Multiple DNA damage recognition factors involved in mammalian nucleotide excision repair

The nucleotide excision repair (NER) subpathway operating throughout the mammalian genome is a versatile DNA repair system that can remove a wide variety of helix-distorting base lesions. This system contributes to prevention of blockage of DNA replication by the lesions, thereby suppressing mutagenesis and carcinogenesis. Therefore, it is of fundamental significance to understand how the huge genome can be surveyed for occurrence of a small number of lesions. Recent studies have revealed that this difficult task seems to be accomplished through sequential actions of multiple DNA damage recognition factors, including UV-DDB, XPC, and TFIIH. Notably, these factors adopt completely different strategies to recognize DNA damage. XPC detects disruption and/or destabilization of the base pairing, which ensures a broad spectrum of substrate specificity for global genome NER. In contrast, UV-DDB directly recognizes particular types of lesions, such as UV-induced photoproducts, thereby vitally recruiting XPC as well as further extending the substrate specificity. After DNA binding by XPC, moreover, the helicase activity associated with TFIIH scans a DNA strand to make a final search for the presence of aberrant chemical modifications of DNA. The combination of these different strategies makes a crucial contribution to simultaneously achieving efficiency, accuracy, and versatility of the entire repair system.     

Hierarchy of DNA damage recognition in Escherichia coli nucleotide excision repair

DNA damage recognition plays a central role in nucleotide excision repair (NER). Here we present evidence that in Escherichia coli NER, DNA damage is recognized through at least two separate but successive steps, with the first focused on distortions from the normal structure of the DNA double helix (initial recognition) and the second specifically recognizing the type of DNA base modifications (second recognition), after an initial local separation of the DNA strands. DNA substrates containing stereoisomeric (+)- or (-)-trans- or (+)- or (-)-cis-BPDE-N(2)-dG lesions in DNA duplexes of known conformations were incised by UvrABC nuclease with efficiencies varying by up to 3-fold. However, these stereoisomeric adducts, when positioned in an opened, single-stranded DNA region, were all incised with similar efficiencies and with enhanced rates (by factors of 1.4-6). These bubble substrates were also equally and efficiently incised by UvrBC nuclease without UvrA. Furthermore, removal of the Watson-Crick partner cytosine residue (leaving an abasic site) in the complementary strand opposite a (+)-cis-BPDE-N(2)-dG lesion led to a significant reduction in both the binding of UvrA and the incision efficiency of UvrABC by a factor of 5. These data suggest that E. coli NER features a dynamic two-stage recognition mechanism.     

Damage recognition in nucleotide excision DNA repair

Nucleotide excision repair (NER) is a highly versatile DNA repair process. Its ability to repair a large number of different damages with the same subset of recognition factors requires structural tools for damage recognition that are both broad and very accurate. Over the past few years detailed structural information on damage recognition factors from eukaryotic and prokaryotic NER has emerged. These structures shed light on the toolkit utilized in the damage recognition process and help explain the broad substrate specificity of NER.     

Damage recognition in nucleotide excision repair of DNA

Nucleotide excision repair (NER) is found throughout nature, in eubacteria, eukaryotes and archaea. In human cells it is the main pathway for the removal of damage caused by UV light, but it also acts on a wide variety of other bulky helix-distorting lesions caused by chemical mutagens. An ongoing challenge is to understand how a site of DNA damage is located during NER and distinguished from non-damaged sites. This article reviews information on damage recognition in mammalian cells and the bacterium Escherichia coli. In mammalian cells the XPC-hHR23B, XPA, RPA and TFIIH factors may all have a role in damage recognition. XPC-hHR23B has the strongest affinity for damaged DNA in some assays, as does the similar budding yeast complex Rad4-Rad23. There is current discussion as to whether XPC or XPA acts first in the repair process to recognise damage or distortions. TFIIH may play a role in distinguishing the damaged strand from the non-damaged one, if translocation along a DNA strand by the TFIIH DNA helicases is interrupted by encountering a lesion. The recognition and incision steps of human NER use 15 to 18 polypeptides, whereas E. coli requires only three proteins to obtain a similar result. Despite this, many remarkable similarities in the NER mechanism have emerged between eukaryotes and bacteria. These include use of a distortion-recognition factor, a strand separating helicase to create an open preincision complex, participation of structure-specific endonucleases and the lack of a need for certain factors when a region containing damage is already sufficiently distorted.     

Long-patch DNA repair synthesis during base excision repair in mammalian cells

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway.     

DNA damage recognition of mutated forms of UvrB proteins in nucleotide excision repair

The DNA repair protein UvrB plays an indispensable role in the stepwise and sequential damage recognition of nucleotide excision repair in Escherichia coli. Our previous studies suggested that UvrB is responsible for the chemical damage recognition only upon a strand opening mediated by UvrA. Difficulties were encountered in studying the direct interaction of UvrB with adducts due to the presence of UvrA. We report herein that a single point mutation of Y95W in which a tyrosine is replaced by a tryptophan results in an UvrB mutant that is capable of efficiently binding to structure-specific DNA adducts even in the absence of UvrA. This mutant is fully functional in the UvrABC incisions. The dissociation constant for the mutant-DNA adduct interaction was less than 100 nM at physiological temperatures as determined by fluorescence spectroscopy. In contrast, similar substitutions at other residues in the beta-hairpin with tryptophan or phenylalanine do not confer UvrB such binding ability. Homology modeling of the structure of E. coli UvrB shows that the aromatic ring of residue Y95 and only Y95 directly points into the DNA binding cleft. We have also examined UvrB recognition of both "normal" bulky BPDE-DNA and protein-cross-linked DNA (DPC) adducts and the roles of aromatic residues of the beta-hairpin in the recognition of these lesions. A mutation of Y92W resulted in an obvious decrease in the efficiency of UvrABC incisions of normal adducts, while the incision of the DPC adduct is dramatically increased. Our results suggest that Y92 may function differently with these two types of adducts, while the Y95 residue plays an unique role in stabilizing the interaction of UvrB with DNA damage, most likely by a hydrophobic stacking.     

Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair

Using only a limited repertoire of recognition subunits, the nucleotide excision repair (NER) system is able to detect a nearly infinite variety of bulky DNA lesions. This extraordinary substrate versatility has generally been ascribed to an indirect readout mechanism, whereby particular distortions of the double helix, induced by a damaged nucleotide, provide the molecular determinants not only for lesion recognition but also for subsequent verification or demarcation processes. Here, we discuss the evidence in support of a bipartite mechanism of substrate discrimination that is initiated by the detection of thermodynamically unstable base pairs followed by direct localization of the lesion through an enzymatic proofreading activity. This bipartite discrimination mechanism is part of a dynamic reaction cycle that confers high levels of selectivity to avoid futile repair events on undamaged DNA and also protect the intact complementary strand from inappropriate cleavage.     

DNA damage induced nucleotide excision repair in Saccharomyces cerevisiae

Nucleotide excision repair (NER) is the most versatile and universal pathway of DNA repair that is capable of repairing virtually any damages other than a double strand break (DSB). This pathway has been shown to be inducible in several systems. However, question of a threshold and the nature of the damage that can signal induction of this pathway remain poorly understood. In this study it has been shown that prior exposure to very low doses of osmium tetroxide enhanced the survival of wild type Saccharomyces cerevisiae when the cells were challenged with UV light. Moreover, it was also found that osmium tetroxide treated rad3 mutants did not show enhanced survival indicating an involvement of nucleotide excision repair in the enhanced survival. To probe this further the actual removal of pyrimidine dimers by the treated and control cells was studied. Osmium tetroxide treated cells removed pyrimidine dimers more efficiently as compared to control cells. This was confirmed by measuring the in vitro repair synthesis in cell free extracts prepared from control and primed cells. It was found that the uptake of active ³²P was significantly higher in the plasmid substrates incubated with extracts of primed cells. This induction is dependent on de novo synthesis of proteins as cycloheximide treatment abrogated this response. The nature of induced repair was found to be essentially error free. Study conclusively shows that NER is an inducible pathway in Saccharomyces cerevisiae and its induction is dependent on exposure to a threshold of a genotoxic stress.     

Biochemical analysis of the damage recognition process in nucleotide excision repair

XPA, XPC-hHR23B, RPA, and TFIIH all are the damage recognition proteins essential for the early stage of nucleotide excision repair. Nonetheless, it is not clear how these proteins work together at the damaged DNA site. To get insight into the molecular mechanism of damage recognition, we carried out a comprehensive analysis on the interaction between damage recognition proteins and their assembly on damaged DNA. XPC physically interacted with XPA, but failed to stabilize the XPA-damaged DNA complex. Instead, XPC-hHR23B was effectively displaced from the damaged DNA by the combined action of RPA and XPA. A mutant RPA lacking the XPA interaction domain failed to displace XPC-hHR23B from damaged DNA, suggesting that XPA and RPA cooperate with each other to destabilize the XPC-hHR23B-damaged DNA complex. Interestingly, the presence of hHR23B significantly increased RPA/XPA-mediated displacement of XPC from damaged DNA, suggesting that hHR23B may modulate the binding of XPC to damaged DNA. Together, our results suggest that damage recognition occurs in a multistep process such that XPC-hHR23B initiates damage recognition, which was replaced by combined action of XPA and RPA. XPA and RPA, once forming a complex at the damage site, would likely work with TFIIH, XPG, and ERCC1-XPF for dual incision.     

Base excision repair of DNA in mammalian cells

Base excision repair (BER) of DNA corrects a number of spontaneous and environmentally induced genotoxic or miscoding base lesions in a process initiated by DNA glycosylases. An AP endonuclease cleaves at the 5' side of the abasic site and the repair process is subsequently completed via either short patch repair or long patch repair, which largely require different proteins. As one example, the UNG gene encodes both nuclear (UNG2) and mitochondrial (UNG1) uracil DNA glycosylase and prevents accumulation of uracil in the genome. BER is likely to have a major role in preserving the integrity of DNA during evolution and may prevent cancer.     

Regulation of nucleotide excision repair by UV-DDB: prioritization of damage recognition to internucleosomal DNA

How tightly packed chromatin is thoroughly inspected for DNA damage is one of the fundamental unanswered questions in biology. In particular, the effective excision of carcinogenic lesions caused by the ultraviolet (UV) radiation of sunlight depends on UV-damaged DNA-binding protein (UV-DDB), but the mechanism by which this DDB1-DDB2 heterodimer stimulates DNA repair remained enigmatic. We hypothesized that a distinctive function of this unique sensor is to coordinate damage recognition in the nucleosome repeat landscape of chromatin. Therefore, the nucleosomes of human cells have been dissected by micrococcal nuclease, thus revealing, to our knowledge for the first time, that UV-DDB associates preferentially with lesions in hypersensitive, hence, highly accessible internucleosomal sites joining the core particles. Surprisingly, the accompanying CUL4A ubiquitin ligase activity is necessary to retain the xeroderma pigmentosum group C (XPC) partner at such internucleosomal repair hotspots that undergo very fast excision kinetics. This CUL4A complex thereby counteracts an unexpected affinity of XPC for core particles that are less permissive than hypersensitive sites to downstream repair subunits. That UV-DDB also adopts a ubiquitin-independent function is evidenced by domain mapping and in situ protein dynamics studies, revealing direct but transient interactions that promote a thermodynamically unfavorable β-hairpin insertion of XPC into substrate DNA. We conclude that the evolutionary advent of UV-DDB correlates with the need for a spatiotemporal organizer of XPC positioning in higher eukaryotic chromatin.     

Nucleotide excision repair functions in the removal of chromium-induced DNA damage in mammalian cells

Some hexavalent chromium (Cr(VI))-containing compounds are human lung carcinogens. While ample information is available on the genetic lesions produced by Cr, surprisingly little is known regarding the cellular mechanisms involved in the removal of Cr-DNA adducts. Nucleotide excision repair (NER) is a highly versatile pathway that is responsive to a variety of DNA helix-distorting lesions. Binary Cr-DNA monoadducts do not produce a significant degree of helical distortion. However, these lesions are unstable due to the propensity of Cr(III) to form DNA adducts (DNA interstrand crosslinks, DNA-protein/amino acid ternary adducts) which may serve as substrates for NER. Therefore, the focus of this study was to determine the role of NER in the processing of Cr-DNA damage using normal (CHO-AA8) and NER-deficient [UV-5 (XP-D); UV-41 (ERCC4/XP-F)] hamster cells. We found that both UV-5 and UV-41 cells exhibited an increased sensitivity towards Cr(VI)-induced clonogenic lethality relative to AA8 cells and were completely deficient in the removal of Cr-DNA adducts. In contrast, repair-complemented UV-5 (expressing hamster XPD) and UV-41 (expressing human ERCC4) cells exhibited similar clonogenic survival and removed Cr-DNA adducts to a similar extent as AA8 cells. In order to extend these findings to the molecular level, we examined the ability of Cr(III)-damaged DNA to induce DNA repair synthesis in cell extracts. Repair synthesis was observed in reactions using extracts derived from AA8, or repair-complemented, but not NER-deficient cells. Cr(III)-induced repair resynthesis was sensitive to inhibition by the DNA polymerase δ/ε inhibitor, aphidicolin, but not 2′,3′-dideoxythymidine triphosphate (ddTTP), a polymerase β inhibitor. These results collectively suggest that NER functions in the protection of cells from Cr(VI) lethality and is essential for the removal of Cr(III)-DNA adducts. Consequently, NER may represent an important mechanism for preventing Cr(VI)-induced mutagenesis and neoplastic transformation.     

Active DNA damage eviction by HLTF stimulates nucleotide excision repair

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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.     

Role of nucleotide excision repair proteins in oxidative DNA damage repair: an updating

DNA repair is a crucial factor in maintaining a low steady-state level of oxidative DNA damage. Base excision repair (BER) has an important role in preventing the deleterious effects of oxidative DNA damage, but recent evidence points to the involvement of several repair pathways in this process. Oxidative damage may arise from endogenous and exogenous sources and may target nuclear and mitochondrial DNA as well as RNA and proteins. The importance of preventing mutations associated with oxidative damage is shown by a direct association between defects in BER (i.e. MYH DNA glycosylase) and colorectal cancer, but it is becoming increasingly evident that damage by highly reactive oxygen species plays also central roles in aging and neurodegeneration. Mutations in genes of the nucleotide excision repair (NER) pathway are associated with diseases, such as xeroderma pigmentosum and Cockayne syndrome, that involve increased skin cancer risk and/or developmental and neurological symptoms. In this review we will provide an updating of the current evidence on the involvement of NER factors in the control of oxidative DNA damage and will attempt to address the issue of whether this unexpected role may unlock the difficult puzzle of the pathogenesis of these syndromes.     

Scanning the DNA for damage by the nucleotide excision repair machinery

Damage detection during nucleotide excision repair requires the action of multiple proteins that probe the DNA for different parameters like disruption of basepairing, DNA bendability and presence of chemical modifications. In a recent study it has been shown that two of these probing events can be spatially separated on the DNA. Upon initial binding of the XPC protein to a region with disrupted basepairing a complex of XPC, TFIIH and XPA is translocated to a CPD lesion even when this chemical modification is located up to 160 nucleotides from the mispaired region.     

Architecture of nucleotide excision repair complexes: DNA is wrapped by UvrB before and after damage recognition

Nucleotide excision repair (NER) is a major DNA repair mechanism that recognizes a broad range of DNA damages. In Escherichia coli, damage recognition in NER is accomplished by the UvrA and UvrB proteins. We have analysed the structural properties of the different protein-DNA complexes formed by UvrA, UvrB and (damaged) DNA using atomic force microscopy. Analysis of the UvrA(2)B complex in search of damage revealed the DNA to be wrapped around the UvrB protein, comprising a region of about seven helical turns. In the UvrB-DNA pre-incision complex the DNA is wrapped in a similar way and this DNA configuration is dependent on ATP binding. Based on these results, a role for DNA wrapping in damage recognition is proposed. Evidence is presented that DNA wrapping in the pre-incision complex also stimulates the rate of incision by UvrC.