Genetic polymorphisms in the nucleotide excision repair pathway and lung cancer risk: a meta-analysis

Various DNA alterations can be caused by exposure to environmental and endogenous carcinogens. Most of these alterations, if not repaired, can result in genetic instability, mutagenesis and cell death. DNA repair mechanisms are important for maintaining DNA integrity and preventing carcinogenesis. Recent lung cancer studies have focused on identifying the effects of single nucleotide polymorphisms (SNPs) in candidate genes, among which DNA repair genes are increasingly being studied. Genetic variations in DNA repair genes are thought to modulate DNA repair capacity and are suggested to be related to lung cancer risk. We identified a sufficient number of epidemiologic studies on lung cancer to conduct a meta-analysis for genetic polymorphisms in nucleotide excision repair pathway genes, focusing on xeroderma pigmentosum group A (XPA), excision repair cross complementing group 1 (ERCC1), ERCC2/XPD, ERCC4/XPF and ERCC5/XPG. We found an increased risk of lung cancer among subjects carrying the ERCC2 751Gln/Gln genotype (odds ratio (OR) = 1.30, 95% confidence interval (CI) = 1.14 - 1.49). We found a protective effect of the XPA 23G/G genotype (OR = 0.75, 95% CI = 0.59 - 0.95). Considering the data available, it can be conjectured that if there is any risk association between a single SNP and lung cancer, the risk fluctuation will probably be minimal. Advances in the identification of new polymorphisms and in high-throughput genotyping techniques will facilitate the analysis of multiple genes in multiple DNA repair pathways. Therefore, it is likely that the defining feature of future epidemiologic studies will be the simultaneous analysis of large samples.     

Sensitivity of group F xeroderma pigmentosum cells to UV and mitomycin C relative to levels of XPF and ERCC1 overexpression

The XPF and ERCC1 proteins form a tight complex and functionas an endonuclease to incise on the 5'-side of pyrimidine dimersin DNA. Levels of both proteins are extremely low in group Fxeroderma pigmentosum (XP-F) cells. We transfected XP-F cellswith the plasmids expressing XPF or ERCC1 and examined levelsof both proteins in the cells. Although XP-F cells are sensitiveto UV and mitomycin C (MMC), cells overexpressing XPF expressedERCCl as well and resistance to UV and MMC was restored to thenormal level. In contrast, cells overexpressing ERCC1 did notexpress XPF and were still sensitive to UV and MMC. These resultsindicate that both the XPF and ERCCl proteins are required torepair UV- and MMC-induced DNA damage. Even though a high levelof ERCC1, which has been presumed to be a catalytic subunitof the endonuclease, is stably present in XP-F cells, ERCC1protein alone cannot carry out excision repair completely. ^*To whom correspondence should be addressed. Tel: +81 75 753 4412; Fax: +81 75 753 4419; Email: c51845{at}sakura.kudpc.kyoto-u.ac.jp     

Structure and conformational change of a replication protein A heterotrimer bound to ssDNA

Replication protein A (RPA) is the main eukaryotic ssDNA-binding protein with essential roles in DNA replication, recombination, and repair. RPA maintains the DNA as single-stranded and also interacts with other DNA-processing proteins, coordinating their assembly and disassembly on DNA. RPA binds to ssDNA in two conformational states with opposing affinities for DNA and proteins. The RPA–protein interactions are compatible with a low DNA affinity state that involves DNA-binding domain A (DBD-A) and DBD-B but not with the high DNA affinity state that additionally engages DBD-C and DBD-D. The structure of the high-affinity RPA–ssDNA complex reported here shows a compact quaternary structure held together by a four-way interface between DBD-B, DBD-C, the intervening linker (BC linker), and ssDNA. The BC linker binds into the DNA-binding groove of DBD-B, mimicking DNA. The associated conformational change and partial occlusion of the DBD-A–DBA-B protein–protein interaction site establish a mechanism for the allosteric coupling of RPA–DNA and RPA–protein interactions.     

The N-terminal domain of the large subunit of human replication protein A binds to Werner syndrome protein and stimulates helicase activity

Werner syndrome (WS) is a recessive inherited human disease characterized by the early onset of aging. The gene mutated in WS encodes a DNA helicase that unwinds the double helical structure of DNA in the 3'-->5' direction as well as a 3'-->5' exonuclease. Our previous studies indicated that the activity of Werner syndrome helicase (WRN) could be stimulated by human replication protein A (hRPA), a heterotrimeric single-stranded DNA binding protein. We now localize the interaction between WRN and hRPA by measuring the stimulation of helicase activity and the binding of WRN by hRPA and its derivatives. The large subunit of hRPA (hRPA70) stimulates WRN helicase to the same extent as the hRPA heterotrimer, whereas the dimer of the two smaller subunits (hRPA 32.14) does not stimulate. By examining hRPA70 mutants with progressive deletions from either the C- or N-terminus, we found that the domain responsible for stimulation lies in the N-terminal half of the protein. By using enzyme-linked immunosorbent assay (ELISA) to examine physical interaction between WRN and the same deletion mutants, we found that the WRN-binding motif is located within amino acids 100-300 and overlaps with the single-stranded DNA binding domain (amino acids 150-450). We suggest that hRPA, by engaging in both protein-protein and protein-DNA interactions, facilitates unwinding events catalyzed by WRN helicase during DNA synthetic processes. These data should help further elucidation of the molecular mechanisms of genetic instability and premature aging phenotypes manifested by WS.     

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5′ flap DNA: basis of interstrand cross-link repair by FAN1

Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI–FANCD2 (ID) complex and participates in ICL repair. Here, we determined the crystal structure of Pseudomonas aeruginosa FAN1 (PaFAN1) lacking the UBZ (ubiquitin-binding zinc) domain in complex with 5′ flap DNA. All four domains of the right-hand-shaped PaFAN1 are involved in DNA recognition, with each domain playing a specific role in bending DNA at the nick. The six-helix bundle that binds the junction connects to the catalytic viral replication and repair (VRR) nuclease (VRR nuc) domain, enabling FAN1 to incise the scissile phosphate a few bases distant from the junction. The six-helix bundle also inhibits the cleavage of intact Holliday junctions. PaFAN1 shares several conserved features with other flap structure-selective nucleases despite structural differences. A clamping motion of the domains around the wedge helix, which acts as a pivot, facilitates nucleolytic cleavage. The PaFAN1 structure provides insights into how archaeal Holliday junction resolvases evolved to incise 5′ flap substrates and how FAN1 integrates with the FA complex to participate in ICL repair.     

Functional interaction between telomere protein TPP1 and telomerase

Human chromosome end-capping and telomerase regulation require POT1 (Protection of Telomeres 1) and TPP1 proteins, which bind to the 3′ ssDNA extension of human telomeres. POT1–TPP1 binding to telomeric DNA activates telomerase repeat addition processivity. We now provide evidence that this POT1–TPP1 activation requires specific interactions with telomerase, rather than it being a DNA substrate-specific effect. First, telomerase from the fish medaka, which extends the same telomeric DNA primer as human telomerase, was not activated by human POT1–TPP1. Second, mutation of a conserved glycine, Gly100 in the TEN (telomerase essential N-terminal) domain of TERT, abolished the enhancement of telomerase processivity by POT1–TPP1, in contrast to other single amino acid mutations. Chimeric human–fish telomerases that contained the human TEN domain were active but not stimulated by POT1–TPP1, showing that additional determinants of processivity lie outside the TEN domain. Finally, primers bound to mouse POT1A and human TPP1 were activated for extension by human telomerase, whereas mPOT1A–mTPP1 was most active with mouse telomerase, indicating that these mammalian telomerases have specificity for their respective TPP1 proteins. We suggest that a sequence-specific interaction between TPP1 in the TPP1–POT1–telomeric DNA complex and the G100 region of the TEN domain of TERT is necessary for high-processivity telomerase action.     

Mechanism of Holliday junction resolution by the human GEN1 protein

Holliday junction (HJ) resolution is essential for chromosome segregation at meiosis and the repair of stalled/collapsed replication forks in mitotic cells. All organisms possess nucleases that promote HJ resolution by the introduction of symmetrically related nicks in two strands at, or close to, the junction point. GEN1, a member of the Rad2/XPG nuclease family, was isolated recently from human cells and shown to promote HJ resolution in vitro and in vivo. Here, we provide the first biochemical/structural characterization of GEN1, showing that, like the Escherichia coli HJ resolvase RuvC, it binds specifically to HJs and resolves them by a dual incision mechanism in which nicks are introduced in the pair of continuous (noncrossing) strands within the lifetime of the GEN1–HJ complex. In contrast to RuvC, but like other Rad2/XPG family members such as FEN1, GEN1 is a monomeric 5′-flap endonuclease. However, the unique feature of GEN1 that distinguishes it from other Rad2/XPG nucleases is its ability to dimerize on HJs. This functional adaptation provides the two symmetrically aligned active sites required for HJ resolution.     

ERCC1 mutations in UV-sensitive Chinese hamster ovary (CHO) cell lines

In mammalian nucleotide excision repair (NER), the ERCC1 protein is known to act as a complex with ERCC4 (XPF) protein, which is necessary for stability of ERCC1, and this complex introduces an incision on the 5′ side of a damaged site in DNA. ERCC1 also binds to XPA protein to make a large protein complex at the site of DNA damage. Since no human disease associated with ERCC1 has been identified, Chinese hamster ovary (CHO) cell lines defective in ERCC1 are a unique source for characterization of ERCC1 deficiency in mammalian cells. We have isolated the full length ERCC1 cDNA from a wild-type CHO cell line and analyzed mutations in two CHO cell lines which fall into complementation group 1 of UV-sensitive rodent cell lines. One cell line, 43-3B, has a missense mutation at the 98th residue (V98E). The in vitro translated mutant protein of 43-3B is unable to bind to XPA protein. Although the mutant protein is able to bind to XPF protein in vitro, the mutant protein is highly unstable in vivo. These defects presumably cause the NER deficiency of this cell line. Another mutant, UV-4, has an insertion mutation in the middle of the coding sequence, resulting in a truncated protein due to a nonsense codon arising from the frameshift. Thus, these two mutant cell lines are deficient in the function of the ERCC1 gene for NER.     

AdnAB: a new DSB-resecting motor–nuclease from mycobacteria

The resection of DNA double-strand breaks (DSBs) in bacteria is a motor-driven process performed by a multisubunit helicase–nuclease complex: either an Escherichia coli-type RecBCD enzyme or a Bacillus-type AddAB enzyme. Here we identify mycobacterial AdnAB as the founder of a new family of heterodimeric helicase–nucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal nuclease module. The AdnAB ATPase is triggered by dsDNA with free ends and the energy of ATP hydrolysis is coupled to DSB end resection by the AdnAB nuclease. The mycobacterial nonhomologous end-joining (NHEJ) protein Ku protects DSBs from resection by AdnAB. We find that AdnAB incises ssDNA by measuring the distance from the free 5′ end to dictate the sites of cleavage, which are predominantly 5 or 6 nucleotides (nt) from the 5′ end. The “molecular ruler” of AdnAB is regulated by ATP, which elicits an increase in ssDNA cleavage rate and a distal displacement of the cleavage sites 16–17 nt from the 5′ terminus. AdnAB is a dual nuclease with a clear division of labor between the subunits. Mutations in the nuclease active site of the AdnB subunit ablate the ATP-inducible cleavages; the corresponding changes in AdnA abolish ATP-independent cleavage. Complete suppression of DSB end resection requires simultaneous mutation of both subunit nucleases. The nuclease-null AdnAB is a helicase that unwinds linear plasmid DNA without degrading the displaced single strands. Mutations of the phosphohydrolase active site of the AdnB subunit ablate DNA-dependent ATPase activity, DSB end resection, and ATP-inducible ssDNA cleavage; the equivalent mutations of the AdnA subunit have comparatively little effect. AdnAB is a novel signature of the Actinomycetales taxon. Mycobacteria are exceptional in that they encode both AdnAB and RecBCD, suggesting the existence of alternative end-resecting motor–nuclease complexes.     

Polymorphism in nucleotide excision repair gene XPC correlates with bleomycin-induced chromosomal aberrations

Chromosomal aberrations (CAs) are important genetic alterations in the development and progression of the majority of human cancers. The frequency with which such alterations occur depends to a large extent on polymorphisms of DNA-repair genes and in genes coding for xenobiotic metabolizing enzymes, which are involved in the processes of activation and inactivation of xenobiotics. The frequency of bleomycin (BLM)-induced CAs is an indirect measure of the effectiveness of DNA repair mechanisms, and a predictor of environment-related risk of cancer. Our study was conducted on the human peripheral blood lymphocytes of 82 healthy volunteers. The aim of the study was to elucidate whether the frequency of BLM-induced CAs is correlated with polymorphisms of selected genes involved in different mechanisms of DNA repair such as: XRCC1 [base excision repair]; XPA, XPC, XPG, XPD, XPF, ERCC1 [nucleotide excision repair], NBS1, RAD51, XRCC2, XRCC3, RAD51, and BRCA1 [homologous recombination], as well as in genes encoding xenobiotic metabolizing enzymes, such as CYP1A, CYP2E1, NAT2, GSTT1, and EPHX (mEH). Our study indicated that, of the polymorphisms studied, only XPC (exon 15 and intron 11) is associated with BLM-induced CAs, suggesting a role of the NER pathway in the repair of BLM-induced chromosomal aberrations.     

Novel XPG (ERCC5) Mutations Affect DNA Repair and Cell Survival after Ultraviolet but not Oxidative Stress

Nucleotide excision repair (NER) is the most flexible of all known DNA‐repair mechanisms, and XPG is a 3′‐endonuclease that participates in NER. Mutations in this gene (ERCC5) may result in the human syndrome xeroderma pigmentosum (XP) and, in some cases, in the complex phenotype of Cockayne syndrome (CS). Two Brazilian XP siblings, who were mildly affected, were investigated and classified into the XP‐G group. The cells from these patients were highly ultraviolet (UV) sensitive but not sensitive to photosensitized methylene blue, an agent that causes oxidative stress. This phenotype is in contrast to XP‐G/CS cells, which are highly sensitive to this oxidative agent. Sequencing revealed a compound heterozygous genotype with two novel missense mutations: c.83C>A (p.Ala28Asp) and c.2904G>C (p.Trp968Cys). The first mutation maps to the catalytic site of the XPG protein, whereas the second may compromise binding to DNA. Functional assays indicated that the mutated alleles were unable to perform the complete repair of UV‐irradiated plasmids; however, full correction was observed for oxidatively damaged plasmids. Therefore, the XP phenotype of these patients is caused by novel missense mutations that specifically affect DNA repair for UV‐ but not oxidative‐stress‐induced DNA damage, and implications for XP versus XP/CS phenotype are discussed.     

Human XPF controls TRF2 and telomere length maintenance through distinctive mechanisms

XPF-ERCC1, a structure-specific endonuclease, is involved in nucleotide excision repair, crosslink repair and homologous recombination. XPF-ERCC1 is also found to interact with TRF2, a duplex telomeric DNA binding protein. We have previously shown that XPF-ERCC1 is required for TRF2-promoted telomere shortening. However, whether XPF-ERCC1 by itself has a role in telomere length maintenance has not been determined. Here we report that overexpression of XPF induces telomere shortening in XPF-proficient cells whereas XPF complementation suppresses telomere lengthening in XPF-deficient cells. These results suggest that XPF-ERCC1 can function as a negative mediator of telomere length maintenance. In addition, we find that introduction of wild type XPF into XPF-deficient cells leads to over 40% reduction in TRF2 association with telomeric DNA, indicating that XPF-ERCC1 negatively regulates TRF2 binding to telomeric DNA. Furthermore, we show that XPF carrying mutations in the conserved nuclease domain fails to control TRF2 association with telomeric DNA but it is competent for modulating telomere length maintenance. These results imply that XPF-ERCC1 controls TRF2 and telomere length maintenance through two distinctive mechanisms, with the former requiring its nuclease activity. Our results further imply that TRF2 association with telomeres may be deregulated in cells derived from XPF patients.     

Translational reprogramming following UVB irradiation is mediated by DNA-PKcs and allows selective recruitment to the polysomes of mRNAs encoding DNA repair enzymes

UVB-induced lesions in mammalian cellular DNA can, through the process of mutagenesis, lead to carcinogenesis. However, eukaryotic cells have evolved complex mechanisms of genomic surveillance and DNA damage repair to counteract the effects of UVB radiation. We show that following UVB DNA damage, there is an overall inhibition of protein synthesis and translational reprogramming. This reprogramming allows selective synthesis of DDR proteins, such as ERCC1, ERCC5, DDB1, XPA, XPD, and OGG1 and relies on upstream ORFs in the 5′ untranslated region of these mRNAs. Experiments with DNA-PKcs-deficient cell lines and a specific DNA-PKcs inhibitor demonstrate that both the general repression of mRNA translation and the preferential translation of specific mRNAs depend on DNA-PKcs activity, and therefore our data establish a link between a key DNA damage signaling component and protein synthesis.     

A common polymorphism in the 5′ UTR of ERCC5 creates an upstream ORF that confers resistance to platinum-based chemotherapy

We show that a common polymorphic variant in the ERCC5 5′ untranslated region (UTR) generates an upstream ORF (uORF) that affects both the background expression of this protein and its ability to be synthesized following exposure to agents that cause bulky adduct DNA damage. Individuals that harbor uORF1 have a marked resistance to platinum-based agents, illustrated by the significantly reduced progression-free survival of pediatric ependymoma patients treated with such compounds. Importantly, inhibition of DNA-PKcs restores sensitivity to platinum-based compounds by preventing uORF1-dependent ERCC5 expression. Our data support a model in which a heritable 5′ noncoding mRNA element influences individuals’ responses to platinum-based chemotherapy.     

Recognition of forked and single-stranded DNA structures by human RAD18 complexed with RAD6B protein triggers its recruitment to stalled replication forks

Post-replication DNA repair facilitates the resumption of DNA synthesis upon replication fork stalling at DNA damage sites. Despite the importance of RAD18 and polymerase η (Polη) for post-replication repair (PRR), the molecular mechanisms by which these factors are recruited to stalled replication forks are not well understood. We present evidence that human RAD18 complexed with RAD6B protein preferentially binds to forked and single-stranded DNA (ssDNA) structures, which are known to be localized at stalled replication forks. The SAP domain of RAD18 (residues 248–282) is crucial for binding of RAD18 complexed with RAD6B to DNA substrates. RAD18 mutated in the SAP domain fails to accumulate at DNA damage sites in vivo and does not guide DNA Polη to stalled replication forks. The SAP domain is also required for the efficient mono-ubiquitination of PCNA. The SAP domain mutant fails to suppress the ultraviolet (UV)-sensitivity of Rad18 -knockout cells. These results suggest that RAD18 complexed with RAD6B is recruited to stalled replication forks via interactions with forked DNA or long ssDNA structures, a process that is required for initiating PRR.