Endonuclease activity of Pseudomonas aeruginosa MutL protein

E. M. E. CORREA; CARLOS E. ARGARAÑA; JOSE L. BARRA

Villa Carlos Paz, Córdoba, Argentina

Workshop; II Argentine Workshop in Current Topics in Pseudomonas and Burkholderia Research; 2009

Replication errors can be corrected by the mismatch repair system found in most organisms. In Escherichia coli, and a reduced number of bacteria, MutH protein nicks the non-methylated DNA at a hemi-methylated GATC site, providing the signal to distinguish the parental strand from the newly synthesized strand. Most genomes do not contain a MutH homologue. Therefore, most organisms must use a different nicking endonuclease. It was recently described that some MutL proteins have an endonuclease activity. The endonuclease active site probably involve a DQHA(X)2E(X)4E and a C(P/N)HGRP motif, which are conserved in some eukaryotic homologs and in MutL proteins from a number of bacterial species, but is absent in MutL proteins from bacteria that have a MutH homologue. We started the analysis of the first conserved motif of P. aeruginosa MutL protein by mutating its D and first E amino acids. Both mutant proteins are inactive in vivo. We also analyzed the endonuclease activity of the C-terminal region (250 aa) of the wild type and mutant proteins. In vitro analysis, using supercoiled plasmid DNA as substrate, showed that the wild type and mutant proteins display an endonuclease activity. These results suggest that although the D and the first E amino acids of the conserved region are essential for in vivo mismatch repair, they are not essential for the MutL endonuclease activity, suggesting that structural modifications of the mutant proteins affecting some other function of MutL may be responsible for their in vivo lack of function.