Repair of oxidative DNA damage

Cellular genomes suffer extensive damage from exogenous agents and reactive oxygen species formed during normal metabolism. The MutT homologs (MutT/MTH) remove oxidized nucleotide precursors so that they cannot be incorporated into DNA during replication. Among many repair pathways, the base excision repair (BER) pathway is the most important cellular protection mechanism responding to oxidative DNA damage. The 8-oxoG glycosylases (Fpg or MutM/OGG) and the MutY homologs (MutY/MYH) glycosylases along with MutT/MTH protect cells from the mutagenic effects of 8-oxoG, the most stable and deleterious product known caused by oxidative damage to DNA. The key enzymes in the BER process are DNA glycosylases, which remove different damaged bases by cleavage of the N-glycosylic bonds between the bases and the deoxyribose moieties of the nucleotide residues. Biochemical and structural studies have demonstrated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of strated the substrate recognition and reaction mechanism of BER enzymes. Cocrystal structures of several glycosylases show that the substrate base flips out of the sharply bent DNA helix and the minor groove is widened to be accessed by the glycosylases. To complete the repair after glycosylase action, the apurinic/apyrimidinic (AP) site is further processed by an incision step, DNA synthesis, an excision step, and DNA ligation through two alternative pathways. The short-patch BER (1-nucleotide patch size) and long-patch BER (2–6-nucleotide patch size) pathways need AP endonuclease to generate a 3′ hydroxyl group but require different sets of enzymes for DNA synthesis and ligation. Protein-protein interactions have been reported among the enzymes involved in BER. It is possible that the successive players in the repair pathway are assembled in a complex to perform concerted actions. The BER pathways are proposed to protect cells and organisms from mutagenesis and carcinogenesis.     

Chemical approaches toward understanding base excision DNA repair

Despite the importance of DNA repair in protecting the genome, the molecular basis for damage recognition and repair remains poorly understood. In the base excision repair pathway (BER), DNA glycosylases recognize and excise damaged bases from DNA. This review focuses on the recent development of chemical approaches that have been applied to the study of BER enzymes. Several distinctive classes of noncleavable substrate analogs that form stable complexes with DNA glycosylases have recently been designed and synthesized. These analogs have been used for biochemical and structural analyses of protein—DNA complexes involving DNA glycosylases, and for the isolation of a novel DNA glycosylase. An approach to trap covalently a DNA glycosylase-intermediate complex has also been used to elucidate the mechanism of DNA glycosylases.     

Base excision repair

Damage to DNA bases resulting from deamination, oxidation, and alkylation is mainly repaired by base-excision repair. BER is initiated by DNA glycosylases, which recognize damaged bases and excise them from DNA by hydrolyzing the N-glycosidic bond between the base and the sugar phosphate backbone of DNA to generate an abasic site. Different human and E. coli DNA glycosylases have been cloned and characterized, each one with unique substrate specificity. Some of them additionally have AP lyase activity, which enables them to cleave the bond between the sugar and phosphate 3' to the damaged site. BER consist of two repair pathways (short or long) in which one or more nucleotides are introduced respectively. In conclusion, it seems to be likely that BER pathways are essential for genomic repair and stability in living cells.     

Base excision repair and its role in maintaining genome stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10(4) events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

Recent progress in the biology, chemistry and structural biology of DNA glycosylases

Since the discovery in 1974 of uracil DNA glycosylase (UDG), the first member of the family of enzymes involved in base excision repair (BER), considerable progress has been made in the understanding of DNA glycosylases, the polypeptides that remove damaged or mispaired DNA bases from DNA. We also know the enzymes that act downstream of the glycosylases, in the processing of abasic sites, in gap filling and in DNA ligation. This article covers the most recent developments in our understanding of BER, with particular emphasis on the mechanistic aspects of this process, which have been made possible by the elucidation of the crystal structures of several glycosylases in complex with their respective substrates, substrate analogues and products. The biological importance of individual BER pathways is also being appreciated through the inactivation of key BER genes in knockout mouse models.     

Base excision repair in yeast and mammals

Base excision repair (BER), as initiated by at least seven different DNA glycosylases or by enzymes that cleave DNA at abasic sites, executes the repair of a wide variety of DNA damages. Many of these damages arise spontaneously because DNA interacts with the cellular milieu, and so BER profoundly influences spontaneous mutation rates. In addition, BER provides significant protection against the toxic and mutagenic effects of DNA damaging agents present in the external environment, and as such is likely to prevent the adverse health effects of such agents. BER pathways have been studied in a wide variety of organisms (including yeasts) and here we review how these varied studies have shaped our current view of human BER.     

Mechanistic comparisons among base excision repair glycosylases

The mechanisms by which various DNA glycosylases initiate the base excision repair pathways are discussed. Fundamental distinctions are made between "simple glycosylases," that do not form DNA single-strand breaks, and "glycosylases/abasic site lyases," that do form single-strand breaks. Several groupings of BER substrate sites are defined and some interactions between these groupings and glycosylase mechanisms discussed. Two characteristics are proposed to be common among all BER glycosylases: a nucleotide flipping step that serves to expose the scissile glycosyl bond to catalysis, and a glycosylase transition state characterized by substantial tetrahedral character at the base glycosyl atom.     

Base Excision Repair and its Role in Maintaining Genome Stability

For all living organisms, genome stability is important, but is also under constant threat because various environmental and endogenous damaging agents can modify the structural properties of DNA bases. As a defense, organisms have developed different DNA repair pathways. Base excision repair (BER) is the predominant pathway for coping with a broad range of small lesions resulting from oxidation, alkylation, and deamination, which modify individual bases without large effect on the double helix structure. As, in mammalian cells, this damage is estimated to account daily for 10⁴ events per cell, the need for BER pathways is unquestionable. The damage-specific removal is carried out by a considerable group of enzymes, designated as DNA glycosylases. Each DNA glycosylase has its unique specificity and many of them are ubiquitous in microorganisms, mammals, and plants. Here, we review the importance of the BER pathway and we focus on the different roles of DNA glycosylases in various organisms.     

Evolution of Base Excision Repair in Entamoeba histolytica is shaped by gene loss,gene duplication,and lateral gene transfer

During its life cycle, the protist parasite Entamoeba histolytica encounters reactive oxygen and nitrogen species that alter its genome. Base excision repair (BER) is one of the most important pathways for the repair of DNA base lesions. Analysis of the E. histolytica genome revealed the presence of most of the BER components. Surprisingly, this included a gene encoding an apurinic/apyrimidinic (AP) endonuclease that previous studies had assumed was absent. Indeed, our analysis showed that the genome of E. histolytica harbors the necessary genes needed for both short and long-patch BER sub-pathways. These genes include DNA polymerases with predicted 5′-dRP lyase and strand-displacement activities and a sole DNA ligase. A distinct feature of the E. histolytica genome is the lack of several key damage-specific BER glycosylases, such as OGG1/MutM, MDB4, Mag1, MPG, SMUG, and TDG. Our evolutionary analysis indicates that several E. histolytica DNA glycosylases were acquired by lateral gene transfer (LGT). The genes that encode for MutY, AlkD, and UDG (Family VI) are included among these cases. Endonuclease III and UNG (family I) are the only DNA glycosylases with a eukaryotic origin in E. histolytica. A gene encoding a MutT 8-oxodGTPase was also identified that was acquired by LGT. The mixed composition of BER genes as a DNA metabolic pathway shaped by LGT in E. histolytica indicates that LGT plays a major role in the evolution of this eukaryote. Sequence and structural prediction of E. histolytica DNA glycosylases, as well as MutT, suggest that the E. histolytica DNA repair proteins evolved to harbor structural modifications that may confer unique biochemical features needed for the biology of this parasite.     

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.     

Lessons learned from structural results on uracil-DNA glycosylase

Uracil-DNA glycosylase (UDG) functions as a sentry guarding against uracil in DNA. UDG initiates DNA base excision repair (BER) by hydrolyzing the uracil base from the deoxyribose. As one of the best studied DNA glycosylases, a coherent and complete functional mechanism is emerging that combines structural and biochemical results. This functional mechanism addresses the detection of uracil bases within a vast excess of normal DNA, the features of the enzyme that drive catalysis, and coordination of UDG with later steps of BER while preventing the release of toxic intermediates. Many of the solutions that UDG has evolved to overcome the challenges of policing the genome are shared by other DNA glycosylases and DNA repair enzymes, and thus appear to be general.     

Suppression of spontaneous mutagenesis in human cells by DNA base excision–repair

The chemical instability of the covalent structure of DNA, and in vivo exposure of DNA to reactive oxygen species and endogenously produced alkylating agents, has triggered the evolution of several specific DNA repair pathways. A major strategy of repair involves the initial removal of an altered base from DNA by a member of the enzyme family of DNA glycosylases. The currently known enzymes of this type in mammalian cells are reviewed, and the subsequent base excision–repair (BER) steps that achieve restoration of the intact DNA structure are also described. The specific problem of retaining high accuracy in this essentially error-free repair process is discussed.     

XRCC1 interactions with multiple DNA glycosylases: a model for its recruitment to base excision repair

Repair of chemically modified bases in DNA is accomplished through base excision repair (BER). This pathway is initiated by a specific DNA glycosylase that recognizes and excises the altered base to yield an abasic (AP) site. After cleavage of the AP site by APE1, repair proceeds through re-synthesis and ligation steps. In mammalian cells, the XRCC1 protein, essential for the maintenance of genomic stability, is involved in both base excision and single-strand break repair. XRCC1 participates in the first step of BER by interacting with the human DNA glycosylases hOGG1 and NEIL1. To analyze the possibility of a general mechanism involving the interaction of XRCC1 with DNA glycosylases we used XRCC1 to pull-down DNA glycosylases activities from human cell extracts. XRCC1 co-purifies with DNA glycosylase activities capable of excising hypoxanthine and dihydrothymine, in addition to 8-oxoguanine, but not uracil. Biochemical analyses with the purified proteins confirmed the interactions between XRCC1 and MPG, hNTH1 or hNEIL2. Furthermore, XRCC1 stimulates the activities of these enzymes. In vivo localization studies show that after genotoxic treatments these DNA glycosylases can be found associated with XRCC1 foci. Our results support a BER model in which XRCC1 is recruited to the repair of alkylated or oxidized bases by the enzyme recognizing the lesion. XRCC1 would then coordinate the subsequent enzymatic steps and modulate the activities of all the proteins involved.     

5-Hydroxy-5-methylhydantoin DNA lesion, a molecular trap for DNA glycosylases

DNA base-damage recognition in the base excision repair (BER) is a process operating on a wide variety of alkylated, oxidized and degraded bases. DNA glycosylases are the key enzymes which initiate the BER pathway by recognizing and excising the base damages guiding the damaged DNA through repair synthesis. We report here biochemical and structural evidence for the irreversible entrapment of DNA glycosylases by 5-hydroxy-5-methylhydantoin, an oxidized thymine lesion. The first crystal structure of a suicide complex between DNA glycosylase and unrepaired DNA has been solved. In this structure, the formamidopyrimidine-(Fapy) DNA glycosylase from Lactococcus lactis (LlFpg/LlMutM) is covalently bound to the hydantoin carbanucleoside-containing DNA. Coupling a structural approach by solving also the crystal structure of the non-covalent complex with site directed mutagenesis, this atypical suicide reaction mechanism was elucidated. It results from the nucleophilic attack of the catalytic N-terminal proline of LlFpg on the C5-carbon of the base moiety of the hydantoin lesion. The biological significance of this finding is discussed.     

Transient protein–protein complexes in base excision repair

Abstract

Transient protein–protein complexes are of great importance for organizing multiple enzymatic reactions into productive reaction pathways. Base excision repair (BER), a process of critical importance for maintaining genome stability against a plethora of DNA-damaging factors, involves several enzymes, including DNA glycosylases, AP endonucleases, DNA polymerases, DNA ligases and accessory proteins acting sequentially on the same damaged site in DNA. Rather than being assembled into one stable multisubunit complex, these enzymes pass the repair intermediates between them in a highly coordinated manner. In this review, we discuss the nature and the role of transient complexes arising during BER as deduced from structural and kinetic data. Almost all of the transient complexes are DNA-mediated, although some may also exist in solution and strengthen under specific conditions. The best-studied example, the interactions between DNA glycosylases and AP endonucleases, is discussed in more detail to provide a framework for distinguishing between stable and transient complexes based on the kinetic data.

Communicated by Ramaswamy H. Sarma     

Suppression of spontaneous mutagenesis in human cells by DNA base excision-repair

The chemical instability of the covalent structure of DNA, and in vivo exposure of DNA to reactive oxygen species and endogenously produced alkylating agents, has triggered the evolution of several specific DNA repair pathways. A major strategy of repair involves the initial removal of an altered base from DNA by a member of the enzyme family of DNA glycosylases. The currently known enzymes of this type in mammalian cells are reviewed, and the subsequent base excision-repair (BER) steps that achieve restoration of the intact DNA structure are also described. The specific problem of retaining high accuracy in this essentially error-free repair process is discussed.     

The mechanism of base excision repair in Chlamydiophila pneumoniae

Repair of damaged DNA is of great importance in maintaining genome integrity, and there are several pathways for repair of damaged DNA in almost all organisms. Base excision repair (BER) is a main process for repairing DNA carrying slightly damaged bases. Several proteins are required for BER; these include DNA glycosylases, AP endonuclease, DNA polymerase, and DNA ligase. In some bacteria the single-stranded specific exonuclease, RecJ, is also involved in BER. In this research, six Chlamydiophila pneumoniae (C. pneumoniae) genes, encoding uracil DNA glycosylase (CpUDG), endonuclease IV (CpEndoIV), DNA polymerase I (CpDNApolI), endonuclease III (CpEndoIII), single-stranded specific exonuclease RecJ (CpRecJ), and DNA ligase (CpDNALig), were inserted into the expression vector pET28a. All proteins, except for CpDNALig, were successfully expressed in E. coli, and purified proteins were characterized in vitro. C. pneumoniae BER was reconstituted in vitro with CpUDG, CpEndoIV, CpDNApolI and E. coli DNA ligase (EcDNALig). After uracil removal by CpUDG, the AP site could be repaired by two BER pathways that involved in the replacement of either one (short patch BER) or multiple nucleotides (long patch BER) at the lesion site. CpEndoIII promoted short patch BER via its 5'-deoxyribophosphodiesterase (5'-dRPase) activity, while CpRecJ had little effect on short patch BER. The flap structure generated during DNA extension could be removed by the 5'-exonuclease activity of CpDNApolI. Based on these observations, we propose a probable mechanism for BER in C. pneumoniae.