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 intermediates as topoisomerase II poisons

Abasic sites are the most commonly formed DNA lesions in the cell and are produced by numerous endogenous and environmental insults. In addition, they are generated by the initial step of base excision repair (BER). When located within a topoisomerase II DNA cleavage site, "intact" abasic sites act as topoisomerase II poisons and dramatically stimulate enzyme-mediated DNA scission. However, most abasic sites in cells are not intact. They exist as processed BER intermediates that contain DNA strand breaks proximal to the damaged residue. When strand breaks are located within a topoisomerase II DNA cleavage site, they create suicide substrates that are not religated readily by the enzyme and can generate permanent double-stranded DNA breaks. Consequently, the effects of processed abasic sites on DNA cleavage by human topoisomerase IIalpha were examined. Unlike substrates with intact abasic sites, model BER intermediates containing 5'- or 3'-nicked abasic sites or deoxyribosephosphate flaps were suicide substrates. Furthermore, abasic sites flanked by 5'- or 3'-nicks were potent topoisomerase II poisons, enhancing DNA scission approximately 10-fold compared with corresponding nicked oligonucleotides that lacked abasic sites. These findings suggest that topoisomerase II is able to convert processed BER intermediates to permanent double-stranded DNA breaks.     

XPA protein as a limiting factor for nucleotide excision repair and UV sensitivity in human cells

Nucleotide excision repair (NER) acts on a variety of DNA lesions, including damage induced by many chemotherapeutic drugs. Cancer therapy with such drugs might be improved by reducing the NER capacity of tumors. It is not known, however to what extent any individual NER protein is rate-limiting for any step of the repair reaction. We studied sensitivity to UV radiation and repair of DNA damage with regard to XPA, one of the core factors in the NER incision complex. About 150,000-200,000 molecules of XPA protein are present in NER proficient human cell lines, and no XPA protein in the XP-A cell line XP12RO. Transfected XP12RO cell lines expressing 50,000 or more XPA molecules/cell showed UV resistance similar to normal cells. Suppression of XPA protein to approximately 10,000 molecules/cell in a Tet-regulatable system modestly but significantly increased sensitivity to UV irradiation. No removal of cyclobutane pyrimidine dimers was detected in the SV40 immortalized cell lines tested. Repair proficient WI38-VA fibroblasts and transfected XP-A cells expressing 150,000 molecules of XPA/cell removed (6-4) photoproducts from the genome with a half-life of 1h. Cells in which XPA protein was reduced to about 10,000 molecules/cell removed (6-4) photoproducts more slowly, with a half-life of 3h. A reduced rate of repair of (6-4) photoproducts thus results in increased cellular sensitivity towards UV irradiation. These data indicate that XPA levels must be reduced to <10% of that present in a normal cell to render XPA a limiting factor for NER and consequent cellular sensitivity. To inhibit NER, it may be more effective to interfere with XPA protein function, rather than reducing XPA protein levels.     

Base flipping in nucleotide excision repair

UvrB, the ultimate damage-binding protein in bacterial nucleotide excision repair is capable of binding a vast array of structurally unrelated lesions. A beta-hairpin structure in the protein plays an important role in damage-specific binding. In this paper we have monitored DNA conformational alterations in the UvrB-DNA complex, using the fluorescent adenine analogue 2-aminopurine. We show that binding of UvrB to a DNA fragment with cholesterol damage moves the base adjacent to the lesion at the 3' side into an extrahelical position. This extrahelical base is not accessible for acrylamide quenching, suggesting that it inserts into a pocket of the UvrB protein. Also the base opposite this flipped base is extruded from the DNA helix. The degree of solvent exposure of both residues varies with the type of cofactor (ADP/ATP) bound by UvrB. Fluorescence of the base adjacent to the damage is higher when UvrB is in the ADP-bound configuration, but concomitantly this UvrB-DNA complex is less stable. In the ATP-bound form the UvrB-DNA complex is very stable and in this configuration the base in the non-damaged strand is more exposed. Hairpin residue Tyr-95 is specifically involved in base flipping in the non-damaged strand. We present evidence that this conformational change in the non-damaged strand is important for 3' incision by UvrC.     

Base excision repair genes XRCC1 and APEX1 and the risk for prostate cancer

Prostate cancer is the second cause of cancer death in Brazilian men. One of the relevant phenomena to the inherited susceptibility is the presence of allelic variants in genes involved with the DNA repair pathway. The aim of this study was to analyze the frequencies of prevalent, heterozygous and rare genotypes of the base excision repair genes APEX1 and XRCC1 in a case?Ccontrol study and relate the genotypes with tumoral aggressiveness. DNA from peripheral blood of 172 patients and 172 controls were analyzed by RFLP-PCR method. The polymorphisms were also evaluated in relation to clinical and pathological parameters. The OR (Odds Ratio) and confidence interval (CI?=?95%) were used in the association study and the Chi-square and ANOVA tests for the evaluation of histopathological parameters. The rare genotypes frequencies of the gene APEX1 increased the risk for the development of prostate cancer (OR?=?1.68 95% CI 1.10?C2.58). No association was found for the gene XRCC1 (OR?=?0.82 95% CI 0.53?C1.27). The combined analysis for both genes did not show association with this neoplasia (OR?=?1.27 95% CI 0.79?C20.5). The relationship of XRCC1 and APEX1 genotypes with cancer aggressiveness through the correlation with histopathological parameters, did not find any association. Our results suggest that the polymorphism in the gene APEX1 may be indicated as a potential marker for prostate cancer risk.     

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.     

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.     

Base pair conformation-dependent excision of benzo[a]pyrene diol epoxide-guanine adducts by human nucleotide excision repair enzymes.

Human nucleotide excision repair processes carcinogen-DNA adducts at highly variable rates, even at adjacent sites along individual genes. Here, we identify conformational determinants of fast or slow repair by testing excision of N2-guanine adducts formed by benzo[a]pyrene diol epoxide (BPDE), a potent and ubiquitous mutagen that induces mainly G x C-->T x A transversions and frameshift deletions. We found that human nucleotide excision repair processes the predominant (+)-trans-BPDE-N2-dG adduct 15 times less efficiently than a standard acetylaminofluorene-C8-dG lesion in the same sequence. No difference was observed between (+)-trans- and (-)-trans-BPDE-N2-dG, but excision was enhanced about 10-fold by changing the adduct configurations to either (+)-cis- or (-)-cis-BPDE-N2-dG. Conversely, excision of (+)-cis- and (-)-cis- but not (+)-trans-BPDE-N2-dG was reduced about 10-fold when the complementary cytosine was replaced by adenine, and excision of these BPDE lesions was essentially abolished when the complementary deoxyribonucleotide was missing. Thus, a set of chemically identical BPDE adducts yielded a greater-than-100-fold range of repair rates, demonstrating that nucleotide excision repair activity is entirely dictated by local DNA conformation. In particular, this unique comparison between structurally highly defined substrates shows that fast excision of BPDE-N2-dG lesions is correlated with displacement of both the modified guanine and its partner base in the complementary strand from their normal intrahelical positions. The very slow excision of carcinogen-DNA adducts located opposite deletion sites reveals a cellular strategy that minimizes the fixation of frameshifts after mutagenic translesion synthesis.     

Base excision repair in nucleosomes lacking histone tails

Recently, we developed an in vitro system using human uracil DNA glycosylase (UDG), AP endonuclease (APE), DNA polymerase beta (pol beta) and rotationally positioned DNA containing a single uracil associated with a 'designed' nucleosome, to test short-patch base excision repair (BER) in chromatin. We found that UDG and APE carry out their catalytic activities with reduced efficiency on nucleosome substrates, showing a distinction between uracil facing 'out' or 'in' from the histone surface, while DNA polymerase beta (pol beta) is completely inhibited by nucleosome formation. In this report, we tested the inhibition of BER enzymes by the N-terminal 'tails' of core histones that take part in both inter- and intra-nucleosome interactions, and contain sites of post-translational modifications. Histone tails were removed by limited trypsin digestion of 'donor' nucleosome core particles and histone octamers were exchanged onto a nucleosome-positioning DNA sequence containing a single G:U mismatch. The data indicate that UDG and APE activities are not significantly enhanced with tailless nucleosomes, and the distinction between rotational settings of uracil on the histone surface is unaffected. More importantly, the inhibition of pol beta activity is not relieved by removal of the histone tails, even though these tails interact with DNA in the G:U mismatch region. Finally, inclusion of X-ray cross complement group protein 1 (XRCC1) or Werner syndrome protein (WRN) had no effect on the BER reactions. Thus, additional activities may be required in cells for efficient BER of at least some structural domains in chromatin.     

Base excision repair of DNA in mammalian cells

Base excision repair (BER) of DNA corrects a number of spontaneous and environmentally induced genotoxic or miscoding base lesions in a process initiated by DNA glycosylases. An AP endonuclease cleaves at the 5' side of the abasic site and the repair process is subsequently completed via either short patch repair or long patch repair, which largely require different proteins. As one example, the UNG gene encodes both nuclear (UNG2) and mitochondrial (UNG1) uracil DNA glycosylase and prevents accumulation of uracil in the genome. BER is likely to have a major role in preserving the integrity of DNA during evolution and may prevent cancer.     

Base excision repair of 8-oxoG in dinucleosomes

In this work we have studied the effect of chromatin structure on the base excision repair (BER) efficiency of 8-oxoG. As a model system we have used precisely positioned dinucleosomes assembled with linker histone H1. A single 8-oxoG was inserted either in the linker or the core particle DNA within the dinucleosomal template. We found that in the absence of histone H1 the glycosylase OGG1 removed 8-oxoG from the linker DNA and cleaved DNA with identical efficiency as in the naked DNA. In contrast, the presence of histone H1 resulted in close to 10-fold decrease in the efficiency of 8-oxoG initiation of repair in linker DNA independently of linker DNA length. The repair of 8-oxoG in nucleosomal DNA was very highly impeded in both absence and presence of histone H1. Chaperone-induced uptake of H1 restored the efficiency of the glycosylase induced removal of 8-oxoG from linker DNA, but not from the nucleosomal DNA. We show, however, that removal of histone H1 and nucleosome remodelling are both necessary and sufficient for an efficient removal of 8-oxoG in nucleosomal DNA. Finally, a model for BER of 8-oxoG in chromatin templates is suggested.     

Base excision repair activities required for yeast to attain a full chronological life span

The chronological life span of yeast, the survival of stationary (G0) cells over time, provides a model for investigating certain of the factors that may influence the aging of non-dividing cells and tissues in higher organisms. This study measured the effects of defined defects in the base excision repair (BER) system for DNA repair on this life span. Stationary yeast survives longer when it is pre-grown on respiratory, as compared to fermentative (glucose), media. It is also less susceptible to viability loss as the result of defects in DNA glycosylase/AP lyases (Ogg1p, Ntg1p, Ntg2p), apurinic/apyrimidinic (AP) endonucleases (Apn1p, Apn2p) and monofunctional DNA glycosylase (Mag1p). Whereas single BER glycosylase/AP lyase defects exerted little influence over such optimized G0 survival, this survival was severely shortened with the loss of two or more such enzymes. Equally, the apn1delta and apn2delta single gene deletes survived as well as the wild type, whereas a apn1delta apn2delta double mutant totally lacking in any AP endonuclease activity survived poorly. Both this shortened G0 survival and the enhanced mutagenicity of apn1delta apn2delta cells were however rescued by the over-expression of either Apn1p or Apn2p. The results highlight the vital importance of BER in the prevention of mutation accumulation and the attainment of the full yeast chronological life span. They also reveal an appreciable overlap in the G0 maintenance functions of the different BER DNA glycosylases and AP endonucleases.     

Base excision repair: AP endonucleases and DNA polymerases

The DNA base lesions in living cells occur permanently and with high frequency as a result of the action of exogenous and endogenous factors. The main mechanism providing removal of such lesions is base excision repair.     

Base excision repair intermediates are mutagenic in mammalian cells

Base excision repair (BER) is the main pathway for repair of DNA damage in mammalian cells. This pathway leads to the formation of DNA repair intermediates which, if still unsolved, cause cell lethality and mutagenesis. To characterize mutations induced by BER intermediates in mammalian cells, an SV-40 derived shuttle vector was constructed carrying a site-specific lesion within the recognition sequence of a restriction endonuclease. The mutation spectra of abasic (AP) sites, 5′-deoxyribose-5-phosphate (5′dRp) and 3′-[2,3-didehydro-2,3-dideoxy-ribose] (3′ddR5p) single-strand breaks (ssb) in mammalian cells was analysed by RFLP/PCR and mutation frequency was estimated by quantitative PCR. Point mutations were the predominant events occurring at all BER intermediates. The AP site-induced mutation spectrum supports evidence for the ‘A-rule’ and is also consistent with the use of the 5′ neighbouring base to instruct nucleotide incorporation (5′-rule). Preferential adenine insertion was also observed after in vivo replication of 5′dRp or 3′ddR5p ssb. We provide original evidence that not only the abasic site but also its derivatives ‘faceless’ BER intermediates are mutagenic, with a similar mutation frequency, in mammalian cells. Our findings support the hypothesis that unattended BER intermediates could be a constant threat for genome integrity as well as a spontaneous source of mutations.     

Ribonucleotides as nucleotide excision repair substrates

The incorporation of ribonucleotides in DNA has attracted considerable notice in recent years, since the pool of ribonucleotides can exceed that of the deoxyribonucleotides by at least 10–20-fold, and single ribonucleotide incorporation by DNA polymerases appears to be a common event. Moreover ribonucleotides are potentially mutagenic and lead to genome instability. As a consequence, errantly incorporated ribonucleotides are rapidly repaired in a process dependent upon RNase H enzymes. On the other hand, global genomic nucleotide excision repair (NER) in prokaryotes and eukaryotes removes damage caused by covalent modifications that typically distort and destabilize DNA through the production of lesions derived from bulky chemical carcinogens, such as polycyclic aromatic hydrocarbon metabolites, or via crosslinking. However, a recent study challenges this lesion-recognition paradigm. The work of Vaisman et al. (2013) [34] reveals that even a single ribonucleotide embedded in a deoxyribonucleotide duplex is recognized by the bacterial NER machinery in vitro. In their report, the authors show that spontaneous mutagenesis promoted by a steric-gate pol V mutant increases in uvrA, uvrB, or uvrC strains lacking rnhB (encoding RNase HII) and to a greater extent in an NER-deficient strain lacking both RNase HI and RNase HII. Using purified UvrA, UvrB, and UvrC proteins in in vitro assays they show that despite causing little distortion, a single ribonucleotide embedded in a DNA duplex is recognized and doubly-incised by the NER complex. We present the hypothesis to explain the recognition and/or verification of this small lesion, that the critical 2′-OH of the ribonucleotide – with its unique electrostatic and hydrogen bonding properties – may act as a signal through interactions with amino acid residues of the prokaryotic NER complex that are not possible with DNA. Such a mechanism might also be relevant if it were demonstrated that the eukaryotic NER machinery likewise incises an embedded ribonucleotide in DNA.     

Common polymorphisms and somatic mutations in human base excision repair genes in ovarian and endometrial cancers

The purpose of this study was to determine whether the human APEX and OGG1 genes, encoding proteins important in base excision repair (BER) of DNA, contain nucleotide sequence polymorphisms or are mutated somatically in tumors from women diagnosed with ovarian or endometrial cancer. Based upon the analysis of germline DNA from 83 individuals, 63 with ovarian cancer and 20 with endometrial cancer, we found two missense polymorphisms in APEX (Q51H and D 148E) and two missense (A3P and S326C) and one intronic (Exon 5-15 bp) polymorphism in OGG1. The frequencies of the various alleles (in the ovarian and endometrial cancer patients combined) were 4.8% for 51-His and 56.2% for 148-Glu in APEX, and 1.0% for 3-Pro and 20.0% for 326-Cys in OGG1. Somatic mutations in APEX (P112L, W188X and R237C) were identified in three of 20 endometrial tumors, but no mutations were identified in APEX in 43 ovarian tumors, or in OGG1 at either tumor site. Given the crucial role of the APEX and OGG1 proteins in BER of oxidative DNA damage, the identified polymorphisms are good candidates for genetic epidemiologic studies of cancer susceptibility, while the finding that three of 20 (15%) endometrial tumors have somatic mutations in APEX suggests that inactivation of the BER pathway is important for the development of endometrial cancer in at least a subset of cases.     

Purification of PCNA as a nucleotide excision repair protein

Human cell free extracts carry out nucleotide excision repair in vitro. The extract is readily separated into two fractions by chromatography on a DEAE column. Neither the low salt (0.1 M KCl) nor the high salt (0.8 M KCl) fractions are capable of repair synthesis but the combination of the two restore the repair synthesis activity. Using the repair synthesis assay we purified a protein of 37 kDa from the high salt fraction which upon addition to the low salt fraction restores repair synthesis activity. Amino acid sequence analysis, amino acid composition and immunobloting with PCNA antibodies revealed that the 37 kDa protein is the proliferating cell nuclear antigen (PCNA) known to stimulate DNA Polymerases δ and ε. By using an assay which specifically measures the excision of thymine dimers we found that PCNA is not required for the actual excision reaction per se but increases the extent of excision by enabling the excision repair enzyme to turn over catalytically.