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

Author:

González de Cózar Jose M1ORCID,Carretero-Junquera Maria1,Ciesielski Grzegorz L123ORCID,Miettinen Sini M4,Varjosalo Markku4,Kaguni Laurie S12,Dufour Eric1,Jacobs Howard T1ORCID

Affiliation:

1. Faculty of Medicine and Health Technology, FI-33014 Tampere University, Finland

2. Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824, USA

3. Department of Chemistry, Auburn University at Montgomery, Montgomery, AL 36117, USA

4. Institute of Biotechnology, FI-00014 University of Helsinki, Finland

Abstract

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.

Funder

Academy of Finland [Centre of Excellence

Academy Professorship

Finland Distinguished Professorship

Finnish Cultural Foundation

Tampere University

Sigrid Juselius Foundation

Publisher

Oxford University Press (OUP)

Subject

Molecular Biology,Biochemistry,General Medicine

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