DNA STRUCTURE SEQUENCE REQUIREMENTS FOR P. aeruginosa MUTL ENDONUCLEASE ACTIVITY

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

Congreso; XLVIII Reunión Anual de SAIB; 2012

The hallmark of the mismatch repair system (MRS) in bacterial and eukaryotic organisms devoid of MutH, is the presence of a MutL homolog containing endonuclease activity. Our group has recently described that P. aeruginosa MutL (PaMutL) has a cation-dependent endonuclease activity. In this study, DNA structure/sequence requirements of PaMutL activity were analyzed. The results showed that PaMutL generated incisions on supercoiled plasmids, but not on homoduplex or heteroduplex linear DNA molecules. Furthermore, PaMutL displayed the same endonuclease activity on highly negative supercoiled and on relaxed plasmids. The fact that PaMutL was able to generate incisions on plasmids independently of its supercoiling degree, but not on linear DNA, suggests that PaMutL requires fixed-ends DNA molecules for its in vitro endonuclease activity. In addition, the analysis of the incision sites generated on supercoiled plasmids indicated that PaMutL, as well as MutL Bacillus Thuringiensis homolog, has no sequence specificity. These results indicate that contrary to MutH, which specifically recognizes and cleaves GATC sequences, MutL homologs with endonuclease activity do not require a specific sequence to make an incision. As a consequence, the MRS in these organisms could be more efficient than in those with MutH, considering that GATC distribution can affect the MRS efficiency.