Oligomerization of MutS from E. coli. Influence of the C-terminal region

MIGUEL V; PEZZA RJ; ARGARAÑA CE

Iguazú, Argentina

Congreso; XL Reunión Anual de SAIB; 2004

Fidelity of DNA replication in eukaryotes and prokaryotes depends on the accuracy of DNA polymerase and the proofreading activity of this enzyme. Errors that escape from this last activity are corrected by the Mismatch Repair System (MMRS). MutS, MutL and MutH are the main factors that constitute this system. MutS, an 853 amino acids (aa) protein, recognizes and binds to mismatched or unpaired bases present in the recently synthesized DNA. It has been shown that in vitro, MutS oligomerizes, being the tetramer the most active conformation. It has also been shown that oligomerization and the interaction with MutL are dependent on the 50 C-terminal aa since deletion of this region abolishes the oligomerization of MutS and diminishes its binding to MutL. In this work, the protein oligomerization was analyzed by turbidemitry at 350 nm and native gel electrophoresis, using a purified his-tag recombinant wild type MutS from E. coli. We found that the oligomerization process occurs only in the presence of Mg++, is maximal at pH 7 and is inhibited by low concentration of glycerol. Using C-terminal deletion versions of E. coli MutS, we found that the deletion of the 24 C-terminal aa are sufficient to restrain both high order oligomerization and tetramer formation. A synthetic  peptide containing the 50 C-terminal aa of MutS did not affect the protein oligomerization suggesting that the structure of this peptide is different to that present in the protein.