Repair of DNA Containing Small Heterologous Sequences by Escherichia Coli: a Dissertation

Plasmid heteroduplexes were constrcted that conta either 1, 4 or 5 unpaied bases within the mnt gene. These were used to assess the effciency of repai of small heterologous sequences ("heterologies ) in DNA by the Escherichia coli Dam-diected repai system. Heteroduplexes in defined states of methylation at d(G-AT-C) sites were transformed into a repai proficient indicator strain (which has a mntlac fusion coding for a nonfunctional mnt repressor) and its isogenic mutH, L and S derivatives. Using this in vivo transformation system, we scored for repai on the basis of colony color: correction in favor of the strand bearng mnt coding information gives rise to colonies that are white, whereas correction in favor of the opposite strand (mnt ) yields colonies that are red when grown on MacConkey agar. Failure to repai a heterology yields colonies that are both red and white ("mied"). The correction efficiencies of two heteroduplexes, each contaning a single GT mismatch within mnt, were also monitored for puroses of comparson. Our results show that the repai of heteroduplexes containing 1, 2 and 3 base deletions is as highly efficient as the repair of GT mismatches. Heteroduplexes contaning a 4 base deletion are marginally repaied and DNA containing a 5 base deletion is not detectably repaired. Like the G-T mispais, correction of the 1 3 and 4 base heterologies is Dam-dependent and requires MutH, Land S function. In addition , we show that purfied MutS protein from Salmonella typhimurium, which can substitute for E. coli MutS in vivo, binds to oligonucleotide duplexes contaning 1, 3 and 4 unpaied bases of the identical sequence as that used for the in vivo studies. Specific binding of MutS to homoduplex DNA and to DNA that had undergone a 5 base deletion was not observed.