Human strand-specific mismatch repair occurs by a bidirectional mechanism similar to that of the bacterial reaction

Abstract

Nuclear extracts prepared from a HeLa cell line have been previously shown to support strand-specific repair of heteroduplex DNAs containing a site-specific, strand-specific incision (Holmes, J.J., Clark, S., and Modrich, P. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 5837-5841; Thomas, D.C., Roberts, J.D., and Kunkel, T.A. (1991) J. Biol. Chem. 266, 3744-3751). Further analysis of the substrate specificity of the reaction has shown that in addition to G-T, A-C, G-G, and C-C, nuclear extracts also recognize and correct in a strand-specific manner A-A, A-G, T-T, and C-T mismatches, with repair in each case being inhibited by aphidicolin. The rate of repair of a circular G-T heteroduplex was found to decrease monotonically with increasing separation between the mismatch and the strand break that targets repair, as viewed along the shorter path joining the two sites in the circular substrate. This decrease is independent of the polarity of the incised strand, suggesting that the human pathway of mismatch correction may possess a bidirectional excision capability similar to that of the Escherichia coli methyl-directed system. This possibility was confirmed by analysis of excision tracts associated with the reaction. Inhibition of DNA synthesis by aphidicolin or by omission of exogenous dNTPs leads to the mismatch-provoked formation of a single-strand gap that spans the shorter path between the strand break and the mismatch, irrespective of the polarity of the incised strand. Formation of these gaps, which extend from the site of the strand break to terminate at a number of discrete sites in the region 90 to 170 nucleotides beyond the mismatch, is therefore independent of the relative orientation of the two sites. Based on similar mismatch specificities and common features of mechanism, we have concluded that the human strand-specific mismatch repair system is functionally homologous to the bacterial methyl-directed pathway.