CHARACTERIZATION OF THE ENZYMATIC ACTIVITY OF Pseudomonas aeruginosa MUTL PROTEIN

DE TULLIO L; CORREA EME; VÉLEZ PS; ARGARAÑA CE; BARRA JL

Congreso; XLVII Reunión Anual de SAIB; 2011

It was described that in the mismatch repair system of some bacterial and eukaryotic organism devoid of MutH and Dam methylation, MutL has a divalent cation-dependent endonucleolytic activity, which could have an in vivo MutH homolog function. In addition, our group recently described that P. aeruginosa MutL (PaMutL) has endonucleolytic activity. Using supercoiled plasmid, this activity was evidenced by the presence of relax plasmid caused by single strand break and lineal DNA fragment due to a second hydrolysis event. In this work we studied the activity of this enzyme but using as a substrate lineal dsDNA fluorescent labeled at the 5 prima ends. The products of the reaction were detected by automated capillary electrophoresis and denaturing polyacrylamide gels, where DNA is visualized as single strand allowing nicks identification. Double strand breaks detection was performed by native agarose gel electrophoresis. Our results indicated that besides the endonucleolytic activity, PaMutL would also have an exonucleolytic activity, removing one or few nucleotides from the 5 prima end. Whereas the hydrolysis of supercoiled DNA plasmid by PaMutL generated nicked plasmid and lineal DNA products, the treatment of lineal dsDNA with PaMutL did not produce double strand breaks or internal nicks. This could indicate that PaMutL enzymatic activity is regulated by DNA topology or sequence context.