Abstract:
The bacterial ATP-dependent restriction enzymes use energy derived from the hydrolysis of ATP to cleave double-stranded DNA (dsDNA), usually between two specific target sequences. Nucleolytic cleavage of the DNA by the enzyme restricts entry of foreign DNA into the host, thus acting as a bacterial innate immune system. ATP hydrolysis is performed by an ATPase domain present in the enzyme, which evolutionarily belongs to the same family as the ATPases present in other enzymes such as helicases and ATP-dependent chromatin remodelers. In certain class of restriction enzymes, such as Type I and Type ISP restriction enzymes, the ATPase hydrolyze ATP continuously to power active directional movement of the enzyme along the DNA, which is referred to as translocation. In certain other class of restriction enzymes, the ATPase acts as a switch that causes change in state of the enzyme on ATP hydrolysis to facilitate long-range bi-directional diffusion of the enzyme along the DNA. The active translocation or the bi-directional diffusion along the DNA facilitates convergence of two enzymes bound to the target sites, causing DNA cleavage at the site of convergence. Interestingly, active translocation is what drives the eukaryotic ATP-dependent chromatin remodelers to remodel nucleosomes, by either sliding the histone octamers or by other means, to expose the DNA for nucleic acid transactions. My research work focuses on finding if the active translocation mechanism of the bacterial ATP-dependent restriction enzyme can remodel a eukaryotic nucleosome assembly. Using detailed biochemical studies, I demonstrate the remodeling of a nucleosome assembly by the ATP-dependent Type ISP restriction enzyme LlaBIII in vitro. I also show that an ATP-dependent restriction enzyme executing bi-directional diffusion cannot carry out nucleosome remodeling. My preliminary data also indicates that LlaBIII can also remodel the nucleoprotein complex formed by the bacterial histone-like protein HU. Furthermore, preliminary studies on expressing LlaBIII in a human cell line, shows that the protein can express in the nucleus. My work paves the way for exploring the use of ATP-dependent restriction enzymes as synthetic chromatin remodelers for genome regulation.
