Abstract
Using the technique of delayed oligonucleotide migration through polyacrylamide gels, we have demonstrated that cell-free extracts of the human Burkitt's lymphoma cell line Raji contain proteins which can recognize and bind to mismatched single base pairs in short fragments of DNA. One of these binding proteins resembles an activity previously reported in HeLa cells (Jiricny, J., Hughes, M., Corman, N., and Rudkin, B. B. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 8860-8864) and recognizes DNA containing G.T mismatches. Extracts of Raji cells contain an additional activity which recognizes A.C, T.C, or T.T mismatches in DNA. This second binding protein can be distinguished from the G.T binding activity by its size, substrate specificity, and its fractionation properties. In addition to Raji cells, the new mismatch binding protein is present in extracts of human lymphoblastoid cell lines from a normal individual and a xeroderma pigmentosum patient as well as the SV40-transformed human fibroblast cell line MRC5V1. It seems likely that this novel activity is involved in a broad specificity DNA repair pathway for the correction of single base mismatches in human cells.
Highlights
From the Imuerial Cancer Research Fund, Clare Hall Laboratories, SouthMimms, Potters Bar, Heitfordshire, EN6 3LD, United Kingdom
Neveractivity which recognizes A*C, T*C, or TOTmis- theless, transfection of SV40 DNA heteroduplexes combined matches in DNA. This second binding protein can be with restriction site analysis of progeny molecules has indidistinguished from the G*Tbinding activity by its size,cated that mismatch correction occurs in mammalian cells
C, T .T, and T .C mispairs, represents a hitherto unrecognized activity which is distinguished from the previously reported G .T bindingprotein by its different size, substrate specificity, and biochemical properties
Summary
From the Imuerial Cancer Research Fund, Clare Hall Laboratories, SouthMimms, Potters Bar, Heitfordshire, EN6 3LD, United Kingdom. As a preliminary step to characterizing pthroecess genes [4] and requires the productof the mutY+ gene[5].The of mismatchrepairanditsrelationtothe cytotoxicity of second pathway, characterized ongenetic grounds by a short methylating agents in human cells, we have investigated the excision tract (very short patch repair)(6),acts ina unidirectional fashion to convert G .T mismatches in DNA to G.C base pairs [7] This system is independent of the mutH andmutU genes but requires functional MutS and MutL ability of extracts of human cells to bind to defined single base mismatches in a standard oligonucleotide sequence.
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