Research in the Section on Formation of RNA is directed toward understanding and utilization of processes involved in cellular DNA replication, the relationship of HIV replication to these cellular events, and how to use this information for therapeutic purposes. We are examining the formation and resolution of RNA/DNA hybrids occuring during DNA replication and/or transcription. Ribonucleases H are important enzymes participating in removal of the RNA of the RNA/DNA hybrids and are intimately related to DNA replication in cells and in HIV replication, during the conversion of the RNA genome of this virus to DNA. RNases H of cells and HIV share common enzymatic mechanisms of cleavage of RNA utilizing similar protein architectures. Drugs to alter levels of specific disease-related genes are being developed 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 employed in these efforts. During transcription, RNA/DNA hybrids can form and are usually cleaved by RNase H. RNase H recognizes and specifically cleaves RNA of hybrids by several contacts to the RNA and interactions with the DNA strand using a two metal ion mechanism. We now know that the eukaryotic RNases H are more complex than the bacterial enzymes, and that the enzymatic activity of eukaryotic RNases H1 form a complex when binding to an RNA/DNA hybrid that enables the enzyme to be more efficient when degrading certain types of RNA/DNA hybrids. In collaboration with the groups at NCI-Frederick, we have been examined inhibitors of RNases H that could prove useful in HIV-AIDS therapy. From our earlier work showing that the mouse RNase H1 is critical for development, we know that it is important to have drugs that specifically target the HIV-AIDS RNase H not the cellular enzyme. Screening of potential drugs now includes use of the human RNase H1 isolated in our laboratory. Mouse RNase H1 is translated from a single messenger RNA to produce the same protein to reside in nuclei and in mitochondria. Production of the two forms depends on a translation process that we have found to be conserved in many eukaryotic organisms and in othe DNA transacting proteins targeted to both the nucleus and mitochondria. AGS is a syndrome is characterized as a severe neurological disorder that mimics in utero viral infection resulting in loss of white matter in the brain and producing high levels of interferon alpha in the cerebral spinal fluid. The disorder in many of the AGS patients results from mutations in any of the three subunits of human RNase H2. Our work on the homologous enzyme from Saccharomyces cerevisiae was the first describing the three subunit composition of eukaryotic RNases H2. Deletion of any genes encoding the three subunits of RNase H2 in S. cerevisiae does not result in any major phenotype unless the mutant is combined with any of several other gene deletions, a condition known as synthetic lethality. We are currently examining the biochemical properties of human and S. cerevisiae RNase H2, and, in particular, are examining the role of RNase H2 in DNA replication, repair and recombination. Our in vitro studies show that most of the proteins with mutations found in AGS patients have nearly normal enzymatic activity but it seems likely that these mutations affect interaction with other cellular proteins or components and from recent structural analysis, some weakening of the subunit interactions may occur leading to more severe problems in brain than in other organs. Recent work at NIEHS has found that S. cerevisiae DNA polymerases mistakenly incorporate ribonucleotides rather than deoxynucleotides into DNA during replication. Removal of the ribonucleotides depends on RNase H2 cleavage.
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