DNA glycosylases: in DNA repair and beyond

The base excision repair machinery protects DNA in cells from the damaging effects of oxidation, alkylation, and deamination; it is specialized to fix single-base damage in the form of small chemical modifications. Base modifications can be mutagenic and/or cytotoxic, depending on how they interfere with the template function of the DNA during replication and transcription. DNA glycosylases play a key role in the elimination of such DNA lesions; they recognize and excise damaged bases, thereby initiating a repair process that restores the regular DNA structure with high accuracy. All glycosylases share a common mode of action for damage recognition; they flip bases out of the DNA helix into a selective active site pocket, the architecture of which permits a sensitive detection of even minor base irregularities. Within the past few years, it has become clear that nature has exploited this ability to read the chemical structure of DNA bases for purposes other than canonical DNA repair. DNA glycosylases have been brought into context with molecular processes relating to innate and adaptive immunity as well as to the control of DNA methylation and epigenetic stability. Here, we summarize the key structural and mechanistic features of DNA glycosylases with a special focus on the mammalian enzymes, and then review the evidence for the newly emerging biological functions beyond the protection of genome integrit

SMC1 coordinates DNA double-strand break repair pathways

The SMC1/SMC3 heterodimer acts in sister chromatid cohesion, and recent data indicate a function in DNA double-strand break repair (DSBR). Since this role of SMC proteins has remained largely elusive, we explored interactions between SMC1 and the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways for DSBR in Saccharomyces cerevisiae. Analysis of conditional single- and double mutants of smc1-2 with rad52Δ, rad54Δ, rad50Δ or dnl4Δ illustrates a significant contribution of SMC1 to the overall capacity of cells to repair DSBs. smc1 but not smc2 mutants show increased hypersensitivity of HR mutants to ionizing irradiation and to the DNA crosslinking agent cis-platin. Haploid, but not diploid smc1-2 mutants were severely affected in repairing multiple genomic DNA breaks, suggesting a selective role of SMC1 in sister chromatid recombination. smc1-2 mutants were also 15-fold less efficient and highly error-prone in plasmid end-joining through the NHEJ pathway. Strikingly, inactivation of RAD52 or RAD54 fully rescued efficiency and accuracy of NHEJ in the smc1 background. Therefore, we propose coordination of HR and NHEJ processes by Smc1p through interaction with the RAD52 pathwa

Reversible Top1 cleavage complexes are stabilized strand-specifically at the ribosomal replication fork barrier and contribute to ribosomal DNA stability

Various topological constraints at the ribosomal DNA (rDNA) locus impose an extra challenge for transcription and DNA replication, generating constant torsional DNA stress. The topoisomerase Top1 is known to release such torsion by single-strand nicking and re-ligation in a process involving transient covalent Top1 cleavage complexes (Top1cc) with the nicked DNA. Here we show that Top1ccs, despite their usually transient nature, are specifically targeted to and stabilized at the ribosomal replication fork barrier (rRFB) of budding yeast, establishing a link with previously reported Top1 controlled nicks. Using ectopically engineered rRFBs, we establish that the rRFB sequence itself is sufficient for induction of DNA strand-specific and replication-independent Top1ccs. These Top1ccs accumulate only in the presence of Fob1 and Tof2, they are reversible as they are not subject to repair by Tdp1- or Mus81-dependent processes, and their presence correlates with Top1 provided rDNA stability. Notably, the targeted formation of these Top1ccs accounts for the previously reported broken replication forks at the rRFB. These findings implicate a novel and physiologically regulated mode of Top1 action, suggesting a mechanism by which Top1 is recruited to the rRFB and stabilized in a reversible Top1cc configuration to preserve the integrity of the rDN

The versatile thymine DNA‐glycosylase: a comparative characterization of the human, Drosophila and fission yeast orthologs

Human thymine‐DNA glycosylase (TDG) is well known to excise thymine and uracil from G·T and G·U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5‐methylcytosine and cytosine. In this study, we characterized two newly discovered orthologs of TDG, the Drosophila melanogaster Thd1p and the Schizosaccharomyces pombe Thp1p proteins, with an objective to address the function of this subfamily of uracil‐DNA glycosylases from an evolutionary perspective. A systematic biochemical comparison of both enzymes with human TDG revealed a number of biologically significant facts. (i) All eukaryotic TDG orthologs have broad and species‐specific substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most efficiently processed substrates of all are uracil and 3,N4‐ ethenocytosine opposite guanine and 5‐fluorouracil in any double‐stranded DNA context; (iii) 5‐methylcytosine and thymine derivatives are processed with an appreciable efficiency only by the human and the Drosophila enzymes; (iv) none of the proteins is able to hydrolyze a non‐damaged 5′‐methylcytosine opposite G; and (v) the double strand and mismatch dependency of the enzymes varies with the substrate and is not a stringent feature of this subfamily of DNA glycosylases. These findings advance our current view on the role of TDG proteins and document that they have evolved with high structural flexibility to counter a broad range of DNA base damage in accordance with the specific needs of individual specie

Cell cycle regulation as a mechanism for functional separation of the apparently redundant uracil DNA glycosylases TDG and UNG2

Human Thymine-DNA Glycosylase (TDG) is a member of the uracil DNA glycosylase (UDG) superfamily. It excises uracil, thymine and a number of chemical base lesions when mispaired with guanine in double-stranded DNA. These activities are not unique to TDG; at least three additional proteins with similar enzymatic properties are present in mammalian cells. The successful co-evolution of these enzymes implies the existence of non-redundant biological functions that must be coordinated. Here, we report cell cycle regulation as a mechanism for the functional separation of apparently redundant DNA glycosylases. We show that cells entering S-phase eliminate TDG through the ubiquitin-proteasome system and then maintain a TDG-free condition until G2. Incomplete degradation of ectopically expressed TDG impedes S-phase progression and cell proliferation. The mode of cell cycle regulation of TDG is strictly inverse to that of UNG2, which peaks in and throughout S-phase and then declines to undetectable levels until it appears again just before the next S-phase. Thus, TDG- and UNG2-dependent base excision repair alternates throughout the cell cycle, and the ubiquitin-proteasome pathway constitutes the underlying regulatory syste

7,8-Dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases

Hydroxyl radicals predominantly react with the C(8) of purines forming 7,8-dihydro-8-oxoguanine (8oxoG) and 7,8-dihydro-8-oxoadenine (8oxoA) adducts, which are highly mutagenic in mammalian cells. The majority of oxidized DNA bases are removed by DNA glycosylases in the base excision repair pathway. Here, we report for the first time that human thymine-DNA glycosylase (hTDG) and Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) can remove 8oxoA from 8oxoA*T, 8oxoA*G and 8oxoA*C pairs. Comparison of the kinetic parameters of the reaction indicates that full-length hTDG excises 8oxoA, 3,N(4)-ethenocytosine (epsilonC) and T with similar efficiency (k(max) = 0.35, 0.36 and 0.16 min(-1), respectively) and is more proficient as compared with its bacterial homologue MUG. The N-terminal domain of the hTDG protein is essential for 8oxoA-DNA glycosylase activity, but not for epsilonC repair. Interestingly, the TDG status had little or no effect on the proliferation rate of mouse embryonic fibroblasts after exposure to gamma-irradiation. Nevertheless, using whole cell-free extracts from the DNA glycosylase-deficient murine embryonic fibroblasts and E. coli, we demonstrate that the excision of 8oxoA from 8oxoA*T and 8oxoA*G has an absolute requirement for TDG and MUG, respectively. The data establish that MUG and TDG can counteract the genotoxic effects of 8oxoA residues in vivo

7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases

Hydroxyl radicals predominantly react with the C8 of purines forming 7,8-dihydro-8-oxoguanine (8oxoG) and 7,8-dihydro-8-oxoadenine (8oxoA) adducts, which are highly mutagenic in mammalian cells. The majority of oxidized DNA bases are removed by DNA glycosylases in the base excision repair pathway. Here, we report for the first time that human thymine-DNA glycosylase (hTDG) and Escherichia coli mismatch-specific uracil-DNA glycosylase (MUG) can remove 8oxoA from 8oxoA•T, 8oxoA•G and 8oxoA•C pairs. Comparison of the kinetic parameters of the reaction indicates that full-length hTDG excises 8oxoA, 3,N4-ethenocytosine (εC) and T with similar efficiency (kmax = 0.35, 0.36 and 0.16 min−1, respectively) and is more proficient as compared with its bacterial homologue MUG. The N-terminal domain of the hTDG protein is essential for 8oxoA-DNA glycosylase activity, but not for εC repair. Interestingly, the TDG status had little or no effect on the proliferation rate of mouse embryonic fibroblasts after exposure to γ-irradiation. Nevertheless, using whole cell-free extracts from the DNA glycosylase-deficient murine embryonic fibroblasts and E. coli, we demonstrate that the excision of 8oxoA from 8oxoA•T and 8oxoA•G has an absolute requirement for TDG and MUG, respectively. The data establish that MUG and TDG can counteract the genotoxic effects of 8oxoA residues in viv

Modulation of Age- and Cancer-Associated DNA Methylation Change in the Healthy Colon by Aspirin and Lifestyle

Background Aberrant DNA methylation in gene promoters is associated with aging and cancer, but the circumstances determining methylation change are unknown. We investigated the impact of lifestyle modulators of colorectal cancer (CRC) risk on the stability of gene promoter methylation in the colonic mucosa. Methods We measured genome-wide promoter CpG methylation in normal colon biopsies (n = 1092) from a female screening cohort, investigated the interaction of lifestyle factors with age-dependent increase in methylation with log-linear multivariable regression, and related their modifying effect to hypermethylation in CRC. All statistical tests were two-sided. Results Of 20025 promoter-associated CpGs analyzed, 1713 showed statistically significant age-dependent methylation gains. Fewer CpGs acquired methylation in users of aspirin (≥2 years) and hormonal replacement therapy (HRT age ≥50 years) compared with nonusers (43 vs 1355; 1 vs1377, respectively), whereas more CpGs were affected in smokers (≥20 years) and individuals with a body mass index (BMI) of 25kg/m2 and greater compared with control groups (180 vs 39; 554 vs 144, respectively). Fifty percent of the CpGs showing age-dependent methylation were found hypermethylated in CRC (odds ratio [OR] = 20; 95% confidence interval [CI] = 18 to 23; P < 2×10-16). These loci gained methylation with a higher median rate compared with age-only methylated sites (P = 2×10-76) and were enriched for polycomb regions (OR = 3.67). Importantly, aspirin (P < .001) and HRT use (P < .001) reduced the methylation rate at these cancer-related genes, whereas smoking (P < .001) and high BMI (P = .004) increased it. Conclusions Lifestyle, including aspirin use, modulates age-associated DNA methylation change in the colonic epithelium and thereby impacts the evolution of cancer methylome

Covalent PARylation of DNA base excision repair proteins regulates DNA demethylation.

The intracellular ATP-ribosyltransferases PARP1 and PARP2, contribute to DNA base excision repair (BER) and DNA demethylation and have been implicated in epigenetic programming in early mammalian development. Recently, proteomic analyses identified BER proteins to be covalently poly-ADP-ribosylated by PARPs. The role of this posttranslational modification in the BER process is unknown. Here, we show that PARP1 senses AP-sites and SSBs generated during TET-TDG mediated active DNA demethylation and covalently attaches PAR to each BER protein engaged. Covalent PARylation dissociates BER proteins from DNA, which accelerates the completion of the repair process. Consistently, inhibition of PARylation in mESC resulted both in reduced locus-specific TET-TDG-targeted DNA demethylation, and in reduced general repair of random DNA damage. Our findings establish a critical function of covalent protein PARylation in coordinating molecular processes associated with dynamic DNA methylation

Conserved interactions of the splicing factor Ntr1/Spp382 with proteins involved in DNA double-strand break repair and telomere metabolism

The ligation of DNA double-strand breaks in the process of non-homologous end-joining (NHEJ) is accomplished by a heterodimeric enzyme complex consisting of DNA ligase IV and an associated non-catalytic factor. This DNA ligase also accounts for the fatal joining of unprotected telomere ends. Hence, its activity must be tightly controlled. Here, we describe interactions of the DNA ligase IV-associated proteins Lif1p and XRCC4 of yeast and human with the putatively orthologous G-patch proteins Ntr1p/Spp382p and NTR1/TFIP11 that have recently been implicated in mRNA splicing. These conserved interactions occupy the DNA ligase IV-binding sites of Lif1p and XRCC4, thus preventing the formation of an active enzyme complex. Consistently, an excess of Ntr1p in yeast reduces NHEJ efficiency in a plasmid ligation assay as well as in a chromosomal double-strand break repair (DSBR) assay. Both yeast and human NTR1 also interact with PinX1, another G-patch protein that has dual functions in the regulation of telomerase activity and telomere stability, and in RNA processing. Like PinX1, NTR1 localizes to telomeres and associates with nucleoli in yeast and human cells, suggesting a function in localized control of DSBR