Identification of the first archaeal Type 1 RNase H gene from Halobacterium sp. NRC-1: archaeal RNase HI can cleave an RNA-DNA junction.
All the archaeal genomes sequenced to date contain a single Type 2 RNase H gene. We found that the genome of a halophilic archaeon, Halobacterium sp. NRC-1, contains an open reading frame with similarity to Type 1 RNase H. The protein encoded by the Vng0255c gene, possessed amino acid sequence identities of 33% with Escherichia coli RNase HI and 34% with a Bacillus subtilis RNase HI homologue. The B. subtilis RNase HI homologue, however, lacks amino acid sequences corresponding to a basic protrusion region of the E. coli RNase HI, and the Vng0255c has the similar deletion. As this deletion apparently conferred a complete loss of RNase H activity on the B. subtilis RNase HI homologue protein, the Vng0255c product was expected to exhibit no RNase H activity. However, the purified recombinant Vng0255c protein specifically cleaved an RNA strand of the RNA/DNA hybrid in vitro, and when the Vng0255c gene was expressed in an E. coli strain MIC2067 it could suppress the temperature-sensitive growth defect associated with the loss of RNase H enzymes of this strain. These results in vitro and in vivo strongly indicate that the Halobacterium Vng0255c is the first archaeal Type 1 RNase H. This enzyme, unlike other Type 1 RNases H, was able to cleave an Okazaki fragment-like substrate at the junction between the 3'-side of ribonucleotide and 5'-side of deoxyribonucleotide. It is likely that the archaeal Type 1 RNase H plays a role in the removal of the last ribonucleotide of the RNA primer from the Okazaki fragment during DNA replication.
Ohtani
Naoto
N
Institute for Advanced Biosciences, Keio University, Tsuruoka, Yamagata 997-0017, Japan. nao10_oh@ybb.ne.jp
Yanagawa
Hiroshi
H
Tomita
Masaru
M
Itaya
Mitsuhiro
M
D000595:Amino Acid Sequence / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology; D001426:Bacterial Proteins / Q000378:metabolism; D002413:Cations, Divalent / Q000737:chemistry; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D019143:Evolution, Molecular; D005816:Genetic Complementation Test / Q000379:methods; D020745:Genome, Archaeal; D006217:Halobacterium / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D016914:Ribonuclease H / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics
D001426:Bacterial Proteins; D002413:Cations, Divalent; D009692:Nucleic Acid Heteroduplexes; D012313:RNA; D004247:DNA; D016914:Ribonuclease H