The major human AP endonuclease (Ape1) is involved in the nucleotide incision repair pathway

In nucleotide incision repair (NIR), an endonuclease nicks oxidatively damaged DNA in a DNA glycosylase-independent manner, providing the correct ends for DNA synthesis coupled to the repair of the remaining 5'-dangling modified nucleotide. This mechanistic feature is distinct from DNA glycosylase-mediated base excision repair. Here we report that Ape1, the major apurinic/apyrimidinic endonuclease in human cells, is the damage- specific endonuclease involved in NIR. We show that Ape1 incises DNA containing 5,6-dihydro-2'-deoxyuridine, 5,6-dihydrothymidine, 5-hydroxy-2'-deoxyuridine, alpha-2'-deoxyadenosine and alpha-thymidine adducts, generating 3'-hydroxyl and 5'-phosphate termini. The kinetic constants indicate that Ape1-catalysed NIR activity is highly efficient. The substrate specificity and protein conformation of Ape1 is modulated by MgCl2 concentrations, thus providing conditions under which NIR becomes a major activity in cell-free extracts. While the N-terminal region of Ape1 is not required for AP endonuclease function, we show that it regulates the NIR activity. The physiological relevance of the mammalian NIR pathway is discussed

Novel post-synthetic generation, isomeric resolution, and characterization of Fapy-dG within oligodeoxynucleotides: differential anomeric impacts on DNA duplex properties

Accumulation of damaged guanine nucleobases within genomic DNA, including the imidazole ring opened N6-(2-Deoxy-α,β-D-erythro-pentafuranosyl)-2,6-diamino-4-hydroxy-5-formylamidopyrimidine (Fapy-dG), is associated with progression of age-related diseases and cancer. To evaluate the impact of this mutagenic lesion on DNA structure and energetics, we have developed a novel synthetic strategy to incorporate cognate Fapy-dG site-specifically within any oligodeoxynucleotide sequence. The scheme involves the synthesis of an oligonucleotide precursor containing a 5-nitropyrimidine moiety at the desired lesion site via standard solid-phase procedures. Following deprotection and isolation, the Fapy-dG lesion is generated by catalytic hydrogenation and subsequent formylation. NMR assignment of the Fapy-dG lesion (X) embedded within a TXT trimer reveals the presence of rotameric and anomeric species. The latter have been characterized by synthesizing the tridecamer oligodeoxynucleotide d(GCGTACXCATGCG) harboring Fapy-dG as the central residue and developing a protocol to resolve the isomeric components. Hybridization of the chromatographically isolated fractions with their complementary d(CGCATGCGTACGC) counterpart yields two Fapy-dG·C duplexes that are differentially destabilized relative to the canonical G·C parent. The resultant duplexes exhibit distinct thermal and thermodynamic profiles that are characteristic of α- and β-anomers, the former more destabilizing than the latter. These anomer-specific impacts are discussed in terms of differential repair enzyme recognition, processing and translesion synthesis

Вплив метилювання та взаємодії з карбоксильною групою амінокислот на УФ спектри пуринових нуклеотидних основ та нуклеозидів у диметилсульфоксиді. 2 . Гуанін

Вивчено УФ спектри поглинання у зневодненому ДМСО похідних гуаніну m⁹Gua, m₂²,⁹Gua, m¹Gua, m₂¹,⁷Guat G, dG, m¹G, m²G, m⁷G, а також його аналога isoGua. Досліджено та інтерпретовано зміну УФ поглинання похідних Gua в присутності похідних амінокислот, що містять нейтральну карбоксильну групу ас-Asp, ac-Glu, ac-Gly, ac-Asp-OMe ma f-GlyONa і NaAc з депротонованою карбоксильною групою. Показано, що m¹Gua та m⁷Gua у зневодненому ДМСО існують як таутомери N9H. Встановлено, що переважна більшість досліджених похідних Gua взаємодіє лише з депротонованою карбоксильною групою, за винятком m⁷G, isoGua та m³Gua, здатних утворювати комплекси також із нейтральною карбоксильною групою. Розраховано енергетичні харектеристики прототропних таутомеріє Gua та їхніх комплексів з карбоксилат-іоном напівемпіричним методом MNDO/H у вакуумному наближенніИзучены УФ спектры поглощния в безводном ДМСО произ­водных гуанина m⁹Gua, m₂²,⁹Gua, m¹Gua, m₂¹,⁷Guat G, dG, m¹G, m²G, m⁷G, а также его аналога isoGua Исследовано и интерпретировано изменение УФ поглощения производных Gua в присутствии производных аминокислот, содержащихнейтральную карбоксильную группу – ac-Asp, ac-Glu, ac-Gly, ac-Asp-OMe и f-GlyONa и NaAc с депротонцрованной карбоксильой группой. Показано, что m¹Gua и m⁷Gua в безводном ДMСО существуют как таутомеры N9H. Установлено, что большая часть исследованных производных Gua взаимодейст­вует только с фепротонированрой карбоксильной группой, за исключением m⁷G, isoGua и m³Gua, способных образовывать комплексы также и с нейтральной карбоксильной группой. Рассчитаны энергетические характеристики прототропных таутомеров Gua и их комплексов с карбоксилат-ионом полу­эмпирическим методом MNDO/H в вакуумном приближении.Uy absorption spectra of guanine derivatives m⁹Gua, m₂²,⁹Gua, m¹Gua, m₂¹,⁷Guat G, dG, m¹G, m²G, m⁷G, as well as of its analogue isoGua have been studied in anhydrous DMSO. The changes in UV absorption of the Gua derivatives in the presence of the amino acid derivatives with neutral carboxylic group – ac-Asp, ac-Glu, ac-Gly, ac-Asp-OMe and f-GlyONa and NaAc with deprotonated carboxylic groups have been investigated and interpreted. It has been shown that m⁷Gua and m Gua exist as the N9H tautomers in anhydrous DMSO. It has been established that most part of the Gua derivatives interact only with deprotonated carboxylic group, excluding m⁷G, isoGua and m³Gua which are capable to form the complexes with neutral carboxylic group as well. Energetic charac­teristics of the Gua prototropic tautomers and their complexes with carboxylate ion were calculated by the semiempirical MNDO/H method at vacuum approximation

Base Excision Repair in Chromatin

ABSTRACT DNA in the eukaryotic nucleus is complexed with histone and non-histone proteins into chromatin. Nucleosomes, the basic repeating unit of chromatin, not only package DNA but are also intimately involved the regulation of gene expression. All DNA transactions including replication, transcription, recombination and repair take place in such a chromatin environment. Access to packaged nucleosomal DNA in vivo is mediated at least in part by protein complexes that modify or remodel chromatin. Buried sequences in nucleosomes can also transiently become accessible to DNA binding proteins during cycles of partial, reversible unwrapping of nucleosomal DNA from the histone octamer. We have investigated the ability of the human, bifunctional DNA glycosylase, endonuclease III (hNTH1), to initiate base excision repair (BER) of discretely positioned oxidative lesions in model nucleosomes. hNTH1 was able to process a thymine glycol (Tg) lesion almost as efficiently as naked DNA, when the minor groove of the lesion faced away from the histone octamer. Lesion processing did not require or result in detectable nucleosome disruption, as assayed in gel mobility-shift experiments. Instead, hNTH1 formed a slower migrating enzyme-nucleosome ternary complex that was found to contain processed DNA. Processing of an inward-facing Tg residue located just 5 bp away from the outward-facing lesion was much reduced and processing of a sterically occluded Tg residue positioned closer to the dyad center of the nucleosome was even more reduced. Notably, processing of both inward-facing lesions was found to increase as a function of enzyme concentration. Restriction enzyme protection studies indicated that access to these inward-facing lesions did not entail nucleosomal translocation or sliding. Collectively, these observations are consistent with a model in which hNTH1 binds to lesions during cycles of reversible, partial unwrapping of nucleosomal DNA from the histone octamer core. To further investigate this partial unwrapping hypothesis, we studied the kinetics of hNTH1 processing of sterically occluded lesions in greater detail. Our results suggest that efficiency of processing of inward-facing lesions is a function of both DNA unwrapping and rewrapping rates, and enzyme affinity for the lesion. In addition, we determined that APE1 which catalyzes the second step in BER, exhibited an increasing capacity to process inward-facing furan residues as its concentration was increased. Thus as with hNTH1, we hypothesize that APE1 can capture occluded furan residues during cycles of partial DNA unwrapping. We propose that cellular regulatory factors benefit from this intrinsic, periodic exposure of nucleosomal DNA exposure in vivo, which may be amplified by the downstream recruitment of remodeling and / or modifying proteins to facilitate DNA transactions in the cell