Bidirectional excision in methyl-directed mismatch repair.
Using electron microscopy and indirect end-labeling methods, we have examined excision tracts produced by the Escherichia coli methyl-directed mismatch repair system on a closed circular G-T heteroduplex that contains a single d(GATC) site. Despite differing polarities of the unmodified strand in the two hemimethylated derivatives of the heteroduplex, that portion of the unmethylated strand spanning the shorter path between the d(GATC) site and mismatch is targeted for excision in both cases. Mismatch-provoked excision occurring on both hemimethylated DNAs requires DNA helicase II, but exonuclease requirements for the reaction depend on heteroduplex orientation. When the d(GATC) sequence on the unmodified strand resides 3' to the mismatch as viewed along the shorter path, excision requires exonuclease I. Excision occurring on the alternate hemimethylated heteroduplex depends on the 5'--> 3' hydrolytic activity of exonuclease VII. Coupled with the previous demonstration that repair initiates via the mismatch-provoked, MutHLS-dependent incision of the unmethylated strand at a d(GATC) sequence (Au, K.G., Welsh, K., and Modrich, P. (1992) J. Biol. Chem. 267, 12142-12148), these findings indicate an excision mechanism in which helicase II displacement renders the incised strand sensitive to the appropriate single-strand exonuclease. Our data imply that hydrolysis commences at the d(GATC) site, proceeds to a point beyond the mismatch, and terminates at a number of discrete sites within a 100-nucleotide region just beyond this site. The extent of excision is therefore controlled by one or more components of the repair system.
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Related Subject Headings
- Restriction Mapping
- Oligonucleotide Probes
- Nucleic Acid Heteroduplexes
- MutS DNA Mismatch-Binding Protein
- MutL Proteins
- Molecular Sequence Data
- Models, Genetic
- Microscopy, Electron
- Methylation
- Exodeoxyribonucleases
Citation
Published In
ISSN
Publication Date
Volume
Issue
Start / End Page
Location
Related Subject Headings
- Restriction Mapping
- Oligonucleotide Probes
- Nucleic Acid Heteroduplexes
- MutS DNA Mismatch-Binding Protein
- MutL Proteins
- Molecular Sequence Data
- Models, Genetic
- Microscopy, Electron
- Methylation
- Exodeoxyribonucleases