The Structural and Biochemical Characterization of Human RNase H2 Complex Reveals the Molecular Basis for Substrate Recognition and Aicardi-Goutières Syndrome Defects

Małgorzata Figiel,Hyongi Chon,Susana M Cerritelli,Magdalena Cybulska,Robert J Crouch,Marcin Nowotny

doi:10.1074/jbc.m110.181974

Abstract

RNase H2 cleaves RNA sequences that are part of RNA/DNA hybrids or that are incorporated into DNA, thus, preventing genomic instability and the accumulation of aberrant nucleic acid, which in humans induces Aicardi-Goutières syndrome, a severe autoimmune disorder. The 3.1 Å crystal structure of human RNase H2 presented here allowed us to map the positions of all 29 mutations found in Aicardi-Goutières syndrome patients, several of which were not visible in the previously reported mouse RNase H2. We propose the possible effects of these mutations on the protein stability and function. Bacterial and eukaryotic RNases H2 differ in composition and substrate specificity. Bacterial RNases H2 are monomeric proteins and homologs of the eukaryotic RNases H2 catalytic subunit, which in addition possesses two accessory proteins. The eukaryotic RNase H2 heterotrimeric complex recognizes RNA/DNA hybrids and (5′)RNA-DNA(3′)/DNA junction hybrids as substrates with similar efficiency, whereas bacterial RNases H2 are highly specialized in the recognition of the (5′)RNA-DNA(3′) junction and very poorly cleave RNA/DNA hybrids in the presence of Mg2+ ions. Using the crystal structure of the Thermotoga maritima RNase H2-substrate complex, we modeled the human RNase H2-substrate complex and verified the model by mutational analysis. Our model indicates that the difference in substrate preference stems from the different position of the crucial tyrosine residue involved in substrate binding and recognition.

Highlights

Ribonucleases H (RNases H) are nucleases that cleave RNA/DNA hybrids [1]
The B and C accessory subunits are unique to eukaryotic RNases H2, and their functions are unknown, they are assumed to assist the catalytic subunit
In our hands the expression of RNase H2A alone resulted in mostly insoluble protein, and the small amount of enzyme that could be partially purified in soluble form was inactive for the cleavage of RNA/DNA and RNA-DNA/DNA hybrid substrates

Summary

EXPERIMENTAL PROCEDURES

Protein Preparation—To allow the testing of different combinations of truncated subunits, subunit A was cloned into a pET28 expression vector, and subunits B and C were cloned into a pET15 vector (EMD Biochemicals). The need to rebuild this region was justified by both the observation that the electron density in this area is clearly split in two traces and the fact that the deletion mutant of the B subunit we used for crystallization was C-terminally truncated at residue 233 and, lacked the fragment corresponding to mouse residues B:267–275. Another fragment that was rebuilt in the human structure is the region corresponding to the mouse residues C:83–94 (Fig. 2, B, D, and E). The melting temperature (Tm) was defined as the temperature showing the maximum fluorescence gradient

RESULTS AND DISCUSSION

Space group

Impaired stability of the structure Impaired stability of the structure