Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease

Interstrand DNA cross-link damage is a severe challenge to genomic integrity. Nucleotide excision repair plays some role in the repair of DNA cross-links caused by psoralens and other agents. However, in mammalian cells there is evidence that the ERCC1-XPF nuclease has a specialized additional function during interstrand DNA cross-link repair, beyond its role in nucleotide excision repair. We placed a psoralen monoadduct or interstrand cross-link in a duplex, 4-6 bases from a junction with unpaired DNA. ERCC1-XPF endonucleolytically cleaved within the duplex on either side of the adduct, on the strand having an unpaired 3' tail. Cross-links that were cleaved only on the 5' side were purified and reincubated with ERCC1-XPF. A second cleavage was then observed on the 3' side. Relevant partially unwound structures near a cross-link may be expected to arise frequently, for example at stalled DNA replication forks. The results show that the single enzyme ERCC1-XPF can release one arm of a cross-link and suggest a novel mechanism for interstrand cross-link repair.     

NEIL2-initiated, APE-independent repair of oxidized bases in DNA: Evidence for a repair complex in human cells

DNA glycosylases/AP lyases initiate repair of oxidized bases in the genomes of all organisms by excising these lesions and then cleaving the DNA strand at the resulting abasic (AP) sites and generate 3' phospho alpha,beta-unsaturated aldehyde (3' PUA) or 3' phosphate (3' P) terminus. In Escherichia coli, the AP-endonucleases (APEs) hydrolyze both 3' blocking groups (3' PUA and 3' P) to generate the 3'-OH termini needed for repair synthesis. In mammalian cells, the previously characterized DNA glycosylases, NTH1 and OGG1, produce 3' PUA, which is removed by the only AP-endonuclease, APE1. However, APE1 is barely active in removing 3' phosphate generated by the recently discovered mammalian DNA glycosylases NEIL1 and NEIL2. We showed earlier that the 3' phosphate generated by NEIL1 is efficiently removed by polynucleotide kinase (PNK) and not APE1. Here we show that the NEIL2-initiated repair of 5-hydroxyuracil (5-OHU) similarly requires PNK. We have also observed stable interaction between NEIL2 and other BER proteins DNA polymerase beta (Pol beta), DNA ligase IIIalpha (Lig IIIalpha) and XRCC1. In spite of their limited sequence homology, NEIL1 and NEIL2 interact with the same domains of Pol beta and Lig IIIalpha. Surprisingly, while the catalytically dispensable C-terminal region of NEIL1 is the common interacting domain, the essential N-terminal segment of NEIL2 is involved in analogous interaction. The BER proteins including NEIL2, PNK, Pol beta, Lig IIIalpha and XRCC1 (but not APE1) could be isolated as a complex from human cells, competent for repair of 5-OHU in plasmid DNA.     

DNA polymerase II (polB) is involved in a new DNA repair pathway for DNA interstrand cross-links in Escherichia coli

DNA-DNA interstrand cross-links are the cytotoxic lesions for many chemotherapeutic agents. A plasmid with a single nitrogen mustard (HN2) interstrand cross-link (inter-HN2-pTZSV28) was constructed and transformed into Escherichia coli, and its replication efficiency (RE = [number of transformants from inter-HN2-pTZSV28]/[number of transformants from control]) was determined to be approximately 0.6. Previous work showed that RE was high because the cross-link was repaired by a pathway involving nucleotide excision repair (NER) but not recombination. (In fact, recombination was precluded because the cells do not receive lesion-free homologous DNA.) Herein, DNA polymerase II is shown to be in this new pathway, since the replication efficiency (RE) is higher in a polB+ ( approximately 0. 6) than in a DeltapolB (approximately 0.1) strain. Complementation with a polB+-containing plasmid restores RE to wild-type levels, which corroborates this conclusion. In separate experiments, E. coli was treated with HN2, and the relative sensitivity to killing was found to be as follows: wild type < polB < recA < polB recA approximately uvrA. Because cells deficient in either recombination (recA) or DNA polymerase II (polB) are hypersensitive to nitrogen mustard killing, E. coli appears to have two pathways for cross-link repair: an NER/recombination pathway (which is possible when the cross-links are formed in cells where recombination can occur because there are multiple copies of the genome) and an NER/DNA polymerase II pathway. Furthermore, these results show that some cross-links are uniquely repaired by each pathway. This represents one of the first clearly defined pathway in which DNA polymerase II plays a role in E. coli. It remains to be determined why this new pathway prefers DNA polymerase II and why there are two pathways to repair cross-links.     

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.     

AP endonuclease-independent DNA base excision repair in human cells

The paradigm for repair of oxidized base lesions in genomes via the base excision repair (BER) pathway is based on studies in Escherichia coli, in which AP endonuclease (APE) removes all 3' blocking groups (including 3' phosphate) generated by DNA glycosylase/AP lyases after base excision. The recently discovered mammalian DNA glycosylase/AP lyases, NEIL1 and NEIL2, unlike the previously characterized OGG1 and NTH1, generate DNA strand breaks with 3' phosphate termini. Here we show that in mammalian cells, removal of the 3' phosphate is dependent on polynucleotide kinase (PNK), and not APE. NEIL1 stably interacts with other BER proteins, DNA polymerase beta (pol beta) and DNA ligase IIIalpha. The complex of NEIL1, pol beta, and DNA ligase IIIalpha together with PNK suggests coordination of NEIL1-initiated repair. That NEIL1/PNK could also repair the products of other DNA glycosylases suggests a broad role for this APE-independent BER pathway in mammals.     

Unique and overlapping functions of the Exo1, Mre11 and Pso2 nucleases in DNA repair

The Mre11 and Pso2 nucleases function in homologous recombination and interstrand cross-link (ICL) repair pathways, respectively, while the Exo1 nuclease is involved in homologous recombination and mismatch repair. Characterization of the sensitivity of single, double and triple mutants for these nucleases in Saccharomyces cerevisiae to various DNA damaging agents reveals complex interactions that depend on the type of DNA damage. The pso2 mutant is uniquely sensitive to agents that generate ICLs and mre11-H125N shows the highest sensitivity of the single mutants for ionizing radiation and methyl methane sulfonate. However, elimination of all three nucleases confers higher sensitivity to IR than any of the single or double mutant combinations indicating a high degree of redundancy and versatility in the response to DNA damage. In response to ICL agents, double-strand breaks are still formed in the triple nuclease mutant indicating that none of these nucleases are responsible for unhooking cross-links.     

Recombinational and mutagenic repair of psoralen interstrand cross-links in Saccharomyces cerevisiae

Psoralen photoreacts with DNA to form interstrand cross-links, which can be repaired by both nonmutagenic nucleotide excision repair and recombinational repair pathways and by mutagenic pathways. In the yeast Saccharomyces cerevisiae, psoralen cross-links are processed by nucleotide excision repair to form double-strand breaks (DSBs). In yeast, DSBs are repaired primarily by homologous recombination, predicting that cross-link and DSB repair should induce similar recombination end points. We compared psoralen cross-link, psoralen monoadduct, and DSB repair using plasmid substrates with site-specific lesions and measured the patterns of gene conversion, crossing over, and targeted mutation. Psoralen cross-links induced both recombination and mutations, whereas DSBs induced only recombination, and monoadducts were neither recombinogenic nor mutagenic. Although the cross-link- and DSB-induced patterns of plasmid integration and gene conversion were similar in most respects, they showed opposite asymmetries in their unidirectional conversion tracts: primarily upstream from the damage site for cross-links but downstream for DSBs. Cross-links induced targeted mutations in 5% of the repaired plasmids; all were base substitutions, primarily T --> C transitions. The major pathway of psoralen cross-link repair in yeast is error-free and involves the formation of DSB intermediates followed by homologous recombination. A fraction of the cross-links enter an error-prone pathway, resulting in mutations at the damage site.     

Expression and purification of NEIL3, a human DNA glycosylase homolog

The base excision repair (BER) pathway is mainly responsible for the repair of a vast number of non-bulky lesions produced by alkylation, oxidation or deamination of bases. DNA glycosylases are the key enzymes that recognize damaged bases and initiate BER by catalyzing the cleavage of the N-glycosylic bond between the base and the sugar. Many of the mammalian DNA glycosylases have been identified by a combination of biochemical and bioinformatics analysis. Thus, a mammalian family of three proteins (NEIL1, NEIL2 and NEIL3) that showed homology to the Escherichia coli Fpg/Nei DNA glycosylases was identified. Two of the proteins, NEIL1 and NEIL2 have been thoroughly characterized and shown to initiate BER of a diverse number of oxidized lesions. However, much less is known about NEIL3. The biochemical properties of NEIL3 have not been elucidated. This is mainly due to the difficulty in the expression and purification of NEIL3. Here, we describe the expression and partial purification of full-length human NEIL3 and the expression, purification and characterization of a truncated human core-NEIL3 (amino acids 1–301) that contains the complete E. coli Fpg/Nei-like domain but lacks the C-terminal region.     

Structure, flexibility, and repair of two different orientations of the same alkyl interstrand DNA cross-link

Interstrand DNA cross-links are the principal cytotoxic lesions produced by chemotherapeutic bifunctional alkylating agents. Using an N(4)C-ethyl-N(4)C interstrand DNA cross-link to mimic this class of clinically important cancer chemotherapeutic agents, we have characterized the repair, structure, and flexibility of DNA that contains this cross-link in two different orientations. Plasmid DNAs in which the cytosines of single CpG or GpC steps are covalently linked were efficiently processed by repair proficient and homologous recombination deficient strains of Escherichia coli. Repair in a nucleotide excision repair (NER) deficient strain was less efficient overall and displayed a 4-fold difference between the two cross-link orientations. Both the structure and flexibility of DNA containing these cross-links were examined using a combination of (1)H NMR, restrained molecular dynamics simulations, and atomic force microscopy (AFM). The NMR structure of a decamer containing a CpG interstrand cross-link shows the cross-link easily accommodated within the duplex with no disruption of hydrogen bonding and only minor perturbations of helical parameters. In contrast, disruptions caused by the GpC cross-link produced considerable conformational flexibility that precluded structure determination by NMR. AFM imaging of cross-link-containing plasmid DNA showed that the increased flexibility observed in the GpC cross-link persists when it is embedded into much larger DNA fragments. These differences may account for the different repair efficiencies seen in NER deficient cells.     

DNA repair defects channel interstrand DNA cross-links into alternate recombinational and error-prone repair pathways

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 cross-link-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 error-prone pathways, one NER-dependent and one NER-independent.     

Defining the roles of nucleotide excision repair and recombination in the repair of DNA interstrand cross-links in mammalian cells

The mechanisms by which DNA interstrand cross-links (ICLs) are repaired in mammalian cells are unclear. Studies in bacteria and yeasts indicate that both nucleotide excision repair (NER) and recombination are required for their removal and that double-strand breaks are produced as repair intermediates in yeast cells. The role of NER and recombination in the repair of ICLs induced by nitrogen mustard (HN2) was investigated using Chinese hamster ovary mutant cell lines. XPF and ERCC1 mutants (defective in genes required for NER and some types of recombination) and XRCC2 and XRCC3 mutants (defective in RAD51-related homologous recombination genes) were highly sensitive to HN2. Cell lines defective in other genes involved in NER (XPB, XPD, and XPG), together with a mutant defective in nonhomologous end joining (XRCC5), showed only mild sensitivity. In agreement with their extreme sensitivity, the XPF and ERCC1 mutants were defective in the incision or "unhooking" step of ICL repair. In contrast, the other mutants defective in NER activities, the XRCC2 and XRCC3 mutants, and the XRCC5 mutant all showed normal unhooking kinetics. Using pulsed-field gel electrophoresis, DNA double-strand breaks (DSBs) were found to be induced following nitrogen mustard treatment. DSB induction and repair were normal in all the NER mutants, including XPF and ERCC1. The XRCC2, XRCC3, and XRCC5 mutants also showed normal induction kinetics. The XRCC2 and XRCC3 homologous recombination mutants were, however, severely impaired in the repair of DSBs. These results define a role for XPF and ERCC1 in the excision of ICLs, but not in the recombinational components of cross-link repair. In addition, homologous recombination but not nonhomologous end joining appears to play an important role in the repair of DSBs resulting from nitrogen mustard treatment.     

DNA interstrand cross-links induce futile repair synthesis in mammalian cell extracts

DNA interstrand cross-links are induced by many carcinogens and anticancer drugs. It was previously shown that mammalian DNA excision repair nuclease makes dual incisions 5' to the cross-linked base of a psoralen cross-link, generating a gap of 22 to 28 nucleotides adjacent to the cross-link. We wished to find the fates of the gap and the cross-link in this complex structure under conditions conducive to repair synthesis, using cell extracts from wild-type and cross-linker-sensitive mutant cell lines. We found that the extracts from both types of strains filled in the gap but were severely defective in ligating the resulting nick and incapable of removing the cross-link. The net result was a futile damage-induced DNA synthesis which converted a gap into a nick without removing the damage. In addition, in this study, we showed that the structure-specific endonuclease, the XPF-ERCC1 heterodimer, acted as a 3'-to-5' exonuclease on cross-linked DNA in the presence of RPA. Collectively, these observations shed some light on the cellular processing of DNA cross-links and reveal that cross-links induce a futile DNA synthesis cycle that may constitute a signal for specific cellular responses to cross-linked DNA.     

Initiation of DNA interstrand cross-link repair in humans: the nucleotide excision repair system makes dual incisions 5' to the cross-linked base and removes a 22- to 28-nucleotide-long damage-free strand.

Most DNA repair mechanisms rely on the redundant information inherent to the duplex to remove damaged nucleotides and replace them with normal ones, using the complementary strand as a template. Interstrand cross-links pose a unique challenge to the DNA repair machinery because both strands are damaged. To study the repair of interstrand cross-links by mammalian cells, we tested the activities of cell extracts of wild-type or excision repair-defective rodent cell lines and of purified human excision nuclease on a duplex with a site-specific cross-link. We found that in contrast to monoadducts, which are removed by dual incisions bracketing the lesion, the cross-link causes dual incisions, both 5' to the cross-link in one of the two strands. The net result is the generation of a 22- to 28-nucleotide-long gap immediately 5' to the cross-link. This gap may act as a recombinogenic signal to initiate cross-link removal.     

Molecular and biological roles of Ape1 protein in mammalian base excision repair

Many oxidative DNA lesions are handled well by base excision repair (BER), but some types may be problematic. Recent work indicates that 2-deoxyribonolactone (dL) is such a lesion by forming stable, covalent cross-links between the abasic residue and DNA repair proteins with lyase activity. In the case of DNA polymerase beta, the reaction is potentiated by incision of dL by Ape1, the major mammalian AP endonuclease. When repair is prevented, polymerase beta is the most reactive cross-linking protein in whole-cell extracts. Cross-linking with dL is largely avoided by processing the damage through the "long-patch" (multinucleotide) BER pathway. However, if excess damage leads to the accumulation of unrepaired oxidative lesions in DNA, there may be a danger of polymerase beta-mediated cross-link formation. Understanding how cells respond to such complex damage is an important issue. In addition to its role in defending against DNA damage caused by exogenous agents, Ape1 protein is essential for coping with the endogenous DNA damage in human cells grown in culture. Suppression of Ape1 using RNA-interference technology causes arrest of cell proliferation and activation of apoptosis in various cell types, correlated with the accumulation of unrepaired abasic DNA damage. Notably, all these effects are reversed by expression of the unrelated protein Apn1 of S. cerevisiae, which shares only the enzymatic repair function with Ape1 (AP endonuclease).     

Early steps in the DNA base excision/single-strand interruption repair pathway in mammalian cells

Base excision repair （BER） is an evolutionarily conserved process for maintaining genomic integrity by eliminating several dozen damaged （oxidized or aikylated） or inappropriate bases that are generated endogenously or induced by genotoxicants, predominantly, reactive oxygen species （ROS）. BER involves 4-5 steps starting with base excision by a DNA glycosylase, followed by a common pathway usually involving an AP-endonuclease （APE） to generate 3＇ OH terminus at the damage site, followed by repair synthesis with a DNA polymerase and nick sealing by a DNA iigase. This pathway is also responsible for repairing DNA single-strand breaks with blocked termini directly generated by ROS. Nearly all glycosylases, far fewer than their substrate lesions particularly for oxidized bases, have broad and overlapping substrate range, and could serve as back-up enzymes in vivo. In contrast, mammalian cells encode only one APE, APEI, unlike two APEs in lower organisms. In spite of overall similarity, BER with distinct subpathways in the mammals is more complex than in E. coli. The glycosylases form complexes with downstream proteins to carry out efficient repair via distinct subpathways one of which, responsible for repair of strand breaks with 3＇ phosphate termini generated by the NEIL family glycosylases or by ROS, requires the phosphatase activity of polynucleotide kinase instead of APE1. Different complexes may utilize distinct DNA polymerases and iigases. Mammalian glycosylases have nonconserved extensions at one of the termini, dispensable for enzymatic activity but needed for interaction with other BER and non-BER proteins for complex formation and organeile targeting. The mammalian enzymes are sometimes covalently modified which may affect activity and complex formation. The focus of this review is on the early steps in mammalian BER for oxidized damage.     

Tip60 is required for DNA interstrand cross-link repair in the Fanconi anemia pathway

The disease Fanconi anemia is a genome instability syndrome characterized by cellular sensitivity to DNA interstrand cross-linking agents, manifest by decreased cellular survival and chromosomal aberrations after such treatment. There are at least 13 proteins acting in the pathway, with the FANCD2 protein apparently functioning as a late term effecter in the maintenance of genome stability. We find that the chromatin remodeling protein, Tip60, interacts directly with the FANCD2 protein in a yeast two-hybrid system. This interaction has been confirmed by co-immunoprecipitation and co-localization using both endogenous and epitope-tagged FANCD2 and Tip60 from human cells. The observation of decreased cellular survival after exposure to mitomycin C in normal fibroblasts depleted for Tip60 indicates a direct function in interstrand cross-link repair. The coincident function of Tip60 and FANCD2 in one pathway is supported by the finding that depletion of Tip60 in Fanconi anemia cells does not increase sensitivity to DNA cross-links. However, depletion of Tip60 did not reduce monoubiquitination of FANCD2 or its localization to nuclear foci following DNA damage. The observations indicate that Fanconi anemia proteins act in concert with chromatin remodeling functions to maintain genome stability after DNA cross-link damage.     

Defects in interstrand cross-link uncoupling do not account for the extreme sensitivity of ERCC1 and XPF cells to cisplatin

The anticancer drug cisplatin reacts with DNA leading to the formation of interstrand and intrastrand cross-links that are the critical cytotoxic lesions. In contrast to cells bearing mutations in other components of the nucleotide excision repair apparatus (XPB, XPD, XPG and CSB), cells defective for the ERCC1-XPF structure-specific nuclease are highly sensitive to cisplatin. To determine if the extreme sensitivity of XPF and ERCC1 cells to cisplatin results from specific defects in the repair of either intrastrand or interstrand cross-links we measured the elimination of both lesions in a range of nucleotide excision repair Chinese hamster mutant cell lines, including XPF- and ERCC1-defective cells. Compared to the parental, repair-proficient cell line all the mutants tested were defective in the elimination of both classes of adduct despite their very different levels of increased sensitivity. Consequently, there is no clear relationship between initial incisions at interstrand cross-links or removal of intrastrand adducts and cellular sensitivity. These results demonstrate that the high cisplatin sensitivity of ERCC1 and XPF cells likely results from a defect other than in excision repair. In contrast to other conventional DNA cross-linking agents, we found that the repair of cisplatin adducts does not involve the formation of DNA double-strand breaks. Surprisingly, XRCC2 and XRCC3 cells are defective in the uncoupling step of cisplatin interstrand cross-link repair, suggesting that homologous recombination might be initiated prior to excision of this type of cross-link.