Physical and functional interactions between Escherichia coli MutL and the Vsr repair endonuclease

Abstract

DNA mismatch repair (MMR) and very-short patch (VSP) repair are two pathways involved in the repair of T:G mismatches. To learn about competition and cooperation between these two repair pathways, we analyzed the physical and functional interaction between MutL and Vsr using biophysical and biochemical methods. Analytical ultracentrifugation reveals a nucleotide-dependent interaction between Vsr and the N-terminal domain of MutL. Using chemical crosslinking, we mapped the interaction site of MutL for Vsr to a region between the N-terminal domains similar to that described before for the interaction between MutL and the strand discrimination endonuclease MutH of the MMR system. Competition between MutH and Vsr for binding to MutL resulted in inhibition of the mismatch-provoked MutS- and MutL-dependent activation of MutH, which explains the mutagenic effect of Vsr overexpression. Cooperation between MMR and VSP repair was demonstrated by the stimulation of the Vsr endonuclease in a MutS-, MutL- and ATP-hydrolysis-dependent manner, in agreement with the enhancement of VSP repair by MutS and MutL in vivo. These data suggest a mobile MutS–MutL complex in MMR signalling, that leaves the DNA mismatch prior to, or at the time of, activation of downstream effector molecules such as Vsr or MutH.

Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli

“…This would have the overall effect of creating a shorter repair track. In addition, this would be consistent with previously reported results, indicating that MutL interacts with the Vsr endonuclease and stimulates the binding of this protein to its substrate (24,32,45,46).…”

“…Therefore, MutS and MutL must have some role in the VSP repair pathway, such that these proteins increase the efficiency of VSP repair. Previous results (24,32) have shown that MutL interacts with the Vsr endonuclease and apparently stimulates binding to the DNA substrate. We have confirmed this result that, in our hands, is only apparent at high concentrations of MutL (data not shown).…”

“…MutL has been shown to stimulate the nicking reaction catalyzed by the Vsr endonuclease (24,32,35). Therefore, it is possible that MutL is able to increase the efficiency of VSP repair by stimulating the nicking reaction catalyzed by the Vsr endonuclease.…”

“…8. MutS binds to the T:G mis- (24,35); other protein-protein interactions, if they exist, are hypothetical. The Vsr endonuclease catalyzes the hydrolysis of a phosphodiester bond 5Ј to the mismatched thymidine.…”

“…It is important to note that this sequence is the only place in the E. coli chromosome that would produce a T:G mismatch caused by spontaneous hydrolytic deamination of 5-mC, because this sequence is the only place where the Dcm methyltransferase acts. Thus, a major function of VSP repair is to maintain 5Ј-C 5me CWGG-3Ј sequences within the genome (24).…”