Proteins, which are the main components of living organisms, exhibit their functions only when the polypeptide chains fold to form three-dimensional structures. Proteins, which functionally or structurally resemble one another, are regarded as members of the same group that is generally termed as a family. The number of the protein families which exist in nature is expected to be roughly 1000. RNase H family is one of them. RNase H is an enzyme that specifically hydrolyzes RNA strand of RNA/DNA hybrid, and is thought to be involved in synthesis and removal of RNA primers which are required for DNA replication, and in elimination of RNAs which are miscorporated into DNA strands. Its tertiary structure is called RNase H-fold. This RNase H-fold is thought to represent a structural motif responsible for the functions of phosphoryl transferases, which are widely present in various organisms. In fact, it has been shown that various enzymes, which are different from RNase H in primary structure and function, such as resolvase and integrase, have RNase H-fold as well.

    In order to apply biological functions of organisms for industrial purposes, it is important to understand cellular functions at first. Because the cellular functions are basically controlled by protein functions, it is important to understand the protein functions to understand the cellular functions. Therefore, many proteins have been isolated from the cells and analyzed for their functions and structures in detail. However, the mechanisms by which proteins fold into unique tertiary structures and exhibit unique functions have not been fully understood. Likewise, the structure-function relationships of RNase H have not been fully understood. We are studying on the functions and structures of RNase H to get information which facilitates understanding for structures and functions of proteins in more detail and to establish a method to improve protein functions.

2-1. Molecular diversity of RNase H

    The genome sequences of various organisms including humans are being determined (Genome Project), and the genome sequences of more than ten organisms have already been determined. Since these sequences have been deposited in a data bank, anyone can find a gene of interest from the sequence similarity. By this method, we found that at least two types of RNase H, which have different amino acid sequences, exist in nature. We classified them as Type 1 and Type 2 RNases H. Three kinds of RNases H have been isolated from bacteria. They are RNases HI, HII, and HIII. Among them, RNase HII and RNase HIII resemble each other in the primary structure and are therefore thought to be paralogues. Both of them, which are the members of the Type 2 RNase H family, do not have a sequence similarity to RNase HI, which is a member of the Type 1 RNase H family, suggesting that Type 1 and Type 2 RNases H have different evolutionary origins. In addition, it has been shown that archaea contain only RNase HII, whereas bacteria and eukaryotes often contain both types of RNase H in a single cell. Moreover, it has been shown that Type 1 RNase H is not always contained in bacteria and eukaryotes. Archaea are thought to resemble more closely to eukaryotes than to bacteria evolutionarily. Thus, it is presumed that Type 2 RNase H represent an ancestral form of RNase H. An ancestral form of Type 1 RNase H may be generated after bacteria, archaea, and eukaryotes had diverged from one another, and then transferred horizontally from bacteria to eukaryotes through symbiotic association of bacteria with host cell early in the evolution of eukaryotes. In order to get data to support this hypothesis, we are cloning the genes encoding RNases H from various organisms, and analyzing their structures and functions.

2-2. Studies on the Application of RNase H

    RNase H has been widely used as one of the enzymes required for recombinant DNA technology. For example, it is used to synthesize cDNA, because it efficiently degrades and removes mRNA after mRNA was used as a template for the synthesis of cDNA. In addition, RNase H is used for removal of polyA-tail and editing of RNA. We intend to develop a new application technology for RNase H. For example, we are now attempting to construct an artificial RNA restriction enzyme by covalently attaching DNA oligomers to RNase H. RNA mediates a transfer of the information from DNA to protein. In addition, it shows various functions by forming various structures and receiving various modifications. Therefore, construction of a new RNA restriction enzyme may contribute to the understanding for the structures and functions of RNA. We are also looking for the inhibitor of RNase H by using peptide-library method. It is known that AIDS is caused by the HIV infection. Because the RNase H activity of reverse transcriptase is required for growth (proliferation) of HIV viruses, the inhibitor of RNase H is regarded as a potential candidate of a drug for AIDS therapy.