Single substitution in bacteriophage T4 RNase H alters the ratio between its exo- and endonuclease activities

Publication date: Available online 24 September 2015 Source:Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis Author(s): Natalia Kholod, Dmitry Sivogrivov, Oleg Latypov, Sergey Mayorov, Rafail Kuznitsyn, Andrey V. Kajava, Mikhail Shlyapnikov, Igor Granovsky The article describes substitutions in bacteriophage T4 RNase H which provide so called das-effect. Phage T4 DNA arrest suppression (das) mutations have been described to be capable of partially suppressing the phage DNA arrest phenotype caused by a dysfunction in genes 46 and/or 47 (also known as Mre11/Rad50 complex). Genetic mapping of das13 (one of the das mutations) has shown it to be in the region of the rnh gene encoding RNase H. Here we report that Das13 mutant of RNase H has substitutions of valine 43 and leucine 242 with isoleucines. To investigate the influence of these mutations on RNase H nuclease properties we have designed a novel in vitro assay that allows us to separate and quantify exo- or endonuclease activities of flap endonuclease. The nuclease assay in vitro showed that V43I substitution increased the ratio between exonuclease/endonuclease activities of RNase H whereas L242I substitution did not affect the nuclease activity of RNase H in vitro. However, both mutations were necessary for the full das effect in vivo. Molecular modelling of the nuclease structure suggests that V43I substitution may lead to disposition of H4 helix, responsible for the interaction wit...

Source: Mutation Research Fundamental and Molecular Mechanisms of Mutagenesis - September 25, 2015 Category: Cytology Source Type: research