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

Document Type

Article

Publication Date

11-1-2020

Subject Area

Animals; Catalytic Domain; DNA-Binding Proteins (genetics, metabolism); Drosophila Proteins (genetics, metabolism); Drosophila melanogaster (enzymology, genetics, metabolism); Models, Molecular; Protein Binding; Ribonuclease H (chemistry, genetics, metabolism); Sequence Homology, Amino Acid; Structure-Activity Relationship; Substrate Specificity

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.

Publication Title

Journal of biochemistry

Volume

168

Issue

5

First Page

515

Last Page

533

Digital Object Identifier (DOI)

10.1093/jb/mvaa067

PubMed ID

32589740

E-ISSN

1756-2651

Language

eng

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