INCISION AT DIAMINOPURINE-THYMINE BASE-PAIRS BUT NOT AT GUANINE-O-4-METHYLTHYMINE BASE-PAIRS IN DNA BY EXTRACTS OF HUMAN-CELLS

Cell-free extract from the A1235 human malignant glioma cell line was employed to study the possibility of incision at 2,6-diaminopurine:T ( DiAP:T), 2-amino-6-methylaminopurine:T (AMAP: T), and G:O-4-methylthym ine (G:T-m4) mismatches, each placed in a 45 bp DNA at a defined site. The incision of a 45 bp DNA containing a G:T mispair at the same site was followed to determine the relationship between base pair structur e and repair activity (ies) in the extract. The cell-free extract inci sed DNAs containing DiAP:T, AMAP:T, and G:T pairs similarly. Reminisce nt of the known pattern of incision at G:T mismatches, products from e ach substrate were consistent with two incisions, one immediately 5' a nd one immediately 3' to the mismatched T, and only in the strand cont aining the mismatched T. While DNA with an O-6-methylguanine:T ((m6)G: T) pair was also incised, DNA containing the G:T-m4 pair was not, but was rendered inciseable by pretreatment with O-6-methylguanine-DNA met hyltransferase. Incision of DiAP:T-containing DNA by the extract was l ess in the presence of unlabeled DNA containing G:T mispairs than in t he presence of A:T- or G:A-containing DNA or in the absence of competi ng DNA. We suggest that the mechanism operating on DiAP:T and/or AMAP: T pairs may be the same as the human G:T repair pathway, possibly init iated by the action of a glycosylase as described by Wiebauer & Jiricn y [Wiebauer, K., and Jiricny, J. (1989) Nature 339, 234-236; Wiebauer, K., and Jiricny, J. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5842-584 5]. That related activities recognize DiAP:T, AMAP:T, G:T, and (m6)G:T pairs but not G:T-m4 pairs suggested a unifying mechanism of action.