) are the most cytotoxic lesions to eukaryotic genome and are repaired by both homologous recombination-dependent and -independent mechanisms. To better understand the role of lesion bypass polymerases in ICL repair, we investigated recombination-independent repair of ICLs in REV3 and REV1 deletion mutants constructed in avian DT40 cells and mouse embryonic fibroblast cells. Our results showed that Rev3 plays a major role in recombination-independent ICL repair, which may account for the extreme sensitivity of REV3 mutants to cross-linking agents. This result raised the possibility that the NER gap synthesis, when encountering an adducted base present in the ICL repair intermediate, can lead to recruitment of Rev3, analogous to the recruitment of polymerase eta during replicative synthesis. Indeed, the monoubiquitination-defective Proliferating Cell Nuclear Antigen (PCNA) mutant exhibits impaired recombination-independent ICL repair as well as drastically reduced mutation rate, indicating that the PCNA switch is utilized to enable lesion bypass during DNA repair synthesis. Analyses of a REV1 deletion mutant also revealed a significant reduction in recombination-independent ICL repair, suggesting that Rev1 cooperates with Rev3 in recombination-independent ICL repair. Moreover, deletion of REV3 or REV1 significantly altered the spectrum of mutations resulting from ICL repair, further confirming their involvement in mutagenic repair of ICLs.     

DNA interstrand crosslinks repair in mammalian cells

We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked plasmids and the ICL repair was monitored by following the restoration of the GFP expression. It was accompanied by gamma-H2AX foci formation suggesting that DSBs were formed during the process. However, the same amount of gamma-H2AX foci was observed when cells were transfected with native plasmid, which indicated that gamma-H2AX foci appearance could not be used to determine the amount of DSBs connected with the ICL repair in this system. For this reason we further monitored the DSB formation by determining the amount of linearized plasmids, since having one crosslink per plasmid on average, any ICL-driven DSB formation would lead to plasmid linearization. Native and crosslinked plasmids were incubated in repair-competent cell-free extracts from G1 and S phase HeLa cells. Although a considerable part of the ICLs was repaired, no linearization of the plasmids was observed in the extracts, which was interpreted that DSBs were not formed as intermediates in the process of ICL repair. In another set of experiments HR-proficient HeLa and HR-deficient irs3 cells were transfected with native and crosslinked plasmids, and 6 h and 12 h later the plasmid DNA was isolated and analyzed by electrophoresis. The same amount of linear plasmid molecules was observed in both cell lines, regardless of whether they were transfected with native or crosslinked pEGFP-N1, which further confirmed that DSB formation was not an obligatory step in the process of ICL repair by HR.     

Evidence for multiple imino intermediates and identification of reactive nucleophiles in peptide-catalyzed beta-elimination at abasic sites

Prior investigations have demonstrated that peptides containing a single aromatic residue flanked by basic ones, such as Lys-Trp-Lys, can incise the phosphodiester backbone of duplex DNA at an AP site via beta-elimination. An amine serves as the reactive nucleophile to attack C1' on the ring-open deoxyribose sugar to form a transient peptide-DNA imino (Schiff base) intermediate, which may be isolated as a stable covalent species under reducing conditions. In the current study, we use this methodology to demonstrate that peptide-catalyzed beta-elimination proceeds via the formation of two Schiff base intermediates, one of which was covalently trapped prior to strand incision and the other following strand incision. N-Terminal acetylation of reactive peptides significantly inhibited formation of a trapped Schiff base complex; thus, we demonstrate for the first time that the preferred reactive nucleophile for peptides catalyzing strand incision is the N-terminal alpha-amino group, not an epsilon-amino group located on a lysine residue as previously postulated. Trapping reactions in which the central tryptophan residue was changed to alanine did not have a significant impact on the efficiency of Schiff base formation, indicating that the presence of an aromatic residue is dispensable for the step prior to peptide-catalyzed beta-elimination. Interestingly, the methodology presented here affords a convenient means for covalently attaching an array of peptides onto AP site-containing DNA in a site-specific fashion. We suggest that the generation of such DNA-peptide cross-links may provide utility in studying the repair of biologically significant DNA-protein cross-link damage as DNA-peptide complexes may mimic intermediate structures along a repair pathway for DNA-protein cross-links.     

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.