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

Kholod, Natalia; Sivogrivov, Dmitry E.; Latypov, O. R.; Mayorov, Sergey G.; Kuznitsyn, Rafail; Kajava, Andrey V.; Shlyapnikov, M. G.; Granovsky, Igor

doi:10.1016/j.mrfmmm.2015.09.004

Cited by 1 publication

(1 citation statement)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An exoribonuclease activity associated with an RNaseH has been reported for viral RNaseHs, including the HIV (Schatz et al, 1990), herpes simplex virus 1 (Crute and Lehman, 1989), avian myoblastosis virus (Grandgenett and Green, 1974), and bacteriophage T4 (Kholod et al, 2015) enzymes. The HIV reverse transcriptase and RNaseH domains are spatially arranged such that the terminal ribonucleotides cannot be efficiently hydrolyzed because the polymerase domain is covering them when the reverse transcriptase-RNaseH holoenzyme has migrated to the end of the template.…”

Section: Discussionmentioning

confidence: 99%

Purification and enzymatic characterization of the hepatitis B virus ribonuclease H, a new target for antiviral inhibitors

et al. 2016

View full text Add to dashboard Cite

Hepatitis B virus (HBV) reverse transcription requires coordinated function of the reverse transcriptase and ribonuclease H (RNaseH) activities of the viral polymerase protein. The reverse transcriptase has been biochemically characterized, but technical difficulties have prevented both assessment of the RNaseH and development of high throughput inhibitor screens against the RNaseH. Expressing the HBV RNaseH domain with both maltose binding protein and hexahistidine tags led to stable, high-level accumulation of the RNaseH in bacteria. Nickel-affinity purification in the presence of Mg2+ and ATP removed co-purifying bacterial chaperones and yielded nearly pure monomeric recombinant enzyme. The endonucleolytic RNaseH activity required an DNA:RNA duplex ≥14 nt, could not tolerate a stem-loop in either the RNA or DNA strands, and could tolerate a nick in the DNA strand but not a gap. The RNaseH had no obvious sequence specificity or positional dependence within the RNA, and it cut the RNA at multiple positions even within the minimal 14 nt duplex. The RNaseH also possesses a processive 3’-5’ exoribonuclease activity that is slower than the endonucleolytic reaction. These results are consistent with the HBV reverse transcription mechanism that features an initial endoribonucleolytic cut, 3’-5’ degradation of RNA, and a sequence-independent terminal RNA cleavage. These data provide support for ongoing anti-RNaseH drug discovery efforts.

show abstract

Section: Discussionmentioning

confidence: 99%