Abstract
Several alkylating agents that either occur in the environment or are self-produced can cause DNA-damaging injuries in bacterial cells. Therefore, all microorganisms have developed repair systems that are able to counteract DNA alkylation damage. The adaptive response to alkylation stress in Escherichia coli consists of the Ada operon, which has been widely described; however, the homologous system in Mycobacterium tuberculosis (MTB) has been shown to have a different genetic organization but it is still largely unknown. In order to describe the defense system of MTB, we first investigated the proteins involved in the repair mechanism in the homologous non-pathogenic mycobacterium M. smegmatis. Ogt, Ada-AlkA and FadE8 proteins were recombinantly produced, purified and characterized. The biological role of Ogt was examined using proteomic experiments to identify its protein partners in vivo under stress conditions. Our results suggested the formation of a functional complex between Ogt and Ada-AlkA, which was confirmed both in silico by docking calculations and by gel filtration chromatography. We propose that this stable association allows the complex to fulfill the biological roles exerted by Ada in the homologous E. coli system. Finally, FadE8 was demonstrated to be structurally and functionally related to its E. coli homologous, AidB.
Highlights
The DNA molecule is a crucial target for several alkylating molecules that either occur in the environment or are self-produced
Ogt was demonstrated to be a methyltransferase with DNA binding capabilities [15]. This unusual gene organization prompted us to investigate the respective roles of Ogt and Ada-AlkA and the other proteins involved in the repair mechanism in M. smegmatis, a non-pathogenic mycobacterium homologous to Mycobacterium tuberculosis (MTB)
This work focused on investigating the specific role of proteins involved in the DNA repair mechanism in M. smegmatis, a non-pathogenic mycobacterium
Summary
The DNA molecule is a crucial target for several alkylating molecules that either occur in the environment or are self-produced. They can react with nucleophilic sites on DNA bases and cause covalent modifications, cytotoxic damage and impair cell survival [1]. A number of sites prone to alkylation have been identified on DNA bases including the guanine oxygen site at position 6. Methylation of this oxygen generates O6-methylguanine (O6-MeG), which prevents the correct base pairing and leads to mutations during DNA replication. Other well-known alkylation products are N7-methylguanine (N7-MeG), N3-methyladenine (N3-MeA), N1-methyladenine (N1-MeA), N3-methylcytosine (N3-MeC), and O4-methyltimine (O4-MeT) [3,4]
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