Obtaining mutant forms of Mycobacterial AP endonuclease MtbXthA

Abstract

Apurinic/apyrimidinic (AP) endonucleases are a key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacterium Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo, which are representatives of exonuclease III and endonuclease IV families, respectively. It is known that both enzymes MtbXthA and MtbNfo contain AP endonuclease or 3'→ 5' exonuclease activities. This paper describes the cloning, expression and purification of recombinant protein MtbXthA. The results showed that comprises MtbXthA AP endonuclease, 3'-phosphodiesterase and 3 '→ 5' nonspecific exonuclease activity. To verify that the observed activity of the types of DNA repair are not the result of contamination by endonucleases of E.coli, mutants MtbXthA-D180N and MtbXthA-N182A were made. The analysis showed that the discovered mutations D180N and N182A in MtbXthA are crucial for enzyme activity.