Our research is directed toward understanding processes involved in cellular DNA replication and the relationship of HIV replication to these cellular events and using such information for therapeutic purposes. We are examining the formation and resolution of RNA-DNA hybrids formed during DNA replication or transcription. Ribonucleases H are important enzymes that participate in the removal of RNA from the RNA-DNA hybrids and are intimately involved in DNA replication in cells and, during HIV replication, in the conversion of the RNA genome of this virus to DNA. Using a similar protein architecture, RNases H of cells and HIV share common enzymatic mechanisms of RNA cleavage. Drugs to alter levels of specific disease-related genes are under development to take advantage of RNases H within the cell. Regulated expression of RNases H could enhance the efficacy of the drugs. Molecular genetic, biochemical, and mouse animal models are central to our efforts.

RNase H1 Requirement for Embryogenesis in Mice
Cerritelli, Crouch; in collaboration with Love
Mouse and human RNases H1 have an N-terminal stretch of amino acids that is absent from RNases H1 of simple eukaryotes (e.g., yeasts) and has the properties of a mitochondrial localization sequence. Using transient transfection assays, we demonstrated mitochondrial targeting by the 26 amino acid leader of the mouse RNase H1. We made an Rnaseh1 knockout to test for the effects on mitochondrial function in vivo. We found that growth is arrested in Rnaseh1-/- mice in utero at day E8.5, that the mice have defective respiratory function, and that they exhibit massive apoptotic cell death; the latter two effects are both direct results of a failure in mitochondrial DNA replication. Mitochondrial DNA replication occurs by two mechanisms, and our results suggest that RNase H1 is required in one or both. Mammalian oocytes contain numerous mitochondria, which, upon fertilization, become distributed without amplification to cells of the developing embryos. As a consequence, mitochondrial DNA replication is delayed until midgestation. We suggest that, during development, the two modes of mitochondrial DNA replication are likely to be activated sequentially and that RNase H1 is required for the earliest system, which is established during mouse embryogenesis.

Mechanism of Action of Mouse and Human RNases H1
Gorshkova, Gaidamkov, Cerritelli, Crouch; in collaboration with Schuck
A few years ago, we discovered that eukaryotic RNases H1have an RNase H domain similar in size, amino acid sequence, and structure to that we reported for E. coli RNase HI. Eukaryotic RNases H1 have an additional domain connected to the N-terminus of the RNase H domain, which can bind to duplex RNAs even in the absence of the RNase H domain. We have now shown that this “extra” dsRNA-binding domain aids in dimerization of the full-length protein when an RNA-DNA substrate is present. Moreover, the presence of the dsRNA-binding region transforms the RNase H activity from random to processive (i.e., the enzyme remains bound to the substrate until as much of the substrate as possible is degraded). Most RNA-DNA hybrids used for priming DNA synthesis are relatively short and would not seem to require processivity. We know that one exception is the RNA-DNA formed during mitochondrial DNA replication. Thus, the findings described above in the previous section fit well with the properties of RNase H1 we have uncovered.

Eukaryotic RNase H2 Activation
Jeong, Crouch
Bacterial and archael RNases HII are active as a single polypeptide chain, and several key residues have been shown to be a part of the active site. The eukaryotic RNase H2 enzyme is considerably larger than RNases HII, yet the orthologous eukaryotic polypeptide is similar in size and sequence to the smaller RNases HII. We have expressed the RNase H2 polypeptide encoded by human, mouse, Caenorhabditis elegans, and Saccharomyces cerevisiae in E. coli and uniformly found the protein to have no RNase H enzymatic activity. It seems that eukaryotic RNases H2 require some modification and/or more than one subunit for RNase H2 activity. From S. cerevisiae cells, we have affinity-purified the RNase H2 polypeptide of S. cerevisiae and found that two proteins co-purify in a complex. The identities of these proteins have been determined by using mass spectrometry. RNase H2 activity is absent from S. cerevisiae strains in which either of the two genes encoding these proteins has been deleted, suggesting that both proteins may be components of the active enzyme. The two proteins have been reported to interact with each other, but no evidence has linked them to RNase H2. However, they have been connected with some of the protein kinases involved in cell cycle regulation. Earlier work in our group demonstrated cell cycle regulation of S. cerevisiae RNase H2.