Identification of the ternary complex of ribonuclease HI:RNA/DNA hybrid:metal ions by ESI mass spectrometry

Tomoshige Ando,Nujarin Jongruja,Nobuaki Okumura,Kosuke Morikawa,Shigenori Kanaya,Toshifumi Takao

doi:10.1016/j.jbc.2021.100462

Tomoshige Ando, Nujarin Jongruja + Show 4 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100462

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Abstract

Ribonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme–substrate complex in the presence of divalent metal ions (Mn2+ or Zn2+) using electrospray ionization–mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn2+ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn2+ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn2+], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn2+ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate.

Highlights

Ribonuclease H (RNase H), a ubiquitous enzyme, is found in all organisms from bacteria to mammals [1]
The electrospray ionization–mass spectrometry (ESI-mass spectrometry (MS)) of an equimolar mixture of RNase HI and 8-mer RNA/8-mer DNA gave multiply charged ion peaks (7+ to 9+), which corresponded to a 1:1 complex of RNase HI: RNA/DNA hybrid, implying that the complex was stable in solution and the gaseous phase after being sprayed, and its ion particles were maintained during the mass measurement (Fig. 1A)
It is noteworthy that the intensity of the complex with either the DNA and RNA strand was much less than that with the RNA/DNA hybrid (Fig. S2), which could be partly attributed to a lack of synergetic binding force between the strands or either of the strands and the enzyme

Summary

Introduction

Ribonuclease H (RNase H), a ubiquitous enzyme, is found in all organisms from bacteria to mammals [1]. We report, for the first time, the detection of the ternary complex of native RNase HI: RNA/ DNA hybrid: divalent metal ions (Mn2+ and Zn2+).

Results

Conclusion