PROOFREADING DNA - RECOGNITION OF ABERRANT DNA TERMINI BY THE KLENOW FRAGMENT OF DNA-POLYMERASE-I

Fluorescence depolarization decays were measured for 5-dimethylaminona phthalene-1-sulfonyl (dansyl) probes attached internally to 17-mer.27- mer oligonucleotides bound to Klenow fragment of DNA polymerase I. The time-resolved motions of the dansyl probes were sensitive indicators of DNA-protein contacts, showing that the protein binds to DNA with tw o footprints, corresponding to primer termini at either the polymerase or 3'-5' exonuclease sites. We examined complexes of Klenow fragment with DNAs containing various base mismatches. Single mismatches at the primer terminus caused a 3- to 4-fold increase in the equilibrium par titioning of DNA into the exonuclease site; the largest effects were o bserved for purine-purine mismatches. Two or more consecutive G.G mism atches caused the DNA to bind exclusively at the exonuclease site, wit h a partitioning constant at least 250-fold greater than that of the c orresponding matched DNA sequence. Internal single mismatches produced larger effects than the same mismatch at the primer terminus, with a Delta Delta G relative to the matched sequence of -1.1 to -1.3 kcal/mo l for mismatches located 2, 3, or 4 bases from the primer terminus. Al though part of the observed effects may be attributed to the increased melting capacity of the DNA, it appears that the polymerase site also promotes movement of DNA into the exonuclease site by rejecting aberr ant primer termini. These effects suggest that the polymerase and exon uclease sites act together to recognize specific errors that distort t he primer terminus, such as frameshifts, in addition to proofreading m isincorporated bases.