The Mycobacterial TopA Interacts With Its 3-MeA DNA Glycosylases And Ribokinase

DNA topoisomerases are ubiquitous nucleic acid metabolic enzymes that play a vital role in variety of cellular processes by maintaining the superhelical density of DNA. Many anti-cancer drugs interfere DNA replication, recombination and gene expression through affecting their topoisomerases. Therefore, DNA topoisomerase has become an important potential drug targets. In the current study, we screened and identified two proteins that interacted with the mycobacterial topoisomerase I, and further studied the regulation relationship between them. The results are as follows:(1) We confirmed the mycobacterial (M. tuberculosis and M. smegmatis) 3-MeA DNA glycosylase and ribokinase (RK) two proteins had physical interactions with TopA by B2H and SPR. Further random mutation assays characterized several essential amino acid residues for the interactions. (2) In vitro enzyme function analysis found that TopA could enhance the glycosylase activity on the cleavage of the damage DNA substrates which contains hypoxanthine. In contrast,3-MeA DNA glycosylase inhibited the function activity of TopA. The functional interactions were confirmed to depend on the physical interactions between these proteins. (3) TopA could enhance the D-ribose phosphorylation activity of RK. However, the interaction inhibited the activity of TopA relaxtion activity. Similarly, the functional regulation also depends on physical interactions between two proteins. (4) Co-Immunoprecipitation experiments confirmed that 3-MeA DNA glycosylase and ribokinase (RK) interacted with TopA in M. smegmatis cells, indicating that these protein interactions have physiological importance.3-MeA DNA glycosylase, ribokinase, and TopA are important nucleic acid metabolic enzyme and play an indispensable role in DNA transcription, replication, and repair processes. The current study identified the physical and functional interaction of these proteins, which provide important clues on further understanding the function and regulation mechanisms of TopA. The new characterized interaction sites are also the potential new targets for anti-tuberculosis drug design.