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Catalytic center of an archaeal type 2 ribonuclease H as revealed by X-ray crystallographic and mutational analyses

¹ Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka 565-0871, Japan
² Department of Structural Biology, Biomolecular Engineering Research Institute, Osaka 565-0874, Japan

Reprint requests to: Dr. Kosuke Morikawa, Department of Structural Biology, Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565-0874, Japan; e-mail: morikawa{at}beri.co.jp; fax: 81-6-6872-8219.

The catalytic center of an archaeal Type 2 RNase H has been identified by a combination of X-ray crystallographic and mutational analyses. The crystal structure of the Type 2 RNase H from Thermococcus kodakaraensis KOD1 has revealed that the N-terminal major domain adopts the RNase H fold, despite the poor sequence similarity to the Type 1 RNase H. Mutational analyses showed that the catalytic reaction requires four acidic residues, which are well conserved in the Type 1 RNase H and the members of the polynucleotidyl transferase family. Thus, the Type 1 and Type 2 RNases H seem to share a common catalytic mechanism, except for the requirement of histidine as a general base in the former enzyme. Combined with the results from deletion mutant analyses, the structure suggests that the C-terminal domain of the Type 2 RNase H is involved in the interaction with the DNA/RNA hybrid.

Keywords: Ribonuclease H; DNA/RNA hybrid; polynucleotidyl transferase family; crystal structure; mutational analysis

Abbreviations: RNase, ribonuclease • DNA, deoxyribonucleic acid • RNA, ribonucleic acid • MIR, multiple isomorphous replacement • PEG, polyethylene glycol • MES, 2-(N-morpholino)ethanesulfonic acid • MALDI, matrix-assisted laser desorption ionization • TOF, time-of-flight

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