Abstract
Restriction enzyme (REase) RM.BpuSI can be described as a Type IIS/C/G REase for its cleavage site outside of the recognition sequence (Type IIS), bifunctional polypeptide possessing both methyltransferase (MTase) and endonuclease activities (Type IIC) and endonuclease activity stimulated by S-adenosyl-L-methionine (SAM) (Type IIG). The stimulatory effect of SAM on cleavage activity presents a major paradox: a co-factor of the MTase activity that renders the substrate unsusceptible to cleavage enhances the cleavage activity. Here we show that the RM.BpuSI MTase activity modifies both cleavage substrate and product only when they are unmethylated. The MTase activity is, however, much lower than that of M1.BpuSI and is thought not to be the major MTase for host DNA protection. SAM and sinefungin (SIN) increase the Vmax of the RM.BpuSI cleavage activity with a proportional change in Km, suggesting the presence of an energetically more favorable pathway is taken. We further showed that RM.BpuSI undergoes substantial conformational changes in the presence of Ca2+, SIN, cleavage substrate and/or product. Distinct conformers are inferred as the pre-cleavage/cleavage state (in the presence of Ca2+, substrate or both) and MTase state (in the presence of SIN and substrate, SIN and product or product alone). Interestingly, RM.BpuSI adopts a unique conformation when only SIN is present. This SIN-bound state is inferred as a branch point for cleavage and MTase activity and an intermediate to an energetically favorable pathway for cleavage, probably through increasing the binding affinity of the substrate to the enzyme under cleavage conditions. Mutation of a SAM-binding residue resulted in altered conformational changes in the presence of substrate or Ca2+ and eliminated cleavage activity. The present study underscores the role of the MTase domain as facilitator of efficient cleavage activity for RM.BpuSI.
Highlights
Restriction endonucleases (REases) have been central to cloning and mapping of DNA sequences [1]
Type I R-M systems are large protein complexes consisting of three unique subunits each contributing to a functional property of the enzymes: an M subunit (HsdM) that contributes to the methylation activity, an S subunit (HsdS) that contributes to the sequence specificity and an R subunit (HsdR) that contributes to the DNA cleavage activity
SAM has been shown to affect the cleavage activity of Type I and Type III REases [27,28,29]. It induces conformational changes required for cleavage activity in EcoKI, a prototypical Type I REases [5,27], and acts as a positive allosteric effector for the cleavage activity of EcoP1I, a prototypical Type III REase [29]
Summary
Restriction endonucleases (REases) have been central to cloning and mapping of DNA sequences [1]. Type I R-M systems are large protein complexes consisting of three unique subunits each contributing to a functional property of the enzymes: an M subunit (HsdM) that contributes to the methylation activity, an S subunit (HsdS) that contributes to the sequence specificity and an R subunit (HsdR) that contributes to the DNA cleavage activity. Type II R-M systems normally contain separate MTases and REases which cleave at defined sites [2]. Type III R-M systems consist of Res and Mod subunits that associate to form a complex in order for DNA cleavage to occur, the Mod subunits modify target bases on their own [3,4,5]. Type IV R-M systems encode proteins that recognize and cleave methylated cytosinecontaining DNA [6,7]
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