Biochemical Analysis of Chromatin Remodelling Activity Of Type-ISP Restriction Modification Enzyme LlabIII

Abstract

Type ISP restriction-modification (RM) enzymes are bacterial defense systems that nucleolytically cleave foreign DNA entering a bacterial cell (restriction) and protect the host DNA from being restricted by methylating the host DNA (modification). The nucleolytic activity of the enzyme requires hydrolysis of ATP. Cleavage happens when two Type ISP enzymes loaded on their specific target sequence converge subsequent to ATP-dependent translocation (one-dimensional motion) along the DNA (Chand et al., 2015). Interestingly, the ATPase motor that drives DNA translocation is conserved in other enzymes, including chromatin remodelers, such as SWI/SNF and Ino80 (Hopfner et al., 2012; Narlikar et al., 2013), DNA repair enzyme Rad54 (Thoma et al., 2005) etc. The domains(Figure1) of Type-ISP RM Enzymes are TRG (Target recognition domain), MTase domain (for methylating the foreign DNA), SF2 helicase like ATPase domain (for hydrolysis of ATP) and Nuclease domain (for nuclease activity). Chromatin remodelers help in making changes in interaction between DNA and histone octamer. H2A, H2B, H3, H4 dimers are the subunits of the octamers. The aim of the project was to find if Type ISP enzyme can remodel chromatin. To address this question, nucleosome was assembled as roadblocks on path of the Type ISP enzyme. Remodeling of nucleosome was examined using biochemical assays. The assays helped us in arriving at the conclusion that the TypeISP RM enzyme LlaBIII could remove the roadblock (histone octamer bound to a DNA substrate).