Base Excision Repair of DNA: Glycosylases

The review considers the role of base excision repair in maintaining the constancy of genetic information in the cell. The genetic control and biochemical mechanism are described for the first stage of base excision repair, which is catalyzed by specific enzymes, DNA glycosylases.__________Translated from Genetika, Vol. 41, No. 6, 2005, pp. 725–735.Original Russian Text Copyright © 2005 by Korolev.     

Enzymatic repair of an expanded genetic information system

The excision repair machinery of a thermophilic bacterium has been shown to recognize and repair an expanded genetic base pair. Native Thermus aquaticus DNA polymerase will remove a mispaired natural base and replace it with a non-natural base to form an expanded base pair. In addition, DNA ligase will recognize a nick formed by polymerase between two non-natural base pairs and covalently attach the two strands, thus demonstrating complete repair of a bifurcated base-paired model duplex. These results add evidence to the idea that the cellular replication and repair machinery of an organism containing an expanded genetic alphabet could recognize and properly repair a site containing consecutive unnatural bases.     

XRCC1 and DNA strand break repair

DNA single-strand breaks can arise indirectly, as normal intermediates of DNA base excision repair, or directly from damage to deoxyribose. Because single-strand breaks are induced by endogenous reactive molecules such as reactive oxygen species, these lesions pose a continuous threat to genetic integrity. XRCC1 protein plays a major role in facilitating the repair of single-strand breaks in mammalian cells, via an ability to interact with multiple enzymatic components of repair reactions. Here, the protein-protein interactions facilitated by XRCC1, and the repair processes in which these interactions operate, are reviewed. Models for the repair of single-strand breaks during base excision repair and at direct breaks are presented.     

A review of recent experiments on step-to-step "hand-off" of the DNA intermediates in mammalian base excision repair pathways

The current "working model" for mammalian base excision repair involves two sub-pathways termed single-nucleotide base excision repair and long patch base excision repair that are distinguished by their repair patch sizes and the enzymes/co-factors involved. These base excision repair sub-pathways are designed to sequester the various DNA intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the base excision repair intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In this review, we explore the question of whether there is an actual step-to-step "hand-off" of the DNA intermediates during base excision repair in vitro. The results show that when base excision repair enzymes are pre-bound to the initial single-nucleotide base excision repair intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase beta and then to DNA ligase. In the long patch base excision repair sub-pathway, where the 5'-end of the incised strand is blocked, the intermediate after polymerase beta gap filling is not channeled from polymerase beta to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules.     

A review of recent experiments on step-to-step “hand-off” of the DNA intermediates in mammalian base excision repair pathways

The current “working model” for mammalian base excision repair involves two sub-pathways termed single-nucleotide base excision repair and long patch base excision repair that are distinguished by their repair patch sizes and the enzymes/co-factors involved. These base excision repair sub-pathways are designed to sequester the various DNA intermediates, passing them along from one step to the next without allowing these toxic molecules to trigger cell cycle arrest, necrotic cell death, or apoptosis. Although a variety of DNA-protein and protein-protein interactions are known for the base excision repair intermediates and enzymes/co-factors, the molecular mechanisms accounting for step-to-step coordination are not well understood. In this review, we explore the question of whether there is an actual step-to-step “hand-off” of the DNA intermediates during base excision repair in vitro. The results show that when base excision repair enzymes are pre-bound to the initial single-nucleotide base excision repair intermediate, the DNA is channeled from apurinic/apyrimidinic endonuclease 1 to DNA polymerase β and then to DNA ligase. In the long patch base excision repair sub-pathway, where the 5′-end of the incised strand is blocked, the intermediate after polymerase β gap filling is not channeled from polymerase β to the subsequent enzyme, flap endonuclease 1. Instead, flap endonuclease 1 must recognize and bind to the intermediate in competition with other molecules.     

Alternative Excision Repair Pathways

Alternative excision repair (AER) is a category of excision repair initiated by a single nick, made by an endonuclease, near the site of DNA damage, and followed by excision of the damaged DNA, repair synthesis, and ligation. The ultraviolet (UV) damage endonuclease in fungi and bacteria introduces a nick immediately 5′ to various types of UV damage and initiates its excision repair that is independent of nucleotide excision repair (NER). Endo IV-type apurinic/apyrimidinic (AP) endonucleases from Escherichia coli and yeast and human Exo III-type AP endonuclease APEX1 introduce a nick directly and immediately 5′ to various types of oxidative base damage besides the AP site, initiating excision repair. Another endonuclease, endonuclease V from bacteria to humans, binds deaminated bases and cleaves the phosphodiester bond located 1 nucleotide 3′ of the base, leading to excision repair. A single-strand break in DNA is one of the most frequent types of DNA damage within cells and is repaired efficiently. AER makes use of such repair capability of single-strand breaks, removes DNA damage, and has an important role in complementing BER and NER.NER and base excision repair (BER) are the major excision repair pathways present in almost all organisms. In NER, dual incisions are introduced, the damaged DNA between the incised sites is then removed, and DNA synthesis fills the single-stranded gap, followed by ligation. In BER, an AP site, formed by depurination or created by a base damage-specific DNA glycosylase, is recognized by an AP endonuclease that introduces a nick immediately 5′ to the AP site, followed by repair synthesis, removal of the AP site, and final ligation. Besides these two fundamental excision repair systems, investigators have found another category of excision repair—AER—an example of which is the excision repair of UV damage, initiated by an endonuclease called UV damage endonuclease (UVDE). UVDE introduces a single nick immediately 5′ to various types of UV lesions as well as other types of base damage, and this nick leads to the removal of the lesions by an AER process designated as UVDE-mediated excision repair (UVER or UVDR). Genetic analysis in Schizosaccharomyces pombe indicates that UVER provides cells with an extremely rapid removal of UV lesions, which is important for cells exposed to UV in their growing phase.Endo IV–type AP endonucleases from Escherichia coli and budding yeast and the Exo III–type human AP endonuclease APEX1 are able to introduce a nick at various types of oxidative base damage and initiate a form of excision repair that has been designated as nucleotide incision repair (NIR). Endonuclease V (ENDOV) from bacteria to humans recognizes deaminated bases, introduces a nick 1 nucleotide 3′ of the base, and leads to excision repair initiated by the nick. These endonucleases introduce a single nick near the DNA-damage site, leaving 3′-OH termini, and initiate repair of both the DNA damage and the nick. The mechanisms of AER may be similar to those of single-strand break (SSB) repair or BER except for the initial nicking process. However, how DNA damage is recognized determines the repair process within the cell. This article discusses the mechanisms and functional roles of AER. We begin with AER of UV damage, because genetic analysis has shown functional differences between this AER and NER in S. pombe.     

Role of base excision repair in maintaining the genetic and epigenetic integrity of CpG sites

Cytosine methylation at CpG dinucleotides is a central component of epigenetic regulation in vertebrates, and the base excision repair (BER) pathway is important for maintaining both the genetic stability and the methylation status of CpG sites. This perspective focuses on two enzymes that are of particular importance for the genetic and epigenetic integrity of CpG sites, methyl binding domain 4 (MBD4) and thymine DNA glycosylase (TDG). We discuss their capacity for countering C to T mutations at CpG sites, by initiating base excision repair of G·T mismatches generated by deamination of 5-methylcytosine (5mC). We also consider their role in active DNA demethylation, including pathways that are initiated by oxidation and/or deamination of 5mC.     

Polynucleotide kinase/phosphatase, Pnk1, is involved in base excision repair in Schizosaccharomyces pombe

We previously reported that Schizosaccharomyces pombe pnk1 cells are more sensitive than wild-type cells to γ-radiation and camptothecin, indicating that Pnk1 is required for DNA repair. Here, we report that pnk1pku70 and pnk1rhp51 double mutants are more sensitive to γ-radiation than single mutants, from which we infer that Pnk1's primary role is independent of either homologous recombination or non-homologous end joining mechanisms. We also report that pnk1 cells are more sensitive than wild-type cells to oxidizing and alkylating agents, suggesting that Pnk1 is involved in base excision repair. Mutational analysis of Pnk1 revealed that the DNA 3'-phosphatase activity is necessary for repair of DNA damage, whereas the 5'-kinase activity is dispensable. A role for Pnk1 in base excision repair is supported by genetic analyses which revealed that pnk1apn2 is synthetically lethal, suggesting that Pnk1 and Apn2 may function in parallel pathways essential for the repair of endogenous DNA damage. Furthermore, the nth1pnk1apn2 and tdp1pnk1apn2 triple mutants are viable, implying that single-strand breaks with 3'-blocked termini produced by Nth1 and Tdp1 contribute to synthetic lethality. We also examined the sensitivity to methyl methanesulfonate of all single and double mutant combinations of nth1, apn2, tdp1 and pnk1. Together, our results support a model where Tdp1 and Pnk1 act in concert in an Apn2-independent base excision repair pathway to repair 3'-blocked termini produced by Nth1; and they also provide evidence that Pnk1 has additional roles in base excision repair.     

Cell cycle regulation of DNA repair in normal and repair deficient human cells

The regulation of nucleotide excision repair and base excision repair by normal and repair deficient human cells was determined. Synchronous cultures of WI-38 normal diploid fibroblasts and Xeroderma pigmentosum fibroblasts (complementation group D) (XP-D) were used to investigate whether DNA repair pathways were modulated during the cell cycle. Two criteria were used: (1) unscheduled DNA synthesis (UDS) in the presence of hydroxyurea (HU) after exposure to UV light or after exposure to N-acetoxy-acetylaminofluorene (N-AcO-AAF) to quantitate nucleotide excision repair or UDS after exposure to methylmethane sulfonate (MMS) to measure base excision repair; (2) repair replication into parental DNA in the absence of HU after exposure to UV light. Nucleotide excision repair after UV irradiation was induced in WI-38 fibroblasts during the cell cycle reaching a maximum in cultures exposed 14–15 h after cell stimulation. Similar results were observed after exposure to N-AcO-AAF. DNA repair was increased 2–4-fold after UV exposure and was increased 3-fold after N-AcO-AAF exposure. In either instance nucleotide excision repair was sequentially stimulated prior to the enhancement of base excision repair which was stimulated prior to the induction of DNA replication. In contrast XP-D failed to induce nucleotide excision repair after UV irradiation at any interval in the cell cycle. However, base excision repair and DNA replication were stimulated comparable to that enhancement observed in WI-38 cells. The distinctive induction of nucleotide excision repair and base excision repair prior to the onset of DNA replication suggests that separate DNA repair complexes may be formed during the eucaryotic cell cycle.     

Activities involved in base excision repair of bacteriophage T4 and lambda DNA in vivo

Summary The in vivo excision repair functions of Escherichia coli exonuclease III and 3-methyladenine DNA glycosylase I, and bacteriophage T4 pyrimidine dimer-DNA glycosylase were investigated. Following exposure of bacteriophage T4 or lambda to methyl methanesulfonate or ultraviolet irradiation, survival was determined by plating on E. coli have various genetic backgrounds. Although exonuclease III was shown to participate in base excision repair initiated by 3-methyladenine DNA glcosylase I, it had no detectable role in base excision repair initiated by the T4 pyrimidine dimer-DNA glycosylase. Despite its 3 apurinic/apyrimidinic endonuclease activity in vitro, T4 pyrimidine dimer-DNA glycosylase, even in large quantities, did not complement mutants defective in exonuclease III in the repair of apurinic sites generated by 3-methyladenine DNA glycosylase I in vivo.     

DNA base excision repair of uracil residues in reconstituted nucleosome core particles

The human base excision repair machinery must locate and repair DNA base damage present in chromatin, of which the nucleosome core particle is the basic repeating unit. Here, we have utilized fragments of the Lytechinus variegatus 5S rRNA gene containing site-specific U:A base pairs to investigate the base excision repair pathway in reconstituted nucleosome core particles in vitro. The human uracil-DNA glycosylases, UNG2 and SMUG1, were able to remove uracil from nucleosomes. Efficiency of uracil excision from nucleosomes was reduced 3- to 9-fold when compared with naked DNA, and was essentially uniform along the length of the DNA substrate irrespective of rotational position on the core particle. Furthermore, we demonstrate that the excision repair pathway of an abasic site can be reconstituted on core particles using the known repair enzymes, AP-endonuclease 1, DNA polymerase beta and DNA ligase III. Thus, base excision repair can proceed in nucleosome core particles in vitro, but the repair efficiency is limited by the reduced activity of the uracil-DNA glycosylases and DNA polymerase beta on nucleosome cores.     

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.     

Genomic approaches to DNA repair and mutagenesis

DNA damage is a constant threat to cells, causing cytotoxicity as well as inducing genetic alterations. The steady-state abundance of DNA lesions in a cell is minimized by a variety of DNA repair mechanisms, including DNA strand break repair, mismatch repair, nucleotide excision repair, base excision repair, and ribonucleotide excision repair. The efficiencies and mechanisms by which these pathways remove damage from chromosomes have been primarily characterized by investigating the processing of lesions at defined genomic loci, among bulk genomic DNA, on episomal DNA constructs, or using in vitro substrates. However, the structure of a chromosome is heterogeneous, consisting of heavily protein-bound heterochromatic regions, open regulatory regions, actively transcribed genes, and even areas of transient single stranded DNA. Consequently, DNA repair pathways function in a much more diverse set of chromosomal contexts than can be readily assessed using previous methods. Recent efforts to develop whole genome maps of DNA damage, repair processes, and even mutations promise to greatly expand our understanding of DNA repair and mutagenesis. Here we review the current efforts to utilize whole genome maps of DNA damage and mutation to understand how different chromosomal contexts affect DNA excision repair pathways.     

Resistance to Nucleotide Excision Repair of Bulky Guanine Adducts Opposite Abasic Sites in DNA Duplexes and Relationships between Structure and Function

The nucleotide excision repair of certain bulky DNA lesions is abrogated in some specific non-canonical DNA base sequence contexts, while the removal of the same lesions by the nucleotide excision repair mechanism is efficient in duplexes in which all base pairs are complementary. Here we show that the nucleotide excision repair activity in human cell extracts is moderate-to-high in the case of two stereoisomeric DNA lesions derived from the pro-carcinogen benzo[a]pyrene (cis- and trans-B[a]P-N ²-dG adducts) in a normal DNA duplex. By contrast, the nucleotide excision repair activity is completely abrogated when the canonical cytosine base opposite the B[a]P-dG adducts is replaced by an abasic site in duplex DNA. However, base excision repair of the abasic site persists. In order to understand the structural origins of these striking phenomena, we used NMR and molecular spectroscopy techniques to evaluate the conformational features of 11mer DNA duplexes containing these B[a]P-dG lesions opposite abasic sites. Our results show that in these duplexes containing the clustered lesions, both B[a]P-dG adducts adopt base-displaced intercalated conformations, with the B[a]P aromatic rings intercalated into the DNA helix. To explain the persistence of base excision repair in the face of the opposed bulky B[a]P ring system, molecular modeling results suggest how the APE1 base excision repair endonuclease, that excises abasic lesions, can bind productively even with the trans-B[a]P-dG positioned opposite the abasic site. We hypothesize that the nucleotide excision repair resistance is fostered by local B[a]P residue—DNA base stacking interactions at the abasic sites, that are facilitated by the absence of the cytosine partner base in the complementary strand. More broadly, this study sets the stage for elucidating the interplay between base excision and nucleotide excision repair in processing different types of clustered DNA lesions that are substrates of nucleotide excision repair or base excision repair mechanisms.     

Short-patch correction of C/C mismatches in human cells

We examined whether the human nucleotide excision repair complex, which is specialized on the removal of bulky DNA adducts, also displays a correcting activity on base mismatches. The cytosine/cytosine (C/C) lesion was used as a model substrate to monitor the correction of base mismatches in human cells. Fibroblasts with different repair capabilities were transfected with shuttle vectors that contain a site-directed C/C mismatch in the replication origin, accompanied by an additional C/C mismatch in one of the flanking sequences that are not essential for replication. Analysis of the vector progeny obtained from these doubly modified substrates revealed that C/C mismatches were eliminated before DNA synthesis not only in the repair-proficient background, but also when the target cells carried a genetic defect in long-patch mismatch repair, in nucleotide excision repair, or when both pathways were deleted. Furthermore, cells deficient for long-patch mismatch repair as well as a cell line that combines mismatch and nucleotide excision repair defects were able to correct multiple C/C mispairs, placed at distances of 21–44 nt, in an independent manner, such that the removal of each lesion led to individual repair patches. These results support the existence of a concurrent short-patch mechanism that rectifies C/C mismatches.     

Regulation of DNA repair in serum-stimulated xeroderma pigmentosum cells

The regulation of DNA repair during serum stimulation of quiescent cells was examined in normal human cells, in fibroblasts from three xeroderma pigmentosum complementation groups (A, C, and D), in xeroderma pigmentosum variant cells, and in ataxia telangiectasia cells. The regulation of nucleotide excision repair was examined by exposing cells to ultraviolet irradiation at discrete intervals after cell stimulation. Similarly, base excision repair was quantitated after exposure to methylmethane sulfonate. WI-38 normal human diploid fibroblasts, xeroderma pigmentosum variant cells, as well as ataxia telangiectasia cells enhanced their capacity for both nucleotide excision repair and for base excision repair prior to their enhancement of DNA synthesis. Further, in each cell strain, the base excision repair enzyme uracil DNA glycosylase was increased prior to the induction of DNA polymerase using the identical cells to quantitate each activity. In contrast, each of the three xeroderma complementation groups that were examined failed to increase their capacity for nucleotide excision repair above basal levels at any interval examined. This result was observed using either unscheduled DNA synthesis in the presence of 10 mM hydroxyurea or using repair replication in the absence of hydroxyurea to quantitate DNA repair. However, each of the three complementation groups normally regulated the enhancement of base excision repair after methylmethane sulfonate exposure and each induced the uracil DNA glycosylase prior to DNA synthesis. These results suggest that there may be a relationship between the sensitivity of xeroderma pigmentosum cells from each complementation group to specific DNA damaging agents and their inability to regulate nucleotide excision repair during cell stimulation.     

How does a cell repair damaged DNA?

DNA in living cells is constantly subjected to different chemical and physical factors of the environment and to cell metabolites. Some changes altering DNA structure occur spontaneously. This raises the potential danger of harmful mutations that could be transmitted to offspring. To avoid the danger of mutations and changing genetic information, a cell is capable to switch on multiple mechanisms of DNA repair that remove damage and restore native structure. In many cases, removal of the same damage may involve several alternative pathways; this is very important for DNA repair under the most unfavorable conditions. This review summarizes data about all known mechanisms of eukaryotic DNA repair including excision repair (base excision repair and nucleotide excision repair), mismatch repair, repair of double-strand breaks, and cross-link repair. Special attention is given to the regulation of excision repair by different proteins—proliferating cell nuclear antigen (PCNA), p53, and proteasome. The review also highlights problem of bypassing irremovable lesions in DNA.Translated from Biokhimiya, Vol. 70, No. 3, 2005, pp. 341–359.Original Russian Text Copyright © 2005 by Sharova.     

My Journey to DNA Repair

I completed my medical studies at the Karolinska Institute in Stockholm but have always been devoted to basic research. My longstanding interest is to understand fundamental DNA repair mechanisms in the fields of cancer therapy, inherited human genetic disorders and ancient DNA. I initially measured DNA decay, including rates of base loss and cytosine deamination. I have discovered several important DNA repair proteins and determined their mechanisms of action. The discovery of uracil-DNA glycosylase defined a new category of repair enzymes with each specialized for different types of DNA damage. The base excision repair pathway was first reconstituted with human proteins in my group. Cell-free analysis for mammalian nucleotide excision repair of DNA was also developed in my laboratory. I found multiple distinct DNA ligases in mammalian cells, and led the first genetic and biochemical work on DNA ligases Ⅰ, and Ⅳ. I discovered the mammalian exonucleases DNase Ⅲ (TREX1) and IV (FEN1). Interestingly, expression of TREX1 was altered in some human autoimmune diseases. I also showed that the mutagenic DNA adduct O6-methylguanine (O6 mG) is repaired without removing the guanine from DNA, identifying a surprising mechanism by which the methyl group is transferred to a residue in the repair protein itself. A further novel process of DNA repair discovered by my research group is the action of AlkB as an iron-dependent enzyme carrying out oxidative demethylation.