Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis
ReviewBase excision repair in yeast and mammals
Introduction
DNA damage emanates from the inherent chemical instability of nucleic acids, from errors made by polymerase during DNA replication as well as from exposure to DNA damaging agents present in our environment or produced by certain endogenous processes (reviewed in [1], [2]). Such damage has the potential to cause cell death or mutation. All organisms possess a panel of DNA repair mechanisms to repair damaged DNA and in order to maintain viability and genomic stability. Among these DNA repair mechanisms, base excision repair (BER) is one of the most highly conserved.
In general, DNA base damage that causes relatively minor disturbances to the helical DNA structure is repaired by BER. Such damage includes deaminated, oxidized, alkylated and even absent bases (reviewed in [1], [3], [4], [5], [6], [7]). A battery of DNA glycosylases initiate BER by recognizing damaged or abnormal bases and cleaving the glycosylic bond linking the base to the sugar phosphate backbone (Fig. 1). Although all DNA glycosylases cleave glycosylic bonds, they differ in their base substrate specificity and in their reaction mechanisms. Moreover, a subset of DNA glycosylases possess an additional apurinic/apyrimidinic (AP) lyase activity, and one DNA glycosylase, Ogg1, has been shown to have a DNA deoxyribophosphatase (dRPase) activity (Ref. [8] and reviewed in [1], [3]).
It is worth noting that abasic AP sites are themselves a form of DNA damage. In addition to being BER intermediates, AP sites are formed spontaneously and are induced by certain DNA damaging agents. AP sites may be processed in one of two ways. The DNA backbone can be cleaved at abasic sites by an AP endonuclease, resulting in the formation of a 3′-hydroxyl and a 5′-abasic sugar phosphate, deoxyribose phosphate (dRP). Alternatively, the AP site can be cleaved by an AP lyase activity that, as mentioned, is associated with a subset of DNA glycosylases. AP lyases catalyze the formation of a 5′-phosphate and 3′-fragmented deoxyribose (Fig. 1).
In mammalian cells completion of BER following DNA backbone cleavage at an AP site can occur by either short patch BER, in which 1 nucleotide is replaced, or by long patch BER, in which 2–13 nucleotides are replaced. In the former pathway, the 5′-dRP terminus created by AP endonuclease can be removed by the dRPase activity of mammalian polymerase β (Pol β) and the 3′-abasic terminus left by AP lyase can be removed by the 3′-diesterase activity associated with AP endonucleases (Fig. 1). In both cases, the resulting gap can be filled in by Pol β and the remaining DNA strand break sealed by either DNA ligase I or DNA ligase III. The components described here are the required elements of short-patch BER, but it is evident that certain accessory components such as the XRCC1 and XPG proteins enhance DNA repair activity in certain situations, although they are not absolutely required [9], [10]. In long patch BER, extension from the 3′-OH group left by AP endonuclease is catalyzed by either Pol γ or Pol ε and involves displacing the strand containing the 5′-dRP for several nucleotides [11]. The structure-specific flap endonuclease (FEN1) can remove the resulting ‘flap’ structure thus producing a DNA strand break that is ligated by DNA ligase (Fig. 1). In addition to DNA polymerase, FEN1 and DNA ligase, the long-patch BER pathway requires at least two accessory factors, namely proliferating cell nuclear antigen (PCNA) and replication factor-C (RFC) which enhance the activity DNA polymerase γ and ε. Although long patch repair has not yet been demonstrated in yeast, both Saccharomyces cerevisiae and Schizosaccharomyces pombe have homologues to mammalian FEN1, PCNA and RFC [12], [13], [14], [15]).
Due to the marked conservation of BER, model organisms have been particularly valuable for the elucidation of the details of BER. At least seven different DNA glycosylases have been identified, and five of these are known to exist in the budding yeast S. cerevisiae (Table 1). Further, homologues to the two known AP endonuclease types (Exonuclease III-like (Exo III-like) and Endonuclease IV-like (Endo IV)) also exist in S. cerevisiae. Although the fission yeast S. pombe is also a powerful model organism, studies on S. pombe DNA repair and genome sequencing efforts have lagged far behind those in S. cerevisiae. Thus, fewer components of the BER pathway have been identified and characterized and fewer genomic tools are available. Nevertheless, studies of S. pombe BER have also added to our knowledge of BER particularly in terms of interactions with other DNA repair pathways. Here we will review several lines of research using yeast that have furthered understanding of BER in mammals. For more comprehensive reviews of BER see [1], [3], [4], [5], [6], [16].
Section snippets
Sequence similarity among BER enzymes
Sequence similarity among BER enzymes has been used successfully as a cloning strategy (as in the case of certain 8-oxoG DNA glycosylases and AP endonucleases described below). Furthermore, comparisons of similar enzymes from evolutionarily divergent species have highlighted protein structures and amino acid residues that are important for biochemical activity (as in the case of the Helix–hairpin–Helix (HhH) family of DNA glycosylases also described below).
Consequences of imbalanced BER
Studies in yeast have underscored the potentially negative consequences of imbalanced BER. AP endonuclease-deficient apn1 S. cerevisiae have elevated rates of spontaneous mutation and this mutator phenotype is profoundly affected by 3-methyladenine (3MeA) DNA glycosylase levels. The absence of Mag1 substantially suppresses the mutator phenotype, and the overproduction of Mag1 dramatically intensifies the mutator phenotype [41], [42], [43]. Specifically, overexpression of MAG1 in apn1 S.
Interactions between BER and other DNA repair pathways
It has long been known that BER initiated by 3MeA DNA glycosylases is important for providing resistance to the toxicity induced by alkylation damage. Indeed, 3MeA DNA glycosylase-deficient E. coli, S. cerevisiae and mouse embryonic stem cells are sensitive to simple methylating agents such as methyl methanesulfonate (MMS) [52], [53], [54]. Thus, it was somewhat puzzling when disruption of a S. pombe 3MeA DNA glycosylase gene, mag1, did not greatly influence MMS sensitivity [55]. Further study
BER and oxidative damage
In E. coli BER initiated by the Fpg/MutM and MutY glycosylases limits spontaneous mutation that result from misincorporation of adenine opposite 8-oxoG or misincorporation of 8-oxoG opposite adenine [1], [20]. As mentioned above, Fpg/MutM is a bifunctional DNA glycosylase/AP lyase, catalyzing 8-oxoG removal and cleavage of the resulting abasic site. Should Fpg/MutM fail to remove 8-oxoG from DNA, the DNA replication machinery pairs the template 8-oxoG with either cytosine or adenine. If
Dominant negative mutants of mammalian Pol β
As mentioned, mammalian DNA Pol β is involved in short patch BER synthesis. Surprisingly, Pol β gene disruption in mice results in embryonic lethality whereas disruption of the S. cerevisiae Pol β homologue, Pol IV, had no discernible phenotype [75], [76]. However, E. coli and S. cerevisiae have been used as tools to characterize mammalian Pol β. Expression of wildtype Pol β can suppress the temperature-sensitive growth defect (due to impaired lagging strand DNA replication) and the MMS
Concluding remarks
Studying BER in organisms that are easy to genetically manipulate, like S. cerevisiae, S. pombe (and E. coli), has enabled us to rapidly obtain a penetrating view of the in vivo roles of BER, and of its many, often surprising, interactions with other DNA repair pathways. In this review, we discuss the fact that BER intermediates can become substrates for NER, RR and DNA lesion bypass pathways, and how changes in the relative balance between these pathways within the cell can have previously
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Present address: Harvard School of Public Health, 677 Huntington Avenue, Department of Epidemiology, Boston, MA 02115. Tel.: +1-617-432-2262.