Alteration of Sequence Specificity of the Type IIS Restriction Endonuclease BtsI

The Type IIS restriction endonuclease BtsI recognizes and digests at GCAGTG(2/0). It comprises two subunits: BtsIA and BtsIB. The BtsIB subunit contains the recognition domain, one catalytic domain for bottom strand nicking and part of the catalytic domain for the top strand nicking. BtsIA has the rest of the catalytic domain that is responsible for the DNA top strand nicking. BtsIA alone has no activity unless it mixes with BtsIB to reconstitute the BtsI activity. During characterization of the enzyme, we identified a BtsIB mutant R119A found to have a different digestion pattern from the wild type BtsI. After characterization, we found that BtsIB(R119A) is a novel restriction enzyme with a previously unreported recognition sequence CAGTG(2/0), which is named as BtsI-1. Compared with wild type BtsI, BtsI-1 showed different relative activities in NEB restriction enzyme reaction buffers NEB1, NEB2, NEB3 and NEB4 and less star activity. Similar to the wild type BtsIB subunit, the BtsI-1 B subunit alone can act as a bottom nicking enzyme recognizing CAGTG(-/0). This is the first successful case of a specificity change among this restriction endonuclease type

Simulating a burnt-bridges DNA motor with a coarse-grained DNA model

We apply a recently-developed coarse-grained model of DNA, designed to capture the basic physics of nanotechnological DNA systems, to the study of a ‘burnt-bridges’ DNA motor consisting of a single-stranded cargo that steps processively along a track of single-stranded stators. We demonstrate that the model is able to simulate such a system, and investigate the sensitivity of the stepping process to the spatial separation of stators, finding that an increased distance can suppress successful steps due to the build up of unfavourable tension. The mechanism of suppression suggests that varying the distance between stators could be used as a method for improving signal-to-noise ratios for motors that are required to make a decision at a junction of stators