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

Journal Article · Sat May 01 00:00:00 EDT 1993 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.90.9.3983· OSTI ID:6442800

Dosanjh, M K; Singer, B ^[1]; Loechler, E L ^[2]

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1. Lawrence Berkeley Laboratory, CA (United States)
2. Boston Univ. MA (United States)

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.

• OSTI ID: 6442800
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Vol. 90:9; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

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