Base excision repair in a network of defence and tolerance

Survival of a species depends on balanced generation of genetic variation but at the same time on the protection of the genome from changes that cause disease and fitness reduction. DNA repair pathways limit mutations but do not totally eliminate them. In fact, some DNA repair pathways are error prone. DNA repair thus has a central function not only in protecting the genome, but also in the generation of genetic diversity. Expression of DNA repair proteins is subject to a delicate balance where both too few and too many of a type may result in increased cytotoxicity and/or mutation (1–4). DNA repair is integrated with transcription, replication, cell cycle control and apoptosis in complex networks (5), a full discussion of which is beyond the scope of this article, and our abilities. While DNA repair is an ancient and conserved defence mechanism, various pathways, and additions to basic pathways, have evolved during different time periods (6). Furthermore, the pathways have overlapping specificities and function as back-up systems for each other. Thus, cytotoxic and mutagenic abasic sites (AP sites) may be dealt with by the relatively accurate mechanisms nucleotide excision repair (NER) (7,8), base excision repair (BER) and recombination repair, as well as by highly error-prone translesion DNA synthesis (TLS) (Figure 1; 9). In addition, different enzymes may substitute for each other in a specific pathway, or variants of a pathway. Conditions that govern the selection of a pathway in each case are not well understood. Furthermore, components of DNA repair systems, such as error-prone DNA polymerases, may also cause untargeted mutations at sites where no apparent damage is present. In this commentary we will address functional aspects of BER and different complementary functions in the light of recent discoveries with regard to mutations, cancer, evolution and ageing. In summary, recent results from different DNA glycosylase-deficient mice have failed to demonstrate strongly increased mutation rates, increased cancer frequencies or other severely altered phenotypes. This may be due to overlap in functions between DNA glycosylases, as well as repair by alternative pathways. There is evidence that the BER pathway is essential, since deficiencies in the common steps downstream of the DNA glycosylase step are embryonic lethal (10). However, these proteins may have additional functions in development and the evidence is therefore not yet conclusive. The minor effects of deficiencies in DNA glycosylases may seem surprising, given the strong conservation of these enzymes. Possibly, deficiency of each of the enzymes may reduce fitness to an extent that, although small, results in negative selection such that glycosylase mutations are very infrequently observed in populations. We speculate that DNA glycosylases have as one major function to protect the long-term integrity of the genome over several generations. DNA is inherently unstable and decays even in the absence of exogenous challenge from DNA-reactive chemicals and radiation (11,12). By convention, DNA damage has been categorized as being of spontaneous origin or environmentally induced. However, in some cases this separation is not distinct. For example, polyunsaturated fatty acids, giving rise to DNA-reactive lipid peroxidation products, are natural components of cellular membranes, but their amount in the cell depends on the quantity ingested. In addition, the formation of lipid peroxidation products may be modulated by ingested antioxidants. BER is generally believed to constitute the primary defence against lesions formed by endogenous DNA-damaging agents. Figure 2 shows some examples of major base modifications repaired by BER. Water and reactive oxygen species are probably the main endogenous reactants damaging DNA in cells. Water attacks electrophilic centres to generate a number of hydrolysis products. The quantitatively most important arise from hydrolysis of the N-glycosidic bond of purines, resulting in loss of ~10 000 purines/human genome/day (13,14). Pyrimidines are lost at 5% of the rate of purines (11). The resulting AP sites are highly cytotoxic and mutagenic and, if left unrepaired, may rearrange to generate single-strand breaks. Hydrolytic deamination of exocyclic amino groups in cytosine and 5-methylcytosine results in generation of uracil and thymine, respectively, both mismatched to guanine. Similarly, adenine may deaminate to hypoxanthine and guanine to xanthine. The estimated rate of cytosine deamination ranges from 100 to 500 events/cell/day and will result in C→T transition mutations unless corrected by repair processes (15–17). Oxidation of DNA by reactive oxygen species is the other major source of spontaneous DNA damage. Reactive oxygen species are formed as by-products from oxidative metabolism and from γ-irradiation. DNA-reactive fatty acid radicals, aldehydes and other compounds are formed during lipid peroxidation and cause various types of damage, including etheno adducts of pyrimidines and purines. Oxidation of bases occurs mainly at electrophilic carbon centres. Major products are the highly mutagenic 8-hydroxyguanine and ring-opened forms of purines (formamidopyrimidines) and pyrimidine glycols, all of which are cytotoxic and mutagenic (11,12,18–20). Oxidative attack on the DNA backbone may result in the generation of single-strand breaks, which are recombinogenic, cytotoxic and mutagenic. The rate of formation and endogenous levels of oxidative DNA damage in living cells are difficult to measure, but it is clear that oxidation reactions collectively constitute major sources of DNA damage (12,21). Several endogenous products of normal metabolism have been demonstrated or suspected to damage DNA. In addition to lipid peroxidation products and oxidants, these include alkylating agents, oestrogens, chlorinating agents, reactive nitrogen species, glycoxidation products, heme precursors and amino acids (22). The possible significance of all these agents, as well as of the long list of environmental agents (23), is beyond the scope of the present paper. The present review will concentrate on functional aspects of BER. For discussions of more comprehensive biochemical and structural aspects of DNA glycosylases, other recent reviews should be consulted (24–31). The mechanism of BER involves a damage-specific step performed by one of the DNA glycosylases (Table I) followed by damage-general steps involving the sequential action of different proteins which correct DNA by template-directed insertion of one or a few nucleotides, starting at the damaged site (Figure 3). Damage recognition and binding vary with different enzymes, but base flipping prior to excision appears to be a common trait. For monofunctional glycosylases hydrolysis of the glycosylic bond involves nucleophilic attack by an activated water molecule, in turn activated by the caboxyl side chain of an Asp residue. For bifunctional DNA glycosylases the nucleophile is an activated amino group of a Lys residue activated by an Asp residue. The amino group forms a Schiff base with C1' followed by β-elimination to cleave the DNA strand at the 3′-side of deoxyribose (lyase activity). The human monofunctional glycosylases include uracil-DNA glycosylase (UNG) (32–34), thymine/uracil mismatch glycosylase (TDG) (35), SMUG1 (36) and MBD4 (37,38), which have uracil as a common substrate but have generally narrow substrate specificities. In contrast, the monofunctional methylpurine-DNA glycosylase (MPG), also referred to as ANPG or AAG (39,40), removes a wide range of damaged purine bases and, surprisingly, may also remove undamaged purines at low rates (2). The bifunctional glycosylases include hOGG1 (41), which removes oxidized purines, and hNTH1 (42,43), which removes oxidized pyrimidines. The human MutY homologue, hMYH1 (44), removes adenine from 8-oxoguanine/A mismatches and harbours only weak, if any, lyase activity (45). The major AP endonuclease, HAP1 (also referred to as APE1 and Ref1), which is at the intersection of all the BER pathways, introduces a nick in the backbone 5′ of the AP site, thereby generating a 3′-OH end as a substrate for DNA polymerases (12). When BER is initiated by bifunctional glycosylases, the additional action of HAP1 results in a 1 by 3′-OH and BER is initiated by the bifunctional glycosylases since the 1 is relatively to synthesis BER is initiated by monofunctional glycosylases, HAP1 a nick with a 3′-OH and a end When BER is initiated by monofunctional glycosylases it by the or the pathway In the pathway DNA is with HAP1 to 1 The is by the activity of which involves as the Schiff base nucleophile in the of this Several of evidence that the BER pathway is between and of all BER in human cells that or substitute for with regard to DNA synthesis no back-up for the activity and this the to embryonic in mice of the single-strand by DNA in complex with the the repair pathway BER involves the synthesis of at the damaged of only 2 is most and the is therefore This pathway is in cases where the is to excision by synthesis a from which the be as of an synthesis be performed by in addition to or BER not but is cell In contrast, repair synthesis by or is on and BER by at the and activity to the the is to DNA cells to be in BER but in or is believed to be the main in the pathway with and is for DNA synthesis by or in other repair pathways and may but significance in BER DNA and the nick but since the it is that DNA is since it with of is to where it has to that be for repair and with and This the that BER be mainly is also that is between the repair pathways for common of the repair are such as DNA glycosylases and hNTH1 In contrast, is at levels the cell cycle is not present in and it may therefore seem that it is in BER to the may limit to in the cell as has been for the between and and and is to speculate that some may in thus repair a different at these of a lesions may be repaired by different pathways, on the of the of BER deficiency have at been have given results different from Several DNA glycosylases, as well as other BER proteins and proteins in other pathways, have been this for comprehensive on genetic in and it that mutation frequencies and, as a cancer at a relatively as do most mismatch repair mice (10). The of mice in a DNA glycosylase mice in the major activity for of mice and not demonstrate increased cancer Furthermore, mice no in spontaneous mutation and only a in mutation to agents is which mutation frequencies in the but do not seem to cancer at a rate do clear of other disease mice an to of spontaneous mutation in some in these other at mutation frequencies of and and a of altered The of spontaneous mutations as in the in mice shows in C→T in but not in of other that is also in mice of a and between and mutation from the of the of the DNA glycosylases in protection of integrity appears to be in cells with and However, cell from these mice to at some of the biochemical with regard to to DNA-damaging agents of and of uracil In to the glycosylases, deficiencies in the steps are embryonic lethal (10). for this has been that is an of repair by the glycosylases, in cytotoxic AP sites and be that of the major glycosylases in these at in the lethal at the of our these have yet to be is that these are lethal not of BER but due to the of and in pathways. The of HAP1 harbours a function that in of a number of in to oxidative By of HAP1 that have lost function but the or one should be to function or AP activity is for during appears to be the activity for of in cells and this may be the cause of and embryonic of mice However, is also evidence that is in recombination and, one that the of this is due to function in BER. DNA is with and, cells very of this activity are in the step in BER development in cells For DNA is not lethal has been that strand be BER as of a and the to these be the cause of of mice The of BER in development on absence of the conserved and of a in that and of DNA DNA for initiated by the action of a activity in a involving apoptosis but the for in to be by during apoptosis in human cells However, and normal mice may against this since the conserved appears to remove may be even in this pathway and the of BER in a an and of shows a with a for in of oxidative DNA damage in and DNA has been as one cause of cellular in However, the evidence for of oxidative DNA damage, with is In fact, it that repair as the of lyase increased with in to for uracil repair mice in glycosylases oxidative damage do not of increased cellular (10). Furthermore, the in and in but also to oxidative and levels in DNA in we on the possible between BER and are in their and do not is clear that glycosylases are to remove mutagenic bases and thus limit The biochemical are by genetic from and Thus, in each of the glycosylases increased spontaneous mutation to increased spontaneous mutation frequencies and an to of the spontaneous of C→T at sites The between altered and types of mutation is mutation are more since types of mutations, are in The loss of activity to mutations in the between and of mutations glycosylases have also been to limit in and and the and pyrimidine and several compounds in enzymes have been to protect against spontaneous mutations and mutations by oxidative a long time that enzymes substitute for each other in in that of deficiency in Furthermore, repair of pyrimidine is to be by at different pathways repair by and in This of has been and an overlap between repair pathways in the protection of integrity has in addition to well also AP adducts and some mismatches as well as oxidative base damage Furthermore, BER of oxidized pyrimidines initiated by the human hNTH1 and other repair proteins for of are repaired by the pathway BER in the strand of but not in the strand of AP sites has to be complex and involves recombination repair and Furthermore, genetic evidence has demonstrated overlapping specificities of the repair, recombination and pathways for oxidative base damage in of damage also involves more one pathway. Thus, BER is by recombination repair and for repair of damage it appears that BER have a with and recombination repair in to alkylating agents in of is of uracil from the Several enzymes that have uracil-DNA glycosylase activity have been in human including and if any, of these the major activity from deamination of cytosine to be these glycosylases only shows with the highly conserved The is and to one of the the structural and are and the mechanism of is as SMUG1 also common with and one in pyrimidine binding and one in bond hydrolysis In addition, MBD4 has a glycosylase and but is to at the the of these enzymes in uracil in is may in BER in the of several very or enzymes with but not specificities may deficiencies in generally have has a number some other glycosylases and as such a case glycosylases The the is mainly in and are with the of in of during and levels of the genome, but levels probably due to effects on synthesis deficiency or with that with and of uracil DNA. The same to of strand and it has been that these from repair of initiated by deficiency is also a for the development of a number of but appears to as a a We are in the to these In summary, different DNA glycosylases may substitute for each other in BER of base damage and pathways may each other in repair of different The significance of each pathway in repair of a but the that govern of pathway are generally not yet understood. The evidence mutations as not in is major is mutations are mainly due to endogenous chemicals and processes or by exogenous agents. is evidence that endogenous are major The of the in on the mutagenic of agents on environmental as possible major in However, with the of with and the of exogenous to levels of human a of demonstrate wide in cancer in in different and even groups in different that environmental and/or to a of However, the of the to cancer is not mainly the of DNA damage may be to for the mutations in human cancer Thus, the may mutations due to spontaneous DNA damage and by environmental and endogenous as a more common for that each of the many endogenous types of DNA damage relatively to the DNA damage, but collectively this type of damage may be is in this that each type of damage may be difficult to with a specific of this damage will repair and cause types of damage may be relatively but to mutagenic prior to repair as uracil resulting from cytosine deamination and types of damage may be more but uracil resulting from of However, even DNA repair pathways have are not uracil base with mutagenic repair with the error by the DNA synthesis step in BER. of a substrate by BER in estimated to generate 1 error The error rate of is some but depends on several such as and in that uracil resulting from cytosine deamination is mutagenic with to a repair error at this limit of from a normal is by the which that of is more common deamination of Thus, cells from mice that have to remove levels of some uracil activity or the of to by an error rate of seem for repair of base since of be increased and therefore mutagenic. increased of uracil DNA during be mechanisms in addition to is by the increased spontaneous mutation in of in has on the spontaneous mutation that repair of levels of in an to generation of mutations, in an relatively in the repair is not a and it has therefore been that be with BER. number of that have with such a function are including and to be these proteins in has been demonstrated to BER in by a mechanism The of DNA repair as a has been by the that of repair enzymes may to an in spontaneous of may be by of the polymerases by the In the case of glycosylases the may be by of mutagenic repair such as AP sites due to the of this substrate range glycosylase to remove normal bases (2). due to DNA damage DNA repair components for repair, a in one should the that of at repair proteins and DNA repair in undamaged DNA as in the case of repair for the of repair reactions may be by of the DNA repair appears to be highly resulting in of the correct for steps in the absence of DNA damage due to during replication, in or in other is well polymerases generate these with a of but the error rate is to by the action of pathways The recent of human low DNA polymerases in have also to our on mutations are in that a may not be by DNA damage. Thus, of undamaged DNA by to result in one is even more but on the with the error for at is not or other polymerases such as in of normal DNA in important is or both to substitute for in BER. our it has not been demonstrated the have mutations in to cancer as in probably due to by a with or an altered substrate for or these it that DNA is not for a of during replication, as a mutation mutation in a Similarly, of or in cells increased mutation rates to be conserved between species has been estimated that the genome of each human at mutations of which one is to a altered mutation rates are highly the genome and also between the same in different cell types the mutation rate appears to be a given between different species, between and over due to it is not clear mutation rates have evolved to a in mutation their be to be of for the human if not for damage in DNA repair and other in DNA metabolism may also be in even if the is over lesions in the time between damage and repair may result in altered that negative or even mutagenic The of proteins with altered functions has been demonstrated with uracil in the strand to the of the possible origin of spontaneous DNA glycosylases that normal bases and AP sites in have been by involving in the the that mutagenic proteins may arise from base damage. proteins in the cell for or and normal or even or cause When the damage the is repaired no of the damage is left at the DNA but mutations seem difficult to such a mechanism of but given the number of cells and the of DNA damage, it be that such damage is to relatively in is not clear which cellular processes are in the mutation rate and to extent their on the of endogenous or exogenous DNA damage. surprisingly, many error-prone DNA polymerases to be highly in the This a possible alternative for the observed in base mutations in cells believed to from during normal in the light of the is function we that a to the for DNA glycosylases such as the highly conserved of of The of human with the and with the In a of more cell and we have not mutations or in the activity of products of the human Thus, it appears that this shows variation that is to the in a In of the of an apparent of the most is probably that most mutations in proteins a but in fitness to against them. in fitness be to most but may have effects on long-term fitness Thus, one is that and other DNA glycosylases are important for the conservation of long-term integrity of the may the species to a extent the cell in a not to with a of thus selection for fitness important is the mutation rate be to have on fitness or to cancer cancer one in the most in the or in from and more a increased mutation This is a of a as a for cancer as However, it is that the between mutation and cancer development in is complex in increased mutation is not a for cancer development an in mutations cause For cases of have been where negative and an increased mutation in normal but not more and more other Furthermore, do not more with cancer, although are cancer prone. This is in to cancer, where of several The a relatively that is also in cancer, the of over mutation as the rate in that mutation is to cause cancer in all and the of in This is by that the mutation rate is to the of cancer in the human increased mutation rate the but the rate steps in development in case be the to the cell control mechanism of cells a mutation in the of a the of mutations to the number of cells. mutations in or of Furthermore, cells be to by the due to mutations in due to other mutations, may also present that of the cells by the Thus, has very and is a relatively common genetic but it may not be a for We have the function of BER this is the we the it is to repair DNA. We a of the mechanisms of DNA repair and the of in DNA of cancer is not a function of DNA repair, but cancer may be a of In fact, all repair systems well long cancer the in In addition to the we to more the mechanisms of repair and of each mechanism, as well as of the BER is more other DNA repair mechanisms since it is initiated by at different DNA glycosylases and at different In contrast, is initiated by an complex and is initiated by only different on the type of damage in well be that repair of DNA damage is important that a highly of overlapping mechanisms is to important will be to the of the different enzymes with overlapping as well as the of alternative pathways in repair of a given We also to the of damage as well as the cell cycle and of We may also to minor as well as a of is an to for one an in mutation rates, too at the of other and For and be to since both are with which may the between and and therefore also uracil The of DNA repair mechanisms that may several pathways for one specific damage, as well as one specific or pathway for several different types of damage, is to be understood. of the complex of in DNA repair, as well as of DNA repair processes with other cellular are even well understood. The of pathways that each other in the defence against mutations to a cellular an DNA DNA AP sites are mainly by DNA glycosylases and major products and rates of by several of repair of AP sites is by and recombination repair, error-prone DNA polymerases may DNA over the This type of in of damage may be more common of DNA bases damaged by endogenous agents. In all cases exogenous agents may also the of damage The BER pathway is initiated by DNA glycosylases and may a and or a in on the type of DNA The in each step is We to of the DNA repair group for of the and and for with the We also and for to from their This by The of The The at the in and The and for