Obtaining mutant forms of Mycobacterial AP endonuclease MtbXthA
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Keywords:
DNA repair, tuberculosis, AP endonucleases, genetic engineering, genomic stability.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.References
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10. Puri R. V., Singh N., Gupta R. K., Tyagi A. K. Endonuclease IV Is the major apurinic/apyrimidinic endonuclease in Mycobacterium tuberculosis and is important for protection against oxidative damage // PLoS One. - 2013. - T. 8. - № 8. - C. e71535.
11. Tsutakawa S. E., Shin D. S., Mol C. D., Izumi T., Arvai A. S., Mantha A. K., Szczesny B., Ivanov I. N., Hosfield D. J., Maiti B., Pique M. E., Frankel K. A., Hitomi K., Cunningham R. P., Mitra S., Tainer J. A. Conserved structural chemistry for incision activity in structurally non-homologous apurinic/apyrimidinic endonuclease APE1 and endonuclease IV DNA repair enzymes // J Biol Chem. - 2013. - T. 288. - № 12. - C. 8445-55.
12. Carpenter E. P., Corbett A., Thomson H., Adacha J., Jensen K., Bergeron J., Kasampalidis I., Exley R., Winterbotham M., Tang C., Baldwin G. S., Freemont P. AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis // EMBO J. - 2007. - T. 26. - № 5. - C. 1363-72.
13. Abeldenov S., Khassenov B. Cloning, expression and purification of recombinant analog of Taq DNA polymerase // Biotechnology. Theory and Practice. - 2014. - № 1. - C. 12-16.
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Abeldenov, S., Ramanculov, E., Saparbaev, M., & Khassenov, B. (2016). Obtaining mutant forms of Mycobacterial AP endonuclease MtbXthA. Experimental Biology, 65(3), 86–93. Retrieved from https://bb.kaznu.kz/index.php/biology/article/view/1101
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МOLECULAR BIOLOGY AND GENETICS
