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Title: Evidence from in vitro replication O[sup 6]-methylguanine can adopt multiple conformations

Abstract

The effect of O[sup 6]-methylguanine (m[sup 6]G) on replication, in a partially double-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time period, using the Klenow fragment of Escherichia coli DNA polymerase I. The sequence surrounding m[sup 6]G has flanking cytosines (C-m[sup 6]G-C), and the initial steady-state kinetics have been reported. When the primer was annealed so that the first base to be replicated was m[sup 6]G, replication was virtually complete in [approximately]5 min, although the reaction appears biphasic. When annealed with a primer where thymine or cytosine is paired opposite template m[sup 6]G, about half the molecules were replicated in the first 15 sec, and no significant further replication was seen over a 1-hr period. When m[sup 6]G was dealkylated by DNA-O[sup 6]-methylguanine-methyltransferase, replication was rapid with no blockage. These data suggest that there can be two (or more) conformations of m[sup 6]G. In these studies the term syn refers to those allowing such base-pairing. Previous physical studies by others indicate that syn- and anti-conformers of the methyl group relative to the N1 of guanine are possible. Here molecular modeling/computational studies are described, suggesting that syn- and anti-m[sup 6]G can bemore » of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed during in vitro replication. An alternative explanation could be the possibility that the different species may manifest differential interactions of m[sup 6]G with Klenow fragment. These results may provide a rationale for why m[sup 6]G lesions in vivo have been reported to be lethal as well as mutagenic. 31 refs., 5 figs., 1 tab.« less

Authors:
;  [1];  [2]
  1. Lawrence Berkeley Laboratory, CA (United States)
  2. Boston Univ. MA (United States)
Publication Date:
OSTI Identifier:
6442800
Resource Type:
Journal Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America; (United States)
Additional Journal Information:
Journal Volume: 90:9; Journal ID: ISSN 0027-8424
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; GUANINE; MOLECULAR STRUCTURE; BIOCHEMISTRY; CYTOSINE; DNA POLYMERASES; ESCHERICHIA COLI; IN VITRO; MOLECULES; MUTAGENS; OLIGONUCLEOTIDES; THYMINE; AMINES; AROMATICS; AZAARENES; AZINES; BACTERIA; CHEMISTRY; ENZYMES; HETEROCYCLIC COMPOUNDS; HYDROXY COMPOUNDS; MICROORGANISMS; NUCLEIC ACIDS; NUCLEOTIDYLTRANSFERASES; ORGANIC COMPOUNDS; ORGANIC NITROGEN COMPOUNDS; ORGANIC OXYGEN COMPOUNDS; PHOSPHORUS-GROUP TRANSFERASES; POLYMERASES; PROTEINS; PURINES; PYRIMIDINES; TRANSFERASES; URACILS; 550200* - Biochemistry

Citation Formats

Dosanjh, M K, Singer, B, and Loechler, E L. Evidence from in vitro replication O[sup 6]-methylguanine can adopt multiple conformations. United States: N. p., 1993. Web. doi:10.1073/pnas.90.9.3983.
Dosanjh, M K, Singer, B, & Loechler, E L. Evidence from in vitro replication O[sup 6]-methylguanine can adopt multiple conformations. United States. doi:10.1073/pnas.90.9.3983.
Dosanjh, M K, Singer, B, and Loechler, E L. Sat . "Evidence from in vitro replication O[sup 6]-methylguanine can adopt multiple conformations". United States. doi:10.1073/pnas.90.9.3983.
@article{osti_6442800,
title = {Evidence from in vitro replication O[sup 6]-methylguanine can adopt multiple conformations},
author = {Dosanjh, M K and Singer, B and Loechler, E L},
abstractNote = {The effect of O[sup 6]-methylguanine (m[sup 6]G) on replication, in a partially double-stranded defined 25-base oligonucleotide, has been studied under nonlimiting conditions of unmodified dNTPs and over an extended time period, using the Klenow fragment of Escherichia coli DNA polymerase I. The sequence surrounding m[sup 6]G has flanking cytosines (C-m[sup 6]G-C), and the initial steady-state kinetics have been reported. When the primer was annealed so that the first base to be replicated was m[sup 6]G, replication was virtually complete in [approximately]5 min, although the reaction appears biphasic. When annealed with a primer where thymine or cytosine is paired opposite template m[sup 6]G, about half the molecules were replicated in the first 15 sec, and no significant further replication was seen over a 1-hr period. When m[sup 6]G was dealkylated by DNA-O[sup 6]-methylguanine-methyltransferase, replication was rapid with no blockage. These data suggest that there can be two (or more) conformations of m[sup 6]G. In these studies the term syn refers to those allowing such base-pairing. Previous physical studies by others indicate that syn- and anti-conformers of the methyl group relative to the N1 of guanine are possible. Here molecular modeling/computational studies are described, suggesting that syn- and anti-m[sup 6]G can be of similar energy in DNA, and, therefore, these two conformers may explain the two types of species observed during in vitro replication. An alternative explanation could be the possibility that the different species may manifest differential interactions of m[sup 6]G with Klenow fragment. These results may provide a rationale for why m[sup 6]G lesions in vivo have been reported to be lethal as well as mutagenic. 31 refs., 5 figs., 1 tab.},
doi = {10.1073/pnas.90.9.3983},
journal = {Proceedings of the National Academy of Sciences of the United States of America; (United States)},
issn = {0027-8424},
number = ,
volume = 90:9,
place = {United States},
year = {1993},
month = {5}
}