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Jingyong Zhao

Advisor: Malcolm Winkler, Ph.D.

Methyl-Directed Mismatch repair (MDM) pathway in Escherichia coli plays important roles in correcting DNA replication errors. MutS, MutL, MutH and UvrD are major players in this system. The process is initiated by the recognition of the mismatch or small bulge loop by MutS. Driven by ATP hydrolysis, MutS can translocate on DNA to form a loop. Binding to a mismatch may activate binding of MutS to MutL, which has been proposed to act as a “molecular matchmaker”. The binding of MutL may stimulate MutH to join the complex. The endonuclease activity of MutH is activated by the complex and it can cut the unmodified strand at a hemimethylated d(GATC) sequence to form a nick. Because the newly synthesized DNA strand remains unmethylated for a while, MutH can discriminate the daughter DNA strand. MutHLS complex then directs the UvrD helicase to unwind the DNA helix and the unwinding allows ExoI, ExoVII or RecJ to digest DNA from the nick to the mismatch. The gap is filled by DNA polymerase III and sealed by ligase. Inactivation of this system in E. coli results in elevated mutation rate. Mutations of MutS or MutL homologs in human are related to colon cancer.

The amounts of MutS and MutH decreased 10- and 3-fold respectively from exponential growth phase to stationary phase or nutrient-limited state. In this study, we try to understand the physiological meaning of the down-regulation of the MDM proteins. The results showed that overexpression of the MutS and MutL decreased the GCÖTA transversion by 2- and 4-fold respectively of the E. coli cells under nutritionally limited conditions. And this kind of mutation is recA independent, unlike other forms of “adaptive mutation”. The GCÖTA transversions are largely prevented by GO system, another DNA repair pathway which is composed of three proteins, MutM, MutT and MutY. The overexpression of MutS suppressed the mutY mutator phenotype. This implies that MutS might be involved in correction of the GO:A mismatches.

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Research Info

Overexpression of MutL and MutS Suppresses the GC to TA Transversion Mutations in Nutrient-Limited Escherichia coli K-12