Replication bypass and mutagenic effect of alpha-deoxyadenosine site- specifically incorporated into single-stranded vectors

Abstract

alpha-2'-Deoxyadenosine (alpha) is a major adenine lesion produced by gamma-ray irradiation of DNA under anoxic conditions. In this study, single-stranded recombinant M13 vectors containing alpha were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion. The data for alpha were further compared with those obtained with M13 vectors containing normal A or a model abasic site (F) at the same site. The transfection assay revealed that alpha constituted a moderate block to DNA replication. The in vivo replication capacity to pass through alpha was approximately 20% relative to normal A, but 20-fold higher than that of F constituting an almost absolute replication block. Similar data were obtained by in vitro replication of oligonucleotide templates containing alpha or F by E.coli DNA polymerase I. The mutagenic consequence of replicating M13 DNA containing alpha was analyzed by direct DNA sequencing of progeny phage. Mutagenesis was totally targeted at the site of alpha introduced into the vector. Mutation was exclusively a single nucleotide deletion and no base substitutions were detected. The deletion frequency associated alpha was dependent on the 3'-nearest neighbor base: with the 3'-nearest neighbor base T mutation (deletion) frequency was 26%, whereas 1% with the 3'-nearest neighbor base G. A possible mechanism of the single nucleotide deletion associated with alpha is discussed on the basis of the misinsertion-strand slippage model.

Mechanism of Abasic Lesion Bypass Catalyzed by a Y-family DNA Polymerase

“…We had reported previously a similar observation where Dpo4 incorporated nucleotides onto a blunt-end DNA substrate with the following preference: dATP Ͼ dTTP Ͼ dCTP Ͼ dGTP (28). In addition, a previous report demonstrated a nearly equal probability of incorporation of dATP and dTTP opposite this lesion in vivo (33). Ultimately, we do not know the reason for the increased probability of dTTP incorporation into AP-8, but we suspect this trend is likely polymerase-dependent.…”

Consequences and Repair of Oxidative DNA Damage

“…112 a-Deoxyadenosine is a moderate block to DNA synthesis in vitro 113 and in E. coli, one-base deletions are found. 114 Spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh), the further oxidation products of 8-oxoG, are both strong blocks to Klenow fragment and Klenow fragment exo À with A and G being primarily inserted opposite the lesions. 115,116 A crystal structure of Gh with a replicative polymerase shows Gh to be extrahelical and rotated towards the major groove and no longer in a position to serve as a template for an incoming base.…”

Effects of a Guanine-derived Formamidopyrimidine Lesion on DNA Replication

“…More interestingly, the nucleotide forms of FapyG and FapyA epimerize in solution, giving rise to a mixture of ␤-and ␣-anomers with respect to the orientation of the N-glycosidic bond around sugar C-1Ј. We have previously shown that the ␣-anomer of 2Ј-deoxyadenosine (␣dA) site-specifically introduced into oligonucleotide templates or M13 vectors constitutes moderate to fairly strong replication blocks in vitro and in vivo (57,58), exhibiting an effect similar to that of mFapyG. However, there are several differences between ␣dA and mFapyG concerning the responses to DNA polymerase and repair enzyme.…”