DNA damage is an important event in the initiation and progression of cancer. DNA is a highly reactive macromolecule that is a target for both physical and chemical agents that alter the base(s) and/or the phosphate backbone. Lesions in DNA at the time of replication may be mutagenic and can lead to cancer or cell death. All organisms have elaborate cellular responses to DNA-damaging agents, including both tolerance and repair mechanisms. A critical component of the cellular response to DNA damage includes the repair pathways dedicated to correcting damage or errors in DNA. Nucleotide excision repair (excision repair)11We use nucleotide excision repair and excision repair as synonyms, as the latter was used for nucleotide excision repair for many years before the discovery of base excision repair and it has the connotation of nucleotide excision repair in common language. We also prefer not to use NER and BER for nucleotide- and base-excision repair, respectively. We find these acronyms and their derivatives, GG-NER, TC-NER, SP-BER, and LP-BER, unnecessary and counterproductive for making the repair field accessible to other biomedical researchers. is a versatile pathway that recognizes and removes a wide spectrum of DNA lesions. In eukaryotes, components of excision repair are also involved in other repair pathways and in various aspects of DNA metabolism. This chapter gives a general discussion of cellular responses to DNA-damaging agents before presenting a detailed discussion of nucleotide excision repair in Escherichia coli and humans as examples of this repair pathway in prokaryotes and eukaryotes. The basic strategy of this essential repair pathway is conserved from E. coli to humans, but the proteins are not conserved and there are some differences in the mechanistic details. DNA repair is modulated by both transcription and condensation into chromatin. Transcription stimulates excision repair both in E. coli and in humans in a process dependent on proteins called transcription-repair coupling factors. The structural organization of chromosomes into densely compacted nucleosomes has a profound effect on DNA repair. This inhibitory effect of chromatin on repair is alleviated by chromatin remodeling and accessibility factors. Excision repair genes have been found in all free-living organisms. A defect in nucleotide excision repair causes extreme ultraviolet (UV) sensitivity in E. coli, Saccharomyces cerevisiae, and other unicellular organisms. In humans, defects in excision repair cause the inherited disease xeroderma pigmentosum (XP) (1). The mechanism of excision repair in S. cerevisiae, which is quite similar to human excision repair, has been reviewed elsewhere (2) and is not discussed here. Archaebacteria represent the third biological kingdom and DNA repair genes are also found in their genomes; surprisingly, excision repair proteins in Archaea may be either prokaryotic- or eukaryotic-like or a combination of the two (3, 4). E. coli-like excision of a 12-13 nucleotide-long damage-containing oligomer has been observed in Methanobacterium thermoautotrophicum (5); other species have not been tested for excision activity. Overview of Cellular Responses to DNA Damage: Ultraviolet (UV) radiation produces cyclobutane pyrimidine dimers and (6-4) photoproducts. Chemical agents that damage DNA are a structurally diverse group of compounds that bind DNA either directly or following metabolic activation to DNA reactive species. These agents include both known carcinogens [e.g., benzo(a)pyrene and acetylaminofluorene] and chemotherapeutic drugs (e.g., cisplatin, nitrogen mustard, and tamoxifen). Bifunctional chemical agents (e.g., psoralen, nitrogen mustard, and mitomycin C) can generate interstrand DNA cross-links that present a unique problem for the cell becuse both strands of the DNA molecule are damaged. Exogenous ionizing radiation and intracellular oxidative metabolism generate reactive oxygen species that damage bases (e.g., oxidized or reduced bases, ring-opening and fragmentation) and generate both single- and double-strand breaks in the DNA. DNA-protein cross-links may be generated by exogenous agents or during certain DNA transactions involving DNA-reactive intermediates. Mismatched bases occur at a low frequency during DNA replication and recombination or as a result of spontaneous or induced base deamination and are mutagenic if not corrected. Under some circumstances cells do not (or cannot) repair DNA damage prior to the start of replication or cell division; in these cases the damage is tolerated. Tolerance mechanisms include the SOS response, translesion synthesis, and the damage checkpoint response. E. coli and other prokaryotes have a tightly regulated physiological response, called the SOS response, that permits cell survival in the presence of DNA damage (6). In the SOS response the expression of over 30 proteins, including a subset of repair proteins (UvrA, UvrB, UvrD), is induced following exposure to damaging agents, thus increasing the repair capacity. The upregulated proteins also include DNA polymerases responsible for lesion bypass (translesion synthesis), a mechanism that avoids replication arrest and cell death. However, translesion polymerases have reduced fidelity relative to replicative polymerases and cell survival is achieved at the cost of elevated mutagenesis; eukaryotes also have error-prone polymerases involved in translesion synthesis (7, 8). There is no evidence for a true SOS response in eukaryotes, but there are suggestions that the DNA damage- and p53-dependent upregulation/stabilization of proteins may function as an SOS response (9). Eukaryotes do have a checkpoint response in which specific proteins sense the presence of DNA damage and signal the cell to stop the cell cycle, presumably allowing time for repair, before proceeding with replication or mitosis or, in the case of extensive damage or abnormally growing cells, the cells are sent to an apoptotic pathway. Mechanistic details of the checkpoint response are not well defined at present, but in humans the process requires damage sensors (e.g., ATM, ATR, Rad17-RFC, and the Rad9-Rad1-Hus1 complex), mediators (e.g., claspin, BRCA1, and MDC1), transducers (e.g., Chk1 and Chk2), and effectors (e.g., p53, Cdk2, and Cdc2) (10). It has been suggested that the bacterial SOS response constitutes a primitive DNA damage checkpoint (11). Both prokaryotic and eukaryotic cells have multiple repair pathways to correct DNA damage or errors of misincorporation. These pathways may be elegantly simple, requiring a single protein, or exquisitely complex, involving multiple steps and the coordinated activity of many proteins. Repair is direct and error-free when the damage is corrected in situ, but repair involving incision of the DNA backbone to excise the damage results in a gapped DNA structure that is filled in by DNA polymerases; thus there is a potential for misincorporation and mutagenesis during excision repair processes. These repair pathways, including direct reversal of damage, base excision repair, and double-strand break repair, have been the subject of other reviews (10, 12-15) and are not discussed here. Overview of Nucleotide Excision Repair: Nucleotide excision repair (excision repair) eliminates a broad spectrum of DNA damage by dual incisions bracketing the lesion. Damage is removed in the form of a 12-13 nucleotide-long oligomer in prokaryotes and in a 24-32 nucleotide-long oligomer in eukaryotes (16, 17) (Fig. 1). Excision repair is composed of three basic steps: (1) damage recognition, (2) dual incisions and release of the excised oligomer, and (3) resynthesis to fill in the gap and ligation (10, 18, 19). Nucleotide excision repair is the primary repair system for bulky DNA adducts such as the cyclobutane pyrimidine dimer (Pyr<>Pyr), (6-4) photoproduct, benzo(a)pyrene-guanine adduct, acetylaminofluorene-guanine (AAF-G), and cisplatin-d(GpG) diadduct. Because humans and other placental mammals lack photolyases, excision repair is the only known mechanism for the removal of sunlight-induced Pyr<>Pyr and (6-4) photoproducts, the most commonly encountered bulky DNA lesions. Additionally, evidence shows that nucleotide excision repair serves as a backup system for the repair of oxidatively damaged and alkylated bases as thymine glycol and 8-oxoguanine are also substrates for the E. coli and human excision nucleases (20-22). In E. coli, dual incisions are accomplished by three proteins (UvrA, UvrB, and UvrC); in humans, 14-15 polypeptides in six repair factors carry out the same task. In contrast to all other repair systems, the prokaryotic and eukaryotic excision repair factors are not evolutionarily related and show no sequence homology to one another. However, the basic strategies for the prokaryotic and eukaryotic excision nucleases are similar and involve sequential steps coordinated via specific interactions between proteins and DNA. First, damage is recognized by an ATP-independent mechanism to form an unstable DNA-protein complex. Then this complex is converted to a stable preincision form by ATPase subunits that hydrolyze ATP and unwind the duplex to create an open structure and to promote formation of more intimate protein-DNA contacts. Finally, the dual incisions are made in a concerted, but asynchronous, manner such that the 3′ incision precedes the 5′ incision. Before presenting a detailed discussion of excision repair in E. coli and human cells, we address the major problem of damage recognition. This is the first and most crucial step in repair and yet, in excision repair, it is the least understood. © 2005 Elsevier Inc. All rights reserved.
