Activity of muts oligomers on Pseudomonas aeruginosa mismatch repair

MONTI MR; MIGUEL V; ARGARAÑA CARLOS E.

Carlos Paz, Argentina

Congreso; XLIV Reunión Anual de SAIB; 2008

MutS is an ABC ATPase that recognizes misspaired bases and activates downstream events in the postreplicative DNA Mismatch Repair (MMR). P. aeruginosa MutS is an 855 amino acid protein with different oligomeric species. At present, the identification of the in vivo functional oligomeric state of this protein is matter of extensive study. We have previously shown that native P. aeruginosa MutS forms only tetramers in solution and that the 57 C-terminal amino acids are indispensable for the formation of this oligomeric state. Furthermore, we found that R842 and K852 are key residues in MutS oligomerization since R842E mutation impairs tetramerization and K852A mutation alters the oligomerization equilibrium. In the present work, we investigate the in vitro activities and the in vivo performance of both, the dimeric MutSR842E and tetrameric MutSK852A. Both mutants showed ~2-fold reduction in ATPase activity and a lower affinity for heteroduplex DNA (Kd = 544nM and 598nM, respectively) compared with native MutS (Kd =376nM). Analysis of P. aeruginosa strains expressing these mutated versions of MutS showed that the tetramer is more efficient that the dimer to repair mismatches under highly mutagenic conditions induced by the base analogue 2-aminopurine. Our results indicate that the tetrameric species could be considered the active form of MutS in P. aeruginosa MMR.