A second hybrid-binding domain modulates the activity of Drosophila ribonuclease H1.

Jose M González De Cózar,Grzegorz L Ciesielski,Eric Dufour,Maria Carretero-Junquera,Sini M Miettinen,Markku Varjosalo,Laurie S Kaguni,Howard T Jacobs

doi:10.1093/jb/mvaa067

Abstract

In eukaryotes, ribonuclease H1 (RNase H1) is involved in the processing and removal of RNA/DNA hybrids in both nuclear and mitochondrial DNA. The enzyme comprises a C-terminal catalytic domain and an N-terminal hybrid-binding domain (HBD), separated by a linker of variable length, 115 amino acids in Drosophila melanogaster (Dm). Molecular modelling predicted this extended linker to fold into a structure similar to the conserved HBD. Based on a deletion series, both the catalytic domain and the conserved HBD were required for high-affinity binding to heteroduplex substrates, while loss of the novel HBD led to an ∼90% drop in Kcat with a decreased KM, and a large increase in the stability of the RNA/DNA hybrid-enzyme complex, supporting a bipartite-binding model in which the second HBD facilitates processivity. Shotgun proteomics following in vivo cross-linking identified single-stranded DNA-binding proteins from both nuclear and mitochondrial compartments, respectively RpA-70 and mtSSB, as prominent interaction partners of Dm RNase H1. However, we were not able to document direct and stable interactions with mtSSB when the proteins were co-overexpressed in S2 cells, and functional interactions between them in vitro were minor.

Highlights

Structure modelling of Drosophila melanogaster (Dm) RNase ribonuclease H1 (H1) Dm RNase H1 was previously proposed to comprise three structural elements [47]: an N-terminal hybrid-binding domain (HBD) domain similar to a region of caulimovirus ORF VI protein [56], an RNase H catalytic domain located towards the C-terminus and a 115-amino acid extended linker that connects these domains
We evaluated the biochemical properties of Dm RNase H1, determined the functional roles of each region of the protein and probed for interacting proteins from the two cellular compartments in which RNase H1 is localized
We identified strong binding for RNA/DNA hybrid in the conserved HBD (Region I) and weaker hybrid binding to the catalytic domain (Region V)

Summary

Introduction

Ribonuclease H1 (RNase H1) is present both in mitochondria and in the nucleus, as distinct translation products encoded by a single mRNA [1, 2]. The enzyme digests the RNA strand of RNA/ DNA heteroduplexes longer than 4 bp [3] and has been implicated in diverse DNA transactions in one or both cellular compartments, including DNA replication [2, 4,5,6,7,8,9,10], transcription [11,12,13], recombination [14, 15], repair [16, 17] and telomere maintenance [18, 19]. Eukaryotic RNase H1 presents a conserved domain organization [20].

Methods

Results

Conclusion