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(Received for publication, August 7, 1996, and in revised form, January 19, 1997)
From the Department of Pharmacological Sciences, State University
of New York, Stony Brook, New York 11794-8651
Site-specifically modified oligodeoxynucleotides
containing a single natural abasic site or a chemically synthesized
(tetrahydrofuran or deoxyribitol) model abasic site were used as
templates for primer extension reactions catalyzed by the Klenow
fragment of Escherichia coli DNA polymerase I or by calf
thymus DNA polymerase
Volume 272, Number 21,
Issue of May 23, 1997
pp. 13916-13922
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
A MECHANISTIC STUDY OF THE "A RULE"
. Analysis of the fully extended products of
these reactions indicated that both polymerases preferentially
incorporate dAMP opposite the natural abasic site and tetrahydrofuran,
while DNA templates containing the ring-opened deoxyribitol moiety
block translesional synthesis, promoting sequence
context-dependent deletions. The frequency of nucleotide
insertion opposite the three types of abasic sites follows the order
dAMP > dGMP > dCMP > dTMP. The frequency of chain
extension was highest when dAMP was positioned opposite a natural
abasic site. The frequency of translesional synthesis past abasic sites
follows the order tetrahydrofuran > deoxyribose > deoxyribitol. The Klenow fragment promotes blunt end addition of dAMP;
this reaction was much less efficient than insertion of dAMP opposite
an abasic site. We conclude that the miscoding potential of a natural
abasic site in vitro closely resembles that of its
tetrahydrofuran analog. Ring-opened abasic sites favor deletions.
Studies with polymerase in vitro predict preferential
incorporation of dAMP at abasic sites in mammalian cells.
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