Nucleic Acids Research, 2001, Vol. 29, No. 11 2409-2417
© 2001 Oxford University Press
Initiation of strand incision at G:T and O6-methylguanine:T base mismatches in DNA by human cell extracts
Department of Biological and Medical Research (MBC 03), King Faisal Specialist Hospital and Research Center, PO Box 3354, Riyadh 11211, Saudi Arabia and 1Department of Biological Sciences, University College of the Cariboo, Box 3010, Kamloops, British Columbia V2C 5N3, Canada
Extracts of the human glioma cell line A1235 (lacking O6-methylguanine-DNA methyltransferase) are known to restore a G:T mismatch to a normal G:C pair in a G:T-containing model (45 bp) DNA substrate. Herein we demonstrate that substitution of G:T with O6-methylguanine:T (m6G:T) results in extract-induced intra-strand incision in the DNA at an efficiency comparable to that of complete repair of the G:T-containing substrate, although the m6G:T mispair serves as a poor substrate for later repair steps (e.g. gap filling, as judged by defective DNA repair synthesis). The A1235 extract, when supplemented with ATP and the four normal dNTPs, incises 5' to the mismatched T, as inferred by the generation of a single-stranded 20mer fragment. Unlike its parental (A1235) counterpart, an extract of the alkylation-tolerant derivative cell line A1235-MR4 produces no 20mer fragment, even when thymine-DNA glycosylase (TDG) is added to the reaction mixture. In contrast, the A1235 extract, when augmented with TDG, catalyzes enhanced incision at m6G:T in the 45 bp DNA, yielding 5–10-fold greater 20mer than that of either extract or TDG alone. Interestingly, the absence of m6G:T incision activity in the A1235-MR4 extract is similar to that seen for extracts of several known mismatch repair-deficient cell lines of colon tumor origin. Together these results suggest that derivative A1235-MR4 cells are defective in m6G:T incision activity and that the efficiency of this activity in the parental (A1235) cells may depend on the presence of several ill-defined mismatch repair recognition proteins along with TDG and ATP.
* To whom correspondence should be addressed. Tel: +966 1 464 7272; Fax: +966 1 442 7858; Email: lari{at}kfshrc.edu.sa