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
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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)