Biochemistry 1992,31, 4181-4188
4181
In Vitro Mispairing Specificity of @-Ethylthymidine+ Peter C.Grevatt,t Jerome J. Solomon,t and Opinder S.Bhanot*.ff Department of Environmental Medicine, New York University Medical Center, 550 First Avenue, New York, New York 10016, and Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103 Received August 21, 1991; Revised Manuscript Received January 28, 1992 ABSTRACT: The @-position of thymine is a major site of base alkylation by N-nitroso-alkylating agents, and its biological relevance remains obscure. The potential significance of this DNA damage was ascertained by studying in vitro DNA replication properties of @-ethylthymidine (02-Et-dT) site-specifically incorporated into a 36-nucleotide template. DNA replication was initiated eight nucleotides away from the 02-Et-dT lesion by Escherichia coli polymerase I (Klenow fragment) using a 17-nucleotide primer. In the presence of 10 pM dNTP and Mg2+, 02-Et-dT blocked DNA replication predominantly (94%) 3’ to 02-Et-dT, with the remainder (5%) blocked after incorporation of a nucleotide opposite 02-Et-dT (incorporation-dependent blocked product). Postlesion synthesis was negligible (
