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.

A CC-NBS-LRR gene induces hybrid lethality in cotton.

Hybrid lethality forms a reproductive barrier that has been found in many eukaryotes. Most cases follow the Bateson-Dobzhansky-Muller genetic incompatibility model and involve two or more loci. In this study, we demonstrate that a coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) gene is the causal gene underlying the Le4 locus for interspecific hybrid lethality between Gossypium barbadense and G. hirsutum (cotton). Silencing this CC-NBS-LRR gene can restore F1 plants from a lethal to a normal phenotype. A total of 11 099 genes were differentially expressed between the leaves of normal and lethal F1 plants, of which genes related to autoimmune responses were highly enriched. Genes related to ATP-binding and ATPase were up-regulated before the lethal syndrome appeared; this may result in the conversion of Le4 into an active state and hence trigger immune signals in the absence of biotic/abiotic stress. We discuss our results in relation to the evolution and domestication of Sea Island cottons and the molecular mechanisms of hybrid lethality associated with autoimmune responses. Our findings provide new insights into reproductive isolation and may benefit cotton breeding.

The FtsK-like motor TraB is a DNA-dependent ATPase that forms higher-order assemblies.

TraB is an FtsK-like DNA translocase responsible for conjugative plasmid transfer in mycelial Streptomyces Unlike other conjugative systems, which depend on a type IV secretion system, Streptomyces requires only TraB protein to transfer the plasmid as dsDNA. The γ-domain of this protein specifically binds to repeated 8-bp motifs on the plasmid sequence, following a mechanism that is reminiscent of the FtsK/SpoIIIE chromosome segregation system. In this work, we purified and characterized the enzymatic activity of TraB, revealing that it is a DNA-dependent ATPase that is highly stimulated by dsDNA substrates. Interestingly, we found that unlike the SpoIIIE protein, the γ-domain of TraB does not confer sequence-specific ATPase stimulation. We also found that TraB binds G-quadruplex DNA structures with higher affinity than TraB-recognition sequences (TRSs). An EM-based structural analysis revealed that TraB tends to assemble as large complexes comprising four TraB hexamers, which might be a prerequisite for DNA translocation across cell membranes. In summary, our findings shed light on the molecular mechanism used by the DNA-translocating motor TraB, which may be shared by other membrane-associated machineries involved in DNA binding and translocation.