Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair

• Published in: Proceedings of the National Academy of Sciences - PNAS 2007-07, Vol.104 (31), p.12709-12713
• Main Authors: Pluciennik, Anna, Modrich, Paul
• Format: Article
• Language: English
• Subjects: Bacteriophages; Base pair mismatch; Base Sequence; Biochemistry; Biological Sciences; Deoxyribonuclease EcoRI - genetics; Deoxyribonuclease EcoRI - metabolism; Dimers; DNA; DNA - chemistry; DNA Helicases - metabolism; DNA Mismatch Repair; DNA repair; E coli; Enzyme Activation; Enzymes; Escherichia coli; Genetics; Glutamic Acid - genetics; Glutamic Acid - metabolism; Molecules; Mutation - genetics; Nucleic acid heteroduplexes; Oligonucleotides; Proteins; rev genes; Signal transduction; Substrate Specificity
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0705129104

The hemimethylated d(GATC) sequence that directs Escherichia coli mismatch repair can reside on either side of a mismatch at a separation distance of 1,000 bp or more. Initiation of repair involves the mismatch-, MutS-, and MutL-dependent activation of MutH endonuclease, which incises the unmethylated strand at the d(GATC) sequence, with the ensuing strand break serving as the loading site for the appropriate 3'-to-5' or 5'-to-3' excision system. However, the mechanism responsible for the coordinated recognition of the mismatch and a hemimodified d(GATC) site is uncertain. We show that a protein roadblock (EcoRIE₁₁₁Q, a hydrolytically defective form of EcoRI endonuclease) placed on the helix between the two DNA sites inhibits MutH activation by 70-80% and that events that escape inhibition are attributable, at least in part, to diffusion of EcoRIE₁₁₁Q away from its recognition site. We also demonstrate that a double-strand break located within the shorter path linking the mismatch and a d(GATC) site in a circular heteroduplex abolishes MutH activation, whereas a double-strand break within the longer path is without effect. These findings support the idea that initiation of mismatch repair involves signaling along the helix contour.