title: Effect of the disease-causing mutations identified in human ribonuclease (RNase) H2 on the activities and stabilities of yeast RNase H2 and archaeal RNase HII.
abstract: Eukaryotic ribonuclease (RNase) H2 consists of one catalytic and two accessory subunits. Several single mutations in any one of these subunits of human RNase H2 cause Aicardi-Goutières syndrome. To examine whether these mutations affect the complex stability and activity of RNase H2, three mutant proteins of His-tagged Saccharomyces cerevisiae RNase H2 (Sc-RNase H2*) were constructed. Sc-G42S*, Sc-L52R*, and Sc-K46W* contain single mutations in Sc-Rnh2Ap*, Sc-Rnh2Bp*, and Sc-Rnh2Cp*, respectively. The genes encoding the three subunits were coexpressed in Escherichia coli, and Sc-RNase H2* and its derivatives were purified in a heterotrimeric form. All of these mutant proteins exhibited enzymatic activity. However, only the enzymatic activity of Sc-G42S* was greatly reduced compared to that of the wild-type protein. Gly42 is conserved as Gly10 in Thermococcus kodakareansis RNase HII. To analyze the role of this residue, four mutant proteins, Tk-G10S, Tk-G10A, Tk-G10L, and Tk-G10P, were constructed. All mutant proteins were less stable than the wild-type protein by 2.9-7.6 degrees C in T(m). A comparison of their enzymatic activities, substrate binding affinities, and CD spectra suggests that the introduction of a bulky side chain into this position induces a local conformational change, which is unfavorable for both activity and substrate binding. These results indicate that Gly10 is required to make the protein fully active and stable.
authors: Department of Material and Life Science, Graduate School of Engineering, Osaka University, Japan.
authors: Muhammad S
authors: MS
authors: Rohman
authors: Yuichi
authors: Y
authors: Koga
authors: Kazufumi
authors: K
authors: Takano
authors: Hyongi
authors: H
authors: Chon
authors: Robert J
authors: RJ
authors: Crouch
authors: Shigenori
authors: S
authors: Kanaya
mesh_terms: D000595:Amino Acid Sequence; D001480:Basal Ganglia Diseases / Q000235:genetics; D002942:Circular Dichroism; D004795:Enzyme Stability; D005998:Glycine / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016914:Ribonuclease H / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D016415:Sequence Alignment; D019712:Thermococcus / Q000201:enzymology*
chemical_list: C091139:ribonuclease HII; D016914:Ribonuclease H; D005998:Glycine