Nucleotide excision repair efficiencies of bulky carcinogen-DNA adducts are governed by a balance between stabilizing and destabilizing interactions

The nucleotide excision repair (NER) machinery, the primary defense against cancer-causing bulky DNA lesions, is surprisingly inefficient in recognizing certain mutagenic DNA adducts and other forms of DNA damage. However, the biochemical basis of resistance to repair remains poorly understood. To address this problem, we have investigated a series of intercalated DNA-adenine lesions derived from carcinogenic polycyclic aromatic hydrocarbon (PAH) diol epoxide metabolites that differ in their response to the mammalian NER apparatus. These stereoisomeric PAH-derived adenine lesions represent ideal model systems for elucidating the effects of structural, dynamic, and thermodynamic properties that determine the recognition of these bulky DNA lesions by NER factors. The objective of this work was to gain a systematic understanding of the relation between aromatic ring topology and adduct stereochemistry with existing experimental NER efficiencies and known thermodynamic stabilities of the damaged DNA duplexes. For this purpose, we performed 100 ns molecular dynamics studies of the lesions embedded in identical double-stranded 11-mer sequences. Our studies show that, depending on topology and stereochemistry, stabilizing PAH-DNA base van der Waals stacking interactions can compensate for destabilizing distortions caused by these lesions that can, in turn, cause resistance to NER. The results suggest that the balance between helix stabilizing and destabilizing interactions between the adduct and nearby DNA residues can account for the variability of NER efficiencies observed in this class of PAH-DNA lesions.     

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.     

DNA excision repair at telomeres

DNA damage is caused by either endogenous cellular metabolic processes such as hydrolysis, oxidation, alkylation, and DNA base mismatches, or exogenous sources including ultraviolet (UV) light, ionizing radiation, and chemical agents. Damaged DNA that is not properly repaired can lead to genomic instability, driving tumorigenesis. To protect genomic stability, mammalian cells have evolved highly conserved DNA repair mechanisms to remove and repair DNA lesions. Telomeres are composed of long tandem TTAGGG repeats located at the ends of chromosomes. Maintenance of functional telomeres is critical for preventing genome instability. The telomeric sequence possesses unique features that predispose telomeres to a variety of DNA damage induced by environmental genotoxins. This review briefly describes the relevance of excision repair pathways in telomere maintenance, with the focus on base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). By summarizing current knowledge on excision repair of telomere damage and outlining many unanswered questions, it is our hope to stimulate further interest in a better understanding of excision repair processes at telomeres and in how these processes contribute to telomere maintenance.     

The eukaryotic nucleotide excision repair pathway

Nucleotide excision repair (NER) is the most versatile mechanism of DNA repair, recognizing and dealing with a variety of helix-distorting lesions, such as the UV-induced photoproducts cyclobutane pyrimidine dimers (CPDs) and pyrimidine 6-4 pyrimidone photoproducts (6-4 PPs). In this review, we describe the main protein players and the different sequential steps of the eukaryotic NER mechanism in human cells, from lesion recognition to damage removal and DNA synthesis. Studies on the dynamics of protein access to the damaged site, and the kinetics of lesion removal contribute to the knowledge of how the cells respond to genetic insult. DNA lesions as well as NER factors themselves are also implicated in changes in cell metabolism, influencing cell cycle progression or arrest, apoptosis and genetic instability. These changes are related to increased mutagenesis and carcinogenesis. Finally, the recent collection of genomic data allows one to recognize the high conservation and the evolution of eukaryotic NER. The distribution of NER orthologues in different organisms, from archaea to the metazoa, displays challenging observations. Some of NER proteins are widespread in nature, probably representing ancient DNA repair proteins, which are candidates to participate in a primitive NER mechanism.     

Role of the DNA repair nucleases Rad13, Rad2 and Uve1 of Schizosaccharomyces pombe in mismatch correction.

Repair of mismatched DNA occurs mainly by the long-patch mismatch repair (MMR) pathway, requiring Msh2 and Pms1. In Schizosaccharomyces pombe mismatches can be repaired by a short-patch repair system, containing nucleotide excision repair (NER) factors. We studied mismatch correction efficiency in cells with inactivated DNA repair nucleases Rad13, Rad2 or Uve1 in MMR proficient and deficient background. Rad13 incises 3' of damaged DNA during NER. Rad2 has a function in the Uve1-dependent repair of DNA damages and in replication. Loss of Rad13 caused a strong reduction of short-patch processing of mismatches formed during meiotic recombination. Mitotic mutation rates were increased, but not to the same extent as in the NER mutant swi10, which is defective in 5' incision. The difference might be caused by an additional role of Rad13 in base excision repair or due to partial redundancy with other 3' endonucleases. Meiotic mismatch repair was not or only slightly affected in rad2 and uve1 mutants. In addition, inactivation of uve1 caused only weak effects on mutation avoidance. Mutation rates were elevated when rad2 was mutated, but not further increased in swi10 rad2 and rad13 rad2 double mutants, indicating an epistatic relationship. However, the mutation spectra of rad2 were different from that of swi10 and rad13. Thus, the function of Rad2 in mutation avoidance is rather independent of NER. rad13, swi10 and rad2, but not uve1 mutants were sensitive to the DNA-damaging agent methyl methane sulphonate. Cell survival was further reduced in the double mutants swi10 rad2, rad13 rad2 and, surprisingly, swi10 rad13. These data confirm that NER and Rad2 act in distinct damage repair pathways and further indicate that the function of Rad13 in repair of alkylated bases is partially independent of NER.     

Nucleotide excision repair: DNA damage recognition and preincision complex assembly

Nucleotide excision repair (NER) is one of the major DNA repair pathways in eukaryotic cells counteracting genetic changes caused by DNA damage. NER removes a wide set of structurally diverse lesions such as pyrimidine dimers arising upon UV irradiation and bulky chemical adducts arising upon exposure to carcinogens or chemotherapeutic drugs. NER defects lead to severe diseases including some forms of cancer. In view of the broad substrate specificity of NER, it is of interest to understand how a certain set of proteins recognizes various DNA lesions in the context of a large excess of intact DNA. This review focuses on DNA damage recognition and following stages resulting in preincision complex assembly, the key and still most unclear steps of NER. The major models of primary damage recognition and preincision complex assembly are considered. The contribution of affinity labeling techniques in study of this process is discussed.     

Insights into protein - DNA interactions, stability and allosteric communications: a computational study of mutSα-DNA recognition complexes

DNA mismatch repair proteins (MMR) maintain genetic stability by recognizing and repairing mismatched bases and insertion/deletion loops mistakenly incorporated during DNA replication, and initiate cellular response to certain types of DNA damage. Loss of MMR in mammalian cells has been linked to resistance to certain DNA damaging chemotherapeutic agents, as well as to increase risk of cancer. Mismatch repair pathway is considered to involve the concerted action of at least 20 proteins. The most abundant MMR mismatch-binding factor in eukaryotes, MutSα, recognizes and initiates the repair of base-base mismatches and small insertion/deletion. We performed molecular dynamics simulations on mismatched and damaged MutSα-DNA complexes. A comprehensive DNA binding site analysis of relevant conformations shows that MutSα proteins recognize the mismatched and platinum cross-linked DNA substrates in significantly different modes. Distinctive conformational changes associated with MutSα binding to mismatched and damaged DNA have been identified and they provide insight into the involvement of MMR proteins in DNA-repair and DNA-damage pathways. Stability and allosteric interactions at the heterodimer interface associated with the mismatch and damage recognition step allow for prediction of key residues in MMR cancer-causing mutations. A rigorous hydrogen bonding analysis for ADP molecules at the ATPase binding sites is also presented. Due to extended number of known MMR cancer causing mutations among the residues proved to make specific contacts with ADP molecules, recommendations for further studies on similar mutagenic effects were made.     

DNA repair: models for damage and mismatch recognition

Maintaining the integrity of the genome is critical for the survival of any organism. To achieve this, many families of enzymatic repair systems which recognize and repair DNA damage have evolved. Perhaps most intriguing about the workings of these repair systems is the actual damage recognition process. What are the chemical characteristics which are common to sites of nucleic acid damage that DNA repair proteins may exploit in targeting sites? Importantly, thermodynamic and kinetic principles, as much as structural factors, make damage sites distinct from the native DNA bases, and indeed, in many cases, these are the features which are believed to be exploited by repair enzymes. Current proposals for damage recognition may not fulfill all of the demands required of enzymatic repair systems given the sheer size of many genomes, and the efficiency with which the genome is screened for damage. Here we discuss current models for how DNA damage recognition may occur and the chemical characteristics, shared by damaged DNA sites, of which repair proteins may take advantage. These include recognition based upon the thermodynamic and kinetic instabilities associated with aberrant sites. Additionally, we describe how small changes in base pair structure can alter also the unique electronic properties of the DNA base pair pi-stack. Further, we describe photophysical, electrochemical, and biochemical experiments in which mismatches and other local perturbations in structure are detected using DNA-mediated charge transport. Finally, we speculate as to how this DNA electron transfer chemistry might be exploited by repair enzymes in order to scan the genome for sites of damage.     

Mismatch repair participates in error-free processing of DNA interstrand crosslinks in human cells

DNA interstrand crosslinks (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. ICLs are induced in DNA by intercalating compounds such as the widely used therapeutic agent psoralen. In bacteria, both nucleotide excision repair (NER) and homologous recombination are required for the repair of ICLs. The processing of ICLs in mammalian cells is not clearly understood. However, it is known that processing can occur by NER, which for psoralen ICLs can be an error-generating process conducive to mutagenesis. We show here that another repair pathway, mismatch repair (MMR), is also involved in eliminating psoralen ICLs in human cells. MMR deficiency renders cells hypersensitive to psoralen ICLs without diminishing their mutagenic potential, suggesting that MMR does not contribute to error-generating repair, and that MMR may represent a relatively error-free mechanism for processing these lesions in human cells. Thus, enhancement of MMR relative to NER may reduce the mutagenesis caused by DNA ICLs in humans.     

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.     

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.     

DNA repair in response to anthracycline-DNA adducts: a role for both homologous recombination and nucleotide excision repair

Doxorubicin, a widely used anthracycline anticancer agent, acts as a topoisomerase II poison but can also form formaldehyde-mediated DNA adducts. This has led to the development of doxorubicin derivatives such as doxoform, which can readily form adducts with DNA. This work aimed to determine which DNA repair pathways are involved in the recognition and possible repair of anthracycline-DNA adducts. Cell lines lacking functional proteins involved in each of the five main repair pathways, mismatch repair (MMR), base excision repair (BER), nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end-joining (NHEJ) were examined for sensitivity to various anthracycline adduct-forming treatments. The treatments used were doxorubicin, barminomycin (a model adduct-forming anthracycline) and doxoform (a doxorubicin-formaldehyde conjugate). Cells with deficiencies in MMR, BER and NHEJ were equally sensitive to adduct-forming treatments compared to wild type cells and therefore these pathways are unlikely to play a role in the repair of these adducts. Some cells with deficiencies in the NER pathway (specifically, those lacking functional XPB, XPD and XPG), displayed tolerance to adducts induced by both barminomycin and doxoform and also exhibited a decreased level of apoptosis in response to adduct-forming treatments. Conversely, two HR deficient cell lines were shown to be more sensitive to barminomycin and doxoform than HR proficient cells, indicating that this pathway is also involved in the repair response to anthracycline-DNA adducts. These results suggest an unusual damage response pathway to anthracycline adducts involving both NER and HR that could be used to optimise cancer therapy for tumours with either high levels of NER or defective HR. Tumours with either of these characteristics would be predicted to respond particularly well to anthracycline-DNA adduct-forming treatments.     

114 Kinetic and thermodynamic basis for damaged bases excision by 8-oxoguanine-DNA glycosylases

DNA glycosylases play the opening act in a highly conserved process for excision of damaged bases from DNA called the base excision repair pathway. DNA glycosylases attend to a wide variety of lesions arising from both endogenous and exogenous factors. The types of damage include alkylation, oxidation, and hydrolysis. A major DNA oxidation product is 8-oxoguanine (8-oxoG), a base with a high mutagenic potential. In bacteria, this lesion is repaired by formamidopyrimidine-DNA glycosylase (Fpg), while in the case of humans this function belongs to 8-oxoG-DNA glycosylase (OGG1). We have attempted a comprehensive characterization of 8-oxoG recognition by DNA glycosylases. First, we have obtained thermodynamic parameters for melting of DNA duplexes containing 8-oxoG in all possible nucleotide contexts. The energy of stacking interactions of 8-oxoG was in strict dependence on 8-oxoG nucleotide environment, which may affect the recognition of damage and the efficiency of eversion of 8-oxoG from DNA helix by glycosylases. Next, we established how the flexibility of DNA context affects damage recognition by these enzymes (Kirpota et al., 2011). Then, we have found that DNA containing 8-oxoG next to a single-strand break provides a good substrate for Fpg, as soon as all structural phosphate residues are maintained. Using site-directed mutagenesis, we have addressed the functions of many previously unstudied amino acid residuess that were predicted to be important for Fpg activity by molecular dynamics simulation and phylogenetic analysis. Of note, many substitutions abolished the excision of 8-oxoG, but did not affect the cleavage efficiency of abasic substrates. Finally, we investigated the contribution of separated structural domains of Fpg to specific enzyme-substrate interaction. Surprisingly, despite the absence of the catalytic domain, C-terminal domain of Fpg possessed a low- residual ability to recognize and cleave abasic substrates. Our study sheds light on mechanism details of Fpg and OGG1 activity, with the ultimate goal of understanding how binding energy can be spent by these enzymes for catalysis.     

Regulation of endonuclease activity in human nucleotide excision repair

Nucleotide excision repair (NER) is a DNA repair pathway that is responsible for removing a variety of lesions caused by harmful UV light, chemical carcinogens, and environmental mutagens from DNA. NER involves the concerted action of over 30 proteins that sequentially recognize a lesion, excise it in the form of an oligonucleotide, and fill in the resulting gap by repair synthesis. ERCC1-XPF and XPG are structure-specific endonucleases responsible for carrying out the incisions 5' and 3' to the damage respectively, culminating in the release of the damaged oligonucleotide. This review focuses on the recent work that led to a greater understanding of how the activities of ERCC1-XPF and XPG are regulated in NER to prevent unwanted cuts in DNA or the persistence of gaps after incision that could result in harmful, cytotoxic DNA structures.     

Crystal structure of UvrB, a DNA helicase adapted for nucleotide excision repair

Nucleotide excision repair (NER) is a highly conserved DNA repair mechanism. NER systems recognize the damaged DNA strand, cleave it on both sides of the lesion, remove and newly synthesize the fragment. UvrB is a central component of the bacterial NER system participating in damage recognition, strand excision and repair synthesis. We have solved the crystal structure of UvrB in the apo and the ATP-bound forms. UvrB contains two domains related in structure to helicases, and two additional domains unique to repair proteins. The structure contains all elements of an intact helicase, and is evidence that UvrB utilizes ATP hydrolysis to move along the DNA to probe for damage. The location of conserved residues and structural comparisons allow us to predict the path of the DNA and suggest that the tight pre-incision complex of UvrB and the damaged DNA is formed by insertion of a flexible beta-hairpin between the two DNA strands.     

Nucleotide excision repair in higher eukaryotes: Mechanism of primary damage recognition

Nucleotide excision repair (NER) is one of the major DNA repair pathways in eukaryotic cells. NER removes structurally diverse lesions such as pyrimidine dimers, arising upon UV irradiation, and bulky chemical adducts, arising upon exposure to carcinogens and some chemotherapeutic drugs. NER defects lead to severe diseases, including some forms of cancer. In view of the broad substrate specificity of NER, it is of interest to study how a certain set of proteins recognizes DNA lesions in contest of a large excess of intact DNA. The review focuses on DNA damage recognition, the key and, as yet, most questionable step of NER. The main models of primary damage recognition and preincision complex assembly are considered. The model of a sequential loading of repair proteins on damaged DNA seems most reasonable in light of the available data.