Abstract

In all living organisms, DNA repair proteins are essential for maintaining the accuracy of genetic material. In Escherichia coli , methyl-directed mismatch repair (MMR) corrects most types of misinsertion errors that arise during DNA replication. In contrast, very short patch (VSP) repair, corrects T/G mismatches caused by the deamination of 5-methylcytosine to thymine in C(T/G)WGG (W = A or T) sequences. Initial experimental data had revealed that the over production of the Vsr endonuclease resulted in a spectrum of transition and frameshift mutations similar to that produced by MMR deficient strains. This spectrum of mutations was also observed for two mutant Vsr proteins known to be defective in VSP repair. These results strongly suggested that the inhibition in MMR repair was due to an interaction between Vsr and one or more of the MMR proteins. The finding of an interaction between Vsr and MutL using both the yeast and bacterial two-hybrid systems further strengthened this hypothesis. Utilization of the two-hybrid systems further revealed that Vsr mutants, with the amino-terminal truncated, still showed an interaction. Conversely even the smallest deletion of the Vsr carboxyl terminal resulted in loss of interaction. A new purification protocol for Vsr yielded a more stable protein that did not precipitate, nor lose its amino terminus and preserved its activity even after long-term storage at -80°C. In vitro endonuclease assays revealed that the activity is equivalent to established values. Single turnover, time course experiments confirmed that MutL does not have a stimulatory effect on Vsr under these conditions. In contrast, under conditions where Vsr is not limiting, MutL does have a stimulatory effect. In addition, Vsr was found to be stimulated to the same extent by MutL mutants deficient in nucleotide binding and/or hydrolysis as it is by wild type MutL. Vsr stimulation in the presence of MutL was unaffected by the presence or absence of ATP or ADP. These results lead us to conclude that the interaction between Vsr and MutL is not nucleotide dependent.