Processing of thymine glycol in a clustered DNA damage site: mutagenic or cytotoxic (original) (raw)
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8-Oxoguanine DNA damage: at the crossroad of alternative repair pathways
Mutation research, 2003
Radical oxygen species (ROS) generate various modified DNA bases. Among them 8-oxo-7,8-dihydroguanine (8oxoG) is the most abundant and seems to play a major role in mutagenesis and in carcinogenesis. 8oxoG is removed from DNA by the specific glycosylase OGG1. An additional post-replication repair is needed to correct the 8oxoG/A mismatches that are produced by persistent 8oxoG residues. This review is focused on the mechanisms of base excision repair (BER) of this oxidized base. It is shown that, in vitro, efficient and complete repair of 8oxoG/C pairs requires a core of four proteins, namely OGG1, APE1, DNA polymerase (Pol) beta, and DNA ligase I. Repair occurs predominantly by one nucleotide replacement reactions (short-patch BER) and Pol beta is the polymerase of election for the resynthesis step. However, alternative mechanisms can act on 8oxoG residues since Pol beta-null cells are able to repair these lesions. 8oxoG/A mismatches are repaired by human cell extracts via two BER ...
Biopolymers, 2015
The magnitude and nature of lesion-induced energetic perturbations empirically correlate with mutagenicity/cytotoxicity profiles and can be predictive of lesion outcomes during polymerase-mediated replication in vitro. In this study, we assess the sequence and counterbase-dependent energetic impact of the Thymine glycol (T g) lesion on a family of deoxyoligonucleotide duplexes. T g damage arises from thymine and methyl-cytosine exposure to oxidizing agents or radiation-generated free-radicals. The T g lesion blocks polymerase-mediated DNA replication in vitro and the unrepaired site elicits cytotoxic lethal consequences in vivo. Our combined calorimetric and spectroscopic characterization correlates T g-induced energetic perturbations with biological and structural properties. Specifically, we incorporate a 5R-T g isomer centered within the tridecanucleotide sequence 5 0-GCGTACXCATGCG-3 0 (X 5 T g or T) which is hybridized with the corresponding complementary sequence 5 0-CGCATGNGTACGC-3 0 (N 5 A, G, T, C) to generate families of T g-damaged (T g ÁN) and lesion-free (TÁN) duplexes. We demonstrate that the magnitude and nature of the T g destabilizing impact is dependent on counterbase identity (i.e., A $ G < T < C). The observation that a T g lesion is less destabilizing when positioned opposite purines suggests that favorable counterbase stacking interactions may partially compensate lesion-induced perturbations. Moreover, the destabilizing energies of T g ÁN duplexes parallel their respective lesion-free TÁN mismatch counterparts (i.e., G < T < C). Elucidation of T g-induced destabilization relative to the corresponding undamaged mismatch energetics allows resolution of lesion-specific and sequence-dependent impacts. The T g-induced energetic perturbations are consistent with its replication blocking properties and may serve as differential recognition elements for discrimination by the cellular repair machinery.
Journal of Molecular Biology, 1997
We have previously shown that a single N-2-acetylaminofluorene (AAF) 1 Cancérogenèse et Mutagenèse Moleculaire et Structurale adduct bound to the C-8 position of a guanine residue located within Unité Propre de Recherche plasmids containing the unidirectional ColE1 origin of replication induces a 20-fold higher mutation frequency when the adduct is located in the (#9003) du Centre National de la Recherche Scientifique lagging strand as compared to the leading strand. In this study, single 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) lesions have been intro-IRCAD, Hopitaux Universitaires, BP 426 duced in the leading and lagging strand orientation within the same 67091 Strasbourg, Cedex, sequence context as for the AAF adducts. The induced frequency of guanine to thymine transversions has been measured, using a specific France PCR-based quantitative assay, in strains deficient in the repair of the 2 Department of oxidative lesion. The potential involvement of the UvrABC excision repair Environmental Oncology system in the removal of 8-oxodG has also been investigated and ruled out. Institute of Industrial Concerning the mutation frequency asymmetry, in contrast to AAF Ecological Sciences adducts, 8-oxodG adducts induce the same mutation frequency, University of Occupational irrespective of their location in the leading or lagging strands. This striking and Environmental Health difference between 8-oxodG and dGuo-C8-AAF adducts is discussed in 1-1 Iseigaoka, Yahatanishi-ku terms of their differential capacity to block DNA replication.
Journal of Molecular Biology, 1999
When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3 H and 5 H phosphate groups, was positioned one, three or six nucleotides 5 H or 3 H to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5 H or 3 H on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5 H or 3 H to the rAP site. To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3 H phosphate termini, insert thè`m issing'' nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.
Journal of molecular biology, 1997
We have previously shown that a single N-2-acetylaminofluorene (AAF) 1 Cancérogenèse et Mutagenèse Moleculaire et Structurale adduct bound to the C-8 position of a guanine residue located within Unité Propre de Recherche plasmids containing the unidirectional ColE1 origin of replication induces a 20-fold higher mutation frequency when the adduct is located in the (#9003) du Centre National de la Recherche Scientifique lagging strand as compared to the leading strand. In this study, single 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxodG) lesions have been intro-IRCAD, Hopitaux Universitaires, BP 426 duced in the leading and lagging strand orientation within the same 67091 Strasbourg, Cedex, sequence context as for the AAF adducts. The induced frequency of guanine to thymine transversions has been measured, using a specific France PCR-based quantitative assay, in strains deficient in the repair of the 2 Department of oxidative lesion. The potential involvement of the UvrABC excision repair Environmental Oncology system in the removal of 8-oxodG has also been investigated and ruled out. Institute of Industrial Concerning the mutation frequency asymmetry, in contrast to AAF Ecological Sciences adducts, 8-oxodG adducts induce the same mutation frequency, University of Occupational irrespective of their location in the leading or lagging strands. This striking and Environmental Health difference between 8-oxodG and dGuo-C8-AAF adducts is discussed in 1-1 Iseigaoka, Yahatanishi-ku terms of their differential capacity to block DNA replication.
Nucleic Acids Research, 2014
The human thymine-DNA glycosylase (TDG) initiates the base excision repair (BER) pathway to remove spontaneous and induced DNA base damage. It was first biochemically characterized for its ability to remove T mispaired with G in CpG context. TDG is involved in the epigenetic regulation of gene expressions by protecting CpG-rich promoters from de novo DNA methylation. Here we demonstrate that TDG initiates aberrant repair by excising T when it is paired with a damaged adenine residue in DNA duplex. TDG targets the non-damaged DNA strand and efficiently excises T opposite of hypoxanthine (Hx), 1,N 6 -ethenoadenine, 7,8-dihydro-8-oxoadenine and abasic site in TpG/CpX context, where X is a modified residue. In vitro reconstitution of BER with duplex DNA containing Hx•T pair and TDG results in incorporation of cytosine across Hx. Furthermore, analysis of the mutation spectra inferred from single nucleotide polymorphisms in human population revealed a highly biased mutation pattern within CpG islands (CGIs), with enhanced mutation rate at CpA and TpG sites. These findings demonstrate that under experimental conditions used TDG catalyzes sequence context-dependent aberrant removal of thymine, which results in TpG, CpA→CpG mutations, thus providing a plausible mechanism for the putative evolutionary origin of the CGIs in mammalian genomes.
Nucleic Acids Research, 2008
Ionizing radiation induces various clustered DNA lesions, including double-strand breaks (DSBs) accompanied by nearby oxidative base damage. Previous work showed that, in HeLa nuclear extracts, DSBs with partially complementary 3' overhangs and a one-base gap in each strand are accurately rejoined, with the gaps being filled by DNA polymerase j. To determine the possible effect of oxidative base damage on this process, plasmid substrates were constructed containing overhangs with 8-oxoguanine or thymine glycol in base-pairing positions of 3-base (-ACG or-GTA) 3' overhangs. In this context, 8-oxoguanine was well tolerated by the end-joining machinery when present at one end of the break, but not when present at both ends. Thymine glycol was less well tolerated than 8-oxoguanine, reducing gap filling and accurate rejoining by at least 10-fold. The results suggest that complex DSBs can be accurately rejoined despite the presence of accompanying base damage, but that nonplanar bases constitute a major barrier to this process and promote errorprone joining. A chimeric DNA polymerase, in which the catalytic domain of polymerase j was replaced with that of polymerase b, could not substitute for polymerase j in these assays, suggesting that this domain is specifically adapted for gap filling on aligned DSB ends.