Ribonuclease H

Ribonuclease H (RNase H) is an enzyme that specifically hydrolyzes the RNA strand of RNA/DNA hybrids to yield 3′-hydroxyl and 5′-phosphate groups. The ‘H’ of RNase H represents ‘hybrid’. The enzyme is widely present in various organisms, including bacteria, archaea and eukaryotes, and is involved in DNA replication, repair and transcription. The enzyme is also present in retroviruses as the C-terminal domain of reverse transcriptase. This activity is required for the proliferation of retroviruses, and HIV-1 RNase H is therefore regarded as one of the targets for AIDS therapy. The rnh gene encoding RNase H was first cloned from Escherichia coli in 1983. This gene and the encoded protein were designated as rnhA and RNase HI, respectively, when the second rnhB gene encoding RNase HII was also cloned from E. coli in 1990. E. coli RNases HI and HII consist of a single polypeptide chain with 155 and 198 amino acid residues, respectively, and show a poor amino acid sequence similarity with each other. As is the case for many enzymes, these RNases H provide a starting point for comparison of various RNases H. Database searches, using programs such as blast, indicate that all RNases H identified to date are either an E. coli RNase HI or RNase HII homologue. Therefore, RNases H have been classified into two major families, type 1 and type 2, based on the difference in their amino acid sequences. E. coli RNases HI and RNase HII represent type 1 and type 2 RNases H, respectively. Studies on RNase H, including methods related to RNase H and the use of RNase H to inhibit specific gene expression, were reviewed in 1998 in the book Ribonuclease H (edited by R. J. Crouch and J. J. Toulme). Since then, however, our knowledge of the structures and functions of RNases H has increased greatly. For example, the number of the crystal structures of RNases H deposited in the PDB increased fourfold in this decade. Determination of the co-crystal structure of RNase H with the substrate and Mg2+ settled a longstanding controversy as to whether the enzyme requires one or two metal ions for activity; the structure indicated that two metal ions bind to the active site of the protein, such that they directly or indirectly coordinate with acidic active site residues and the scissile phosphate group of the substrate. According to a mechanism currently proposed for the cleavage reaction catalyzed by RNase H, one metal ion is required for substrate-assisted nucleophile formation and product release, and the other is required to destabilize the enzyme–substrate complex and thereby promote the phosphoryl transfer reaction. Generation of RNase H1 knockout mice showed that these mice die during embryogenesis owing to the failure of mitochondrial DNA replication. Combination of positional mapping, homology prediction and functional studies indicated that eukaryotic type 2 RNases H are functional in a heterotrimeric form and mutation in either of these subunits causes an autosomal recessive neurological disorder in humans. These results corroborate the importance of studies on RNases H from various sources. In this minireview series, recent advances are covered in three minireviews which focus on prokaryotic, eukaryotic and retroviral RNases H. Tadokoro and Kanaya provide an overview of the molecular diversities, catalytic mechanism and substrate-binding domains of prokaryotic RNases H. Crouch focuses on eukaryotic RNases H, especially on yeast and human RNases H, and Champoux and Schultz provide an overview of the unique RNase H activity associated with reverse transcriptases from human immunodeficiency virus and Moloney murine leukemia virus.