Functional characterization of a DNA-dependent AAA ATPase in a F-cluster mycobacteriophage.

Mycobacteriophages are viruses of Mycobacterium spp. with promising diagnostic and therapeutic potential. Phage genome exploration and characterization of their proteomes are essential to gaining a better understanding of their role in phage biology. So far, genomes of about 2113 mycobacteriophages have been defined and from among those, 1563 phage protein families (phamilies) are identified. However, the function of only a fraction (about 15%) is known since a majority of ORFs in phage genomes are hypothetical proteins. In this study, we have analyzed Gp65 (AQT25877.1), a putative AAA ATPase (Pham 9410) from a F1 cluster mycobacteriophage SimranZ1 (KY385384.1). Though homology analysis of Gp65-AAA ATPase showed the presence of this gene in 38 mycobacteriophages of the F1 cluster, however its further analysis has not been reported yet in any study. The sequence-based functional annotation predicted Gp65 to belong to the P-loop NTPase superfamily and to have AAA_24 and RecA/RadA domains, which are known to be involved in ATP-dependent DNA recombination/repair/maintenance mechanisms. Molecular docking of Gp65 with ATP identified Gly21 and Ser23 residues to be involved in the specific binding. The experimental validation of the DNA-dependent ATPase activity of Gp65 was done using a microtiter plate assay, where the ATPase activity was observed to increase in the presence of dsDNA. The structural characteristics of the protein are demonstrated by non-denaturing gel electrophoresis, showing Gp65 to exist in oligomeric states, which was confirmed by transmission electron microscopy (TEM). It was revealed to exist as a hexamer with a prominent central pore. In this study, based on the stated structural and functional characterization, we report the AAA ATPase to have a putative role in DNA recombination/repair/maintenance mechanism in mycobacteriophages.