Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 597-603 |
| Number of pages | 7 |
| Journal | Nucleic Acids Research |
| Volume | 25 |
| Issue number | 3 |
| DOIs | |
| Publication status | Published - Jul 30 1997 |
| Externally published | Yes |
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ASJC Scopus subject areas
- Genetics
Cite this
Replication bypass and mutagenic effect of α-deoxyadenosine site-specifically incorporated into single-stranded vectors. / Shimizu, Hironori; Yagi, Ryohei; Kimura, Yoshiharu; Makino, Keisuke; Terato, Hiroaki; Ohyama, Yoshihiko; Ide, Hiroshi.
In: Nucleic Acids Research, Vol. 25, No. 3, 30.07.1997, p. 597-603.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Replication bypass and mutagenic effect of α-deoxyadenosine site-specifically incorporated into single-stranded vectors
AU - Shimizu, Hironori
AU - Yagi, Ryohei
AU - Kimura, Yoshiharu
AU - Makino, Keisuke
AU - Terato, Hiroaki
AU - Ohyama, Yoshihiko
AU - Ide, Hiroshi
PY - 1997/7/30
Y1 - 1997/7/30
N2 - 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.
AB - 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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U2 - 10.1093/nar/25.3.597
DO - 10.1093/nar/25.3.597
M3 - Article
VL - 25
SP - 597
EP - 603
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 3
ER -