Replication Bypass and Mutagenic Effect of α-Deoxyadenosine Site-Specifically Incorporated into Single-Stranded Vectors

Nucleic Acids Research, Feb 1997

α-2′-Deoxyadenosine (α) is a major adenine lesion produced by γ-ray irradiation of DNA under anoxic conditions. In this study, single-stranded recombinant M13 vectors containing α were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion. The data for α 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 α constituted a moderate block to DNA replication. The in vivo replication capacity to pass through α was ∼ 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 α or F by E.coli DNA polymerase I. The mutagenic consequence of replicating M13 DNA containing α was analyzed by direct DNA sequencing of progeny phage. Mutagenesis was totally targeted at the site of α introduced into the vector. Mutation was exclusively a single nucleotide deletion and no base substitutions were detected. The deletion frequency associated α 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 α is discussed on the basis of the misinsertion-strand slippage model.

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Replication Bypass and Mutagenic Effect of α-Deoxyadenosine Site-Specifically Incorporated into Single-Stranded Vectors

Hironori Shimizu 1 Ryohei Yagi 1 Yoshiharu Kimura 1 Keisuke Makino 1 Hiroaki Terato 0 1 Yoshihiko Ohyama 0 1 Hiroshi Ide 0 1 0 Graduate Department of Gene Science, Faculty of Science, Hiroshima University , Kagamiyama, Higashi-Hiroshima 739, Japan 1 Department of Polymer Science and Engineering, Kyoto Institute of Technology , Matsugasaki, Sakyo-ku, Kyoto 606, Japan - a -2-Deoxyadenosine (a ) is a major adenine lesion produced by g -ray irradiation of DNA under anoxic conditions. In this study, single-stranded recombinant M13 vectors containing a were constructed and transfected into Escherichia coli to assess lethal and mutagenic effects of this lesion. The data for a 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 a constituted a moderate block to DNA replication. The in vivo replication capacity to pass through a was ~ 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 a or F by E.coli DNA polymerase I. The mutagenic consequence of replicating M13 DNA containing a was analyzed by direct DNA sequencing of progeny phage. Mutagenesis was totally targeted at the site of a introduced into the vector. Mutation was exclusively a single nucleotide deletion and no base substitutions were detected. The deletion frequency associated a 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 a is discussed on the basis of the misinsertion-strand slippage model. Cellular DNA is continuously exposed to endogenous and exogenous genotoxic agents that generate a wide variety of structural defects in DNA. These structural defects are restored in cells by multiple pathways such as base or nucleotide excision repair pathways (1). However, recent evidence shows that lesions present in DNA are not repaired at an equal rate, but those in transcribed strands in expressed genes are preferentially repaired (for review, see ref. 2). This raises the possibility that the DNA replication fork encounters DNA lesions before they are restored. In this case, DNA replication could be either aborted due to the lesions or proceed through the sites with a risk of mutation. Accumulated data indicate that the response of DNA polymerases to the encountered lesion is not unique and how they cope with it depends on the structure of the lesion, sequence contexts flanking to the lesion, enzymatic properties of DNA polymerases and accessory proteins (3,4). We have been focusing our attention on the structural factors of DNA lesions that perturb hydrogen bonding and base stacking interactions, showing that alteration of these interactions exerts differential effects on DNA replication (57). In a series of these studies, we have recently shown by in vitro experiments that a -2-deoxyadenosine (a ) site-specifically introduced into oligonucleotide templates transiently inhibits DNA synthesis (8). The data also suggest that a is potentially mutagenic. a was originally shown to be produced in g -irradiation of aqueous deoxyadenosine under anoxic conditions (9) and later this product was found in DNA, poly(dAdT) and poly(dA) irradiated under similar conditions (10). a has the following unique structural feature. The N-glycosidic bond linking adenine and deoxyribose moieties is flipped due to the abstraction of the H1 atom by OH radicals, but the base moiety is totally intact. Although basal and induced levels of a in cellular DNA are not known, several lines of evidence imply that this lesion, in particular, or lesions with the a configuration with respect to the N-glycosidic bond might, in general, have some biological relevance. The cell nucleus is a very poorly oxygenated intracellular compartment (11,12), and cell hypoxia is ubiquitously observed in tumor cells and anaerobe. In addition, Escherichia coli and yeast cells have repair enzymes such as endonuclease IV (13) and Apn (Ide et al., unpublished data) that recognize this lesion. The nucleoside of 5-formyluracil, resulting from oxidative damage of thymine, readily undergoes base-catalyzed anomerization with respect to the N-glycosidic bond (14). Molecular mechanics and thermodynamic studies on the duplex DNA containing this lesion have revealed that a paired with pyrimidines generates little distortions in DNA, whereas purines introduce distinct kinks and budges (15). These studies also suggest that the space created in the major groove by the flipped base is key to the specific recognition by endonuclease IV, a repair enzyme from E.coli (13). In this study, we extended our investigations on a to an in vivo system. Single-stranded M13 vectors containing a (...truncated)


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Hironori Shimizu, Ryohei Yagi, Yoshiharu Kimura, Keisuke Makino, Hiroaki Terato, Yoshihiko Ohyama, Hiroshi Ide. Replication Bypass and Mutagenic Effect of α-Deoxyadenosine Site-Specifically Incorporated into Single-Stranded Vectors, Nucleic Acids Research, 1997, pp. 597-603, 25/3, DOI: 10.1093/nar/25.3.597