Mutagenesis Advance Access originally published online on April 13, 2006
Mutagenesis 2006 21(3):173-178; doi:10.1093/mutage/gel020
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Base excision repair fidelity in normal and cancer cells
1 Radiation and Genome Stability Unit, Medical Research Council Harwell, Oxfordshire OX11 0RD, UK 2 Laboratory of Radiation Biology, Graduate School of Sciences, Kyoto University, Kitashirakawa-Oiwakecho Sakyo-ku, Kyoto 606-8502, Japan
In mammalian cells, base excision repair (BER) is the major repair pathway involved in the removal of non-bulky damaged nucleotides. The fidelity of BER is dependent on the polymerization step, where the major BER DNA polymerase (Pol ß) must incorporate the correct Watson–Crick base paired nucleotide into the one nucleotide repair gap. Recent studies have indicated that expression of some Pol ß variants or changes in expression of wild-type Pol ß protein, frequently found in cancer cells, can lead to DNA repair synthesis errors and confers to cells a mutator phenotype.
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Introduction |
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Genome integrity can be threatened by environmental agents such as ultraviolet (UV) light, ionizing radiation, carcinogens and cytotoxic compounds, as well as by endogenous factors, such as reactive oxygen species (1
,2). Consequently, DNA is protected by various DNA repair mechanisms such as base excision repair (BER) and nucleotide excision repair to prevent mutations. The fine balance between genome stability and instability must be tightly regulated since mutations are central to the progression of disease, ageing and evolution.
In mammalian cells, the highly conserved BER pathway is the major cellular defence against non-bulky damaged nucleotides, abasic sites and single-strand breaks generated by reactive oxygen species, alkylating agents and ionizing radiation (2). BER is a multiprotein pathway initiated by damage-specific DNA glycosylases which recognize and cleave the N-glycosylic bond connecting the aberrant base to the sugar–phosphate backbone, creating an abasic or apurinic/apyrimidinic (AP) site (3). Presently, 11 DNA glycosylases have been implicated in mammalian BER and they effectively cover repair of the majority of DNA base lesions. AP sites generated by spontaneous hydrolysis of the N-glycosylic bond (
10 000 per mammalian cell/day) (4) or as a result of DNA glycosylase action are highly mutagenic and cytotoxic as they are non-coding (5–7). Next, AP endonuclease 1 (APE1) recognizes AP site and cleaves the DNA phosphodiester backbone on the 5' side of the AP site, leaving a 3'-hydroxyl group and a 5'-deoxyribose phosphate (dRp) group flanking the nucleotide (nt) gap (8,9). Further repair can proceed via two subpathways that utilize different subsets of enzymes; (i) short-patch BER involves replacement of 1 nt or (ii) long-patch BER that involves gap-filling of 2–6 nt (10). Both subpathways are initiated by DNA polymerase ß (Pol ß), the major BER DNA polymerase, which adds the first nucleotide into the repair gap. During short-patch BER (Figure 1) Pol ß adds 1 nt into the repair gap and simultaneously removes the 5'-dRp moiety by its intrinsic dRp lyase activity (11). Finally, the nick containing 3'-hydroxyl and 5'-phosphate ends are sealed by DNA ligase III
–XRCC1 heterodimer. However, modification of the 5'-dRp moiety by oxidation or reduction prevents Pol ß from excising it and the long-patch BER mechanism, or ‘proliferating cell nuclear antigen (PCNA)-dependent BER’ is employed (12). At this point, Pol ß is replaced by Pol 
/
, which extends the repair patch and displaces several nucleotides to create a 5'-flap junction (13). Flap endonuclease-1 (FEN-1) cleaves the flap at the single-strand/double-strand DNA junction and the DNA ends are then sealed by DNA ligase I. All these reactions have been found to be stimulated by PCNA (10). Among the different enzymatic activities participating in both short- and long-patch BER, the DNA synthesis step is the most likely source of generating mutations during BER. Therefore, the ability of DNA polymerases involved in BER to perform error-free repair synthesis has been addressed in many studies. This review will focus primarily on the fidelity of mammalian BER.
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DNA polymerases involved in BER
Faithful replication by DNA polymerases is essential in maintaining genome integrity during DNA repair and recombination. Each DNA polymerase is presented with the same task of selecting the sole correct Watson–Crick base paired substrate from a pool of four structurally similar deoxynucleotide triphosphates (dNTPs), and successfully incorporating it onto the growing DNA strand.
Currently, over 19 different eukaryotic DNA polymerases have been identified (14). Among those, Pol , and are the major mammalian replicative DNA polymerases. These DNA polymerases have relatively high fidelity. In situations where the replication machinery encounters a DNA lesion, translesion DNA polymerases (Pol 
, 
, 
, 
and Rev1) assist replication bypass of the lesion to avoid collapse of the replication fork (reviewed in ref. 15). Depending on the DNA polymerases involved, translesion synthesis may be quite accurate or error-prone. For example, Pol and are involved in two independent pathways: error-prone and error-free bypass of thymine–thymine cyclobutane (TT) dimers, respectively (16,17).
Three or four DNA polymerases are thought to be involved in different BER pathways; Pol ß, , and possibly Pol 
, where Pol ß is the major BER polymerase (18–20). Pol ß is a 39 kDa monomeric protein comprising two catalytic domains connected by a protease-sensitive hinge region; the N-terminal 8 kDa dRp lyase and DNA binding domain, and the C-terminal 31 kDa DNA polymerase domain (reviewed in ref. 21). The protein also consists of several subdomains necessary for catalysis; the fingers, the thumb and the palm (11,22). In contrast to replicative DNA polymerases, Pol ß lacks any intrinsic 3' to 5' exonuclease activity and is incapable of proofreading. As previously mentioned, Pol ß is the major DNA polymerase involved in short-patch BER (20) but Pol ß also initiates long-patch BER by adding the first nucleotide into the repair gap (12). However, further DNA synthesis during long-patch BER is conducted by Pol / which further incorporate 2–10 more nucleotides (10,18,23).
Mammalian Pol and have similar roles in DNA replication and repair; however, their biochemical properties differ. Pol requires two other factors; PCNA and replication factor C (RF-C) for efficient DNA synthesis (24). In contrast, Pol is highly processive in the absence of PCNA (25). Pol is comprised of four subunits (125, 66, 50 and 12 kDa) (26) and Pol is a heterodimer with a 215 kDa catalytic subunit and 55 kDa subunit with unknown function (27). Both Pol and contain 3' to 5' exonuclease proofreading activity and are able to remove misincorporated nucleotides (28).
The newest member of the X family of DNA polymerases, Pol may also play a backup role in BER (29). Mammalian Pol shares 32% amino acid homology with Pol ß (30) and has an intrinsic dRp lyase activity (31). Pol was suggested to be responsible for background repair activity in Pol ß-deficient cell extracts (19), implicating a possible role in BER. However, the major role of Pol is still undefined since Pol is also involved in processing of DNA ends during non-homologous end joining (32–34).
DNA polymerase synthesis fidelity assays
Three major approaches have been used to address DNA synthesis fidelity of purified DNA polymerases: (i) direct measurement of nucleotide misincorporation frequency using fluorescent or radioactively labelled nucleotide triphosphates; (ii) primer extension reactions using oligonucleotide duplex substrates with a 5'-labelled template; or (iii) reporter gene-based systems where misincorporation into bacteriophage or plasmid DNA during in vitro DNA synthesis is tested as reverse or forward mutations of a reporter gene, e.g. lacZ after transformation of template DNA into bacterial cells.
Initially, synthetic homopolymer polynucleotides were used as DNA polymerase synthesis templates (35). Although the assay was fairly high-throughput, the repeating nature of the polynucleotide promotes slippage of the primer along the template during synthesis. To overcome this problem, assays using synthetic oligonucleotide templates were developed and proved to be efficient substrates for DNA synthesis studies (reviewed in ref. 36).
Biological assays with natural DNA templates were based on analysis of reversions or direct mutations arising during DNA synthesis. Defined single base substitution mutations were introduced into the amber locus (am3) of 
X174 bacteriophage DNA and errors during DNA synthesis were detected after transfection of the template DNA in bacterial cells by determining the reversion frequency of the mutation to wild type (37). This assay was 2–3 times more sensitive than the polynucleotide assays and provided more realistic misincorporation rates; however, analyses were restricted to measuring base substitution errors only.
Spontaneous and induced mutation rates reflect several different types of errors and often occur at different sites within a gene, so in order to monitor a wide spectrum of errors, the assay was adapted and simplified by Kunkel's group (38) who developed an M13mp2/lacZ DNA-based assay to measure DNA synthesis fidelity in vitro. This assay monitors DNA synthesis of a target sequence located within the lacZ gene and is performed using purified DNA polymerase and a gapped DNA substrate. The size of the gap may vary from 5 to 390 nt (38,39). After repair synthesis, substrate DNA is transfected into bacterial cells and any errors causing loss of lacZ gene function can be observed as colourless plaques on indicator plates. This forward mutational assay is capable of detecting frameshift, deletion, duplication and complex errors in addition to a large number of different base substitution errors at many sites and consequently has proved to be a powerful and widely used tool.
More recently, this in vitro forward mutation assay has been further developed by Eckert and co-workers to measure DNA damages on the herpes simplex virus type-1 thymidine kinase (HSV-tk) gene, using chloramphenicol selection (40). This system allows mutagenesis to be monitored in both bacterial and human cells (40).
Fidelity of repair synthesis by purified/recombinant BER DNA polymerases
As previously mentioned, Pol ß is involved in both BER pathways by inserting the first nucleotide into a repair gap. In early studies Pol ß fidelity was measured using substrates with long (up to 300 nt) gaps (38) or in primer extension reactions using duplex oligonucleotide substrates (41). Both the efficiency and fidelity of Pol ß DNA synthesis was found to be reduced (average mutation frequency, 10–4) compared to replicative DNA polymerases (average mutation frequency, 10–6 to 10–8) (42). So, for many years Pol ß was considered as a low fidelity DNA polymerase (36). However, since Pol ß plays the major role in short-patch BER one can assume that Pol ß has evolved as a distributive polymerase designed to fill 1 nt gaps containing 3'-hydroxyl and 5'-phosphate ends. Indeed, Pol ß requires a phosphate group at the 5'-margin of the gap and several studies have shown that 5'-phosphorylated 1 nt gapped DNA is the preferred substrate for Pol ß (41,43,44). Nucleotide misincorporation by Pol ß is 10–100-fold lower on 5'-phosphorylated 1 nt gapped DNA compared with recessed, 6 nt gapped, 5'-phosphorylated 6 nt gapped and unphosphorylated 1 nt gapped substrates (44). As Pol ß has no proofreading activity, its incorporation accuracy results exclusively from the nucleotide selectivity of the enzyme. Unlike most replicative polymerases, which select the correct dNTP through an ‘induced fit’ mechanism, Pol ß fidelity is determined through specific interactions between the enzyme and the template base at the active site. In the induced-fit mechanism, binding of the correct incoming nucleotide and two metal ions within the active site creates a substrate-induced conformational change from an open state to a closed state, which then activates the polymerase for catalysis and hence DNA extension (45). However, nucleotide discrimination by Pol ß appears to be determined by steric exclusion against the incorrect substrate, the precise positioning of the DNA duplex terminus, templating and incoming nucleotides and the catalytic residues within the active site of Pol ß (46). Imprecise positioning of active site residues or DNA can result in misincorporation of nucleotides into DNA. Amino acid residues both distant and near to the Pol ß active site influence its geometry, suggesting that the movements and positioning of subdomains of Pol ß have a significant impact upon its fidelity and consequently its catalytic efficiency (46).
Presently, it is not clear which DNA polymerase, Pol or is contributing more to long-patch BER, since both enzymes can catalyse strand-displacement synthesis which is required for this pathway (18,47,48). However, both Pol and have shown very high fidelity DNA synthesis in the in vitro systems (42).
Mutation spectrum of BER-induced mutations
The M13mp2lacZ forward mutation assay has been widely used to investigate BER fidelity of human cell extracts for a variety of different lesions such as uracil (49), singlet oxygen-induced and gamma radiation-induced lesions (50,51) and gapped DNA where the gap size may vary from 1 to 390 nt (38,39). In the majority of the assays used, the DNA substrate contains site-specific lesion(s) positioned within a coding region of the lacZ gene and after repair in cell extracts any arising changes in the DNA sequence are detected as a loss of gene function, hence loss of ß-galactosidase expression. Repaired bacteriophage DNA substrates are purified and transformed into bacterial cells, mutant phages (colourless) can be isolated and sequenced to identify mutations.
Several independent studies have found that although different types of mutations can arise during short-patch BER, the majority of mutations were single nucleotide deletions (41,52–54).
Models for replication errors involving strand misalignments
Two major models have been utilized to explain the occurrence of 1 nt frameshifts during DNA replication: (i) the majority of sequence-dependent models are based on the ‘template-primer slippage’ model, proposed by Streisinger in 1985 (55), that does not include an active role of DNA polymerase in mutation formation but involves strand displacement during replication of short repeat sequences. Polymerization proceeds through the repetitive sequence and slippage of the template and primer strands produces misaligned intermediates that can be stabilized by correct base pairs, (Figure 2A). (ii) The second group of models include an active role of DNA polymerase during mutation formation. For example, the ‘dNTP-stabilized misalignment’ model has been proposed where the templating base is bypassed by the polymerase and a nucleotide complementary to an adjacent downstream template site is incorporated (56), (Figure 2B).
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Interestingly, DNA sequence context has a profound effect on the resulting mutation spectrum during repair of long gaps (up to 300 nt) and multiple mechanisms of frameshift formation are likely to be involved (38
). However, ‘dNTP-stabilized misalignment’ may be the predominant mechanism during BER since 1 nt frameshifts (the major class of mutations for both BER pathways) were induced independently of sequence context (41,52–54).
DNA polymerase imbalance: effects on replication fidelity and genome stability
We proposed earlier that dNTP-stabilized misalignment is most probably involved in frameshift formation during BER (54). According to this mechanism, incorporation of the new nucleotide to an adjacent template site downstream of the repair gap generates 1 nt frameshifts (Figure 3). As a distributive enzyme, Pol ß dissociates from DNA after addition of this single new misaligned nucleotide, leaving behind an unstable loop that will most probably melt and form an unligatable structure that will be repaired later. However, if an excess of enzyme is present, then Pol ß (even operating as a distributive enzyme) may add more than one nucleotide into the misaligned repair gap, thus increasing the stability of the loop and increasing the probability of frameshift formation. Indeed, overexpression of Pol ß by 2–4-fold in mammalian cells can increase mutation rate and promote tumourigenesis as a consequence (57).
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Early observations indicated that Pol ß is overexpressed in some cancer cells (58
–60). Recently, Albertella and co-workers used expression arrays to systematically study the expression patterns of BER DNA polymerases (Pol ß, , , ) in cancer cells (61). They found that the major BER DNA polymerase, Pol ß, was overexpressed at the mRNA and protein level in approximately one-third of all tumours sampled and Pol ß was most frequently overexpressed in uterus, ovary, prostate and stomach cancer samples. Pol was also overexpressed in a range of tumours, but to a lesser extent than Pol ß. The long-patch BER DNA polymerases (Pol and ) were not commonly overexpressed (61).
Moreover, by inoculating immunodeficient nude mice with various control and Pol ß overexpressing cell lines, Hoffmann and co-workers proved that excess Pol ß can induce cancer. Less than 25% of control inoculum induced the formation of small, slow-growing tumours, whereas >70% of Pol ß overexpressing inoculum induced rapidly growing carcinomas (59).
Pol ß is essential during embryonic development, and thus Pol ß null mice die immediately after birth (62). Interestingly, Pol ß null mice display a tissue-specific reduction of 1 nt frameshift mutations as well as increased apoptosis compared to wild-type embryos (63). Various studies have shown that underexpression of Pol ß increases the rate of chromosomal aberrations by reducing BER activity (64,65), and ultimately the accumulation of DNA damages results in mutagenesis or apoptosis. Indeed, Albertella et al. (61) observed reduced levels of Pol ß mRNA and protein in approximately one-fifth of all tumours sampled, particularly breast and colon cancers, using expression arrays.
DNA polymerase ß variants: effects on replication fidelity and genome stability
Pol ß variants affecting control of incoming nucleotide selection may be a major source of mutations during BER. Direct sequencing of the pol ß gene from different cancer cells identified a number of mutations. To date, 189 various tumours have been characterized and 54 (30%) of these express variants of the Pol ß protein (60,66). pol ß gene mutations have been identified in human prostate, gastric, colorectal, lung, breast and bladder cancers (60,67–73). Single amino acid alterations (48%) were the most prevalent mutations but alternative splice variants of exon 11 (26%), multiple variant forms (14%) and truncated forms (12%) of Pol ß were also observed (66).
To investigate whether these Pol ß mutations are increasing the spontaneous level of mutations, Sweasy and co-workers (74) have extensively studied the fidelity of DNA synthesis and transformation activity of colon (K298M) and prostate (I260M) cancer-associated Pol ß variants. The Ile260 residue is located in the hydrophobic hinge region of Pol ß, which is important for the closing of the polymerase upon interaction with its nucleotide substrate (22). Therefore, amino acid alterations at key Pol ß residues impaired DNA synthesis and resulted in a mutator phenotype. They found that expression of the tumour variants in mouse cells resulted in cellular transformation, which was maintained even after abolition of Pol ß variant expression. In contrast, expression of wild-type Pol ß did not induce a transformed phenotype in mouse cells, thus suggesting that Pol ß variants may induce mutations into cells during the gap-filling step of BER (75). Banerjee and co-workers (68,70) have been investigating an alternative splice variant of Pol ß where 87 nt are deleted (exon 11) in the catalytic domain. Expression of the truncated (36kDa) Pol ß protein has been demonstrated in colorectal and breast cancers, suggesting that dominant negative mutants of Pol ß may interfere with normal repair and as a result induce genetic instability and sensitivity to DNA damaging agents. Indeed, transformation of cells with dominant negative mutants of Pol ß resulted in radiosensitivity (76).
Clearly, alterations to Pol ß whether it is changes in expression or gene mutation have an adverse effect on cells and can induce mutations, possibly by altering the efficiency and/or fidelity of gap-filling during BER.
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Notes |
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*To whom correspondence should be addressed. Tel: +44 1235 841134; Fax: +44 1235 841200; Email: g.dianov{at}har.mrc.ac.uk
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Received on February 10, 2006; revised on March 17, 2006; accepted on March 17, 2006.
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