title: Single substitution in bacteriophage T4 RNase H alters the ratio between its exo- and endonuclease activities.
abstract: 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 with the first base pairs of 5'end of branched DNA. These structural changes may affect unwinding of the first base pairs of gapped or nicked DNA generating a short flap and therefore may stabilize the DNA-enzyme complex. L242I substitution did not affect the structure of RNase H and its role in providing das-effect remains unclear.
authors.affiliation: Laboratory of Genetic Enzymology, Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, 5 Prospect Nauki, Pushchino, Moscow Region 142290, Russia.
authors.forename: Natalia
authors.initials: N
authors.lastname: Kholod
authors.affiliation: Laboratory of Genetic Enzymology, Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, 5 Prospect Nauki, Pushchino, Moscow Region 142290, Russia.
authors.forename: Dmitry
authors.initials: D
authors.lastname: Sivogrivov
authors.affiliation: Laboratory of Genetic Enzymology, Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, 5 Prospect Nauki, Pushchino, Moscow Region 142290, Russia.
authors.forename: Oleg
authors.initials: O
authors.lastname: Latypov
authors.affiliation: Laboratory of Genetic Enzymology, Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, 5 Prospect Nauki, Pushchino, Moscow Region 142290, Russia.
authors.forename: Sergey
authors.initials: S
authors.lastname: Mayorov
authors.affiliation: Laboratory of Genetic Enzymology, Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, 5 Prospect Nauki, Pushchino, Moscow Region 142290, Russia; Federal Government-financed Educational Institution of Higher Professional Education «Vyatka State University», 36 Moskovskaya street, Kirov 610000, Russia.
authors.forename: Rafail
authors.initials: R
authors.lastname: Kuznitsyn
authors.affiliation: Centre de Recherches de Biochimie Macromoléculaire, CNRS, Université Montpellier 1 et 2, 1919 Route de Mende, 34293 Montpellier Cédex 5, France; The Institut de Biologie Computationnelle, 95 rue de la Galéra, 34095 Montpellier, Cédex, France.
authors.forename: Andrey V
authors.initials: AV
authors.lastname: Kajava
authors.affiliation: Laboratory of Genetic Enzymology, Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, 5 Prospect Nauki, Pushchino, Moscow Region 142290, Russia.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Shlyapnikov
authors.affiliation: Laboratory of Genetic Enzymology, Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, 5 Prospect Nauki, Pushchino, Moscow Region 142290, Russia. Electronic address: granovsky@ibpm.pushchino.ru.
authors.forename: Igor
authors.initials: I
authors.lastname: Granovsky
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics*; D017122:Bacteriophage T4 / Q000201:enzymology*; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D005092:Exonucleases / Q000378:metabolism; D045585:Flap Endonucleases / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020125:Mutation, Missense / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013489:Suppression, Genetic / Q000235:genetics
chemical_list: D017931:DNA Primers; D005092:Exonucleases; D045585:Flap Endonucleases; D016914:Ribonuclease H
keywords: Bacteriophage; DNA arrest suppression; Flap endonuclease; RNase H