Protein roadblocks and helix discontinuities are barriers to the initiation of mismatch repair.
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 (EcoRI(E111Q), 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 EcoRI(E111Q) 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.
Pluciennik
Anna
A
Department of Biochemistry and Howard Hughes Medical Institute, Box 3711, Duke University Medical Center, Durham, NC 27710, USA.
Modrich
Paul
P
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D004265:DNA Helicases / Q000378:metabolism; D053843:DNA Mismatch Repair; D015246:Deoxyribonuclease EcoRI / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D013379:Substrate Specificity
D018698:Glutamic Acid; D004247:DNA; D015246:Deoxyribonuclease EcoRI; D004265:DNA Helicases