The Regulatory Mechanism Of3-Methyladenine Glycosylase AAG In Mycobacterium Bovis BCG

Base excision repair (BER) is one kind of DNA repair systems which exists in organisms extensively. It helps cells to tackle with base damage caused by various endogenous and exogenous factors.3-methyladenine DNA glycosylase AAG, which recognizes and excises damaged bases, plays an important role in base excision repair system, In Mycobacterium tuberculosis, the causative microbe for tuberculosis, however, there is not enough knowledge about the regulation of AAG Mycobacterium bovis Bacillus Calmette-Guerina is a live vaccine that has been used as an important weapon for preventing TB worldwide for over7decades. Aud it possesses high genome identity with pathogenic Mycobacterium tuberculosis as well. Therefore, Mycobacterium bovis BCG has been widely used as a model strain for studying gene regulation of Mycobacterium tuberculosis. This study focused on the regulation of AAG in BCG strain with multiple methods. The main results are listed as follows:(1) One TetR family transcription factorm, namely BCG0878c, was found to bind with the promoter of mbaag and its own promoter directly by bacterial one-hybrid, EMSA and ChIP assays. Real-time PCR and β-galactosidase activity assays further confirmed that BCG0878c downregulated the expression of mbaag, while activated the expression of itself. Using EMSA and DNase I footprint assays, we identified a conserved binding motif within the upstream region of mbaag specifically recognized by BCG0878c.(2) Using bacterial two-hybrid, Co-IP, pull-down, in combination with SPR assays, BCG0878c was found to physically interact with MbAAG protein. One the one hand, the interaction, was found to facilitate the cleavage and DNA-binding activity of MbAAG MbAAG, on the other hand, could also stimulate the DNA-binding activity of BCG0878c.(3) Overexpression of BCG0878c and MbAAG respectively inhibited the growth of Mycobacterium bovis BCG to varying degrees. While the expression of mbaag was upregulated when subjected to the stress of nitrous acid, the expression level of BCG0878c changed little under the same condition. Besides, nitrous acid did not affect the binding of BCG0878c to promoters in our experiments.These findings provide novel insights into the regulatory mechanism of transcription factors and enhance our understanding of the regulation of AAG in mycobacteria.