Crystal structure of RNase H3-substrate complex reveals parallel evolution of RNA/DNA hybrid recognition.

Małgorzata Figiel,Marcin Nowotny

doi:10.1093/nar/gku615

Małgorzata Figiel, Marcin Nowotny

Open Access

PDF Available

https://doi.org/10.1093/nar/gku615

Copy DOI

Export

Save

Cite

Abstract
Highlights/Summary
Full-Text PDF
Similar Papers

Abstract

Listen

RNases H participate in the replication and maintenance of genomic DNA. RNase H1 cleaves the RNA strand of RNA/DNA hybrids, and RNase H2 in addition hydrolyzes the RNA residue of RNA–DNA junctions. RNase H3 is structurally closely related to RNases H2, but its biochemical properties are similar to type 1 enzymes. Its unique N-terminal substrate-binding domain (N-domain) is related to TATA-binding protein. Here, we report the first crystal structure of RNase H3 in complex with its RNA/DNA substrate. Just like RNases H1, type 3 enzyme recognizes the 2′-OH groups of the RNA strand and detects the DNA strand by binding a phosphate group and inducing B-form conformation. Moreover, the N-domain recognizes RNA and DNA in a manner that is highly similar to the hybrid-binding domain of RNases H1. Our structure demonstrates a remarkable example of parallel evolution of the elements used in the specific recognition of RNA and DNA.

Highlights

Synthetic genes that encode Thermovibrio ammonificans (Ta), Thermocrinis albus (Tha), and Thermotoga lettingae (Tl) RNase H3 were purchased from Epoch Life Science and subcloned into a pET28 expression vector that carries an N-terminal His-tag and SUMO-tag that is removable by SENP protease
The vectors were transformed into Escherichia coli BL21 Star cells (Ta-RNase H3 and Tha-RNase H3) or RIL cells (Tl-RNase H3)
The D78N variant of Ta-RNase H3 was mixed with the hybrid in a 1:1.2 protein:substrate molar ratio

Summary

Introduction

Synthetic genes that encode Thermovibrio ammonificans (Ta), Thermocrinis albus (Tha), and Thermotoga lettingae (Tl) RNase H3 were purchased from Epoch Life Science and subcloned into a pET28 expression vector that carries an N-terminal His-tag and SUMO-tag that is removable by SENP protease. Pure protein was concentrated to 25-56 mg/ml and stored in 20 mM Tris (pH 8.0), 100 mM NaCl, 5% glycerol, 0.5 mM EDTA, and 1 mM DTT. The collected fraction was concentrated and applied to a Superdex 75 column equilibrated with 20 mM Tris (pH 8.0), 100 mM NaCl, 5% glycerol, 0.5 mM EDTA, and 1 mM DTT.

Results

Conclusion