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 formed during DNA replication 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. Together with Dr. Marc Drolet, we have reported that an increase in RNA/DNA hybrid formation (R-loops) occurs in bacteria when protein synthesis is blocked, thereby freeing to nascent RNA to anneal to the DNA immediately after transcription. In eukaryotes, a similar observation has been reported and that increasing the level of RNase H1 can eliminate some of the problems created by failure to remove the RNA of R-loops. We now know that the eukaryotic RNases H are more complex than the bacterial enzymes, and that the enzymatic activity of mouse RNase H1 (and presumably other eukaryotic RNases H1) form a complex when binding to and RNA/DNA hybrid that enables the enzyme to be more efficient when degrading ceratin types of RNA/DNA hybrids. In collaboration with the groups at NCI- Frederick we have been examining 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.
| Nowotny, Marcin; Gaidamakov, Sergei A; Ghirlando, Rodolfo et al. (2007) Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription. Mol Cell 28:264-76 |
| Budihas, Scott R; Gorshkova, Inna; Gaidamakov, Sergei et al. (2005) Selective inhibition of HIV-1 reverse transcriptase-associated ribonuclease H activity by hydroxylated tropolones. Nucleic Acids Res 33:1249-56 |
| Gaidamakov, Sergei A; Gorshkova, Inna I; Schuck, Peter et al. (2005) Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain. Nucleic Acids Res 33:2166-75 |
| Nowotny, Marcin; Gaidamakov, Sergei A; Crouch, Robert J et al. (2005) Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis. Cell 121:1005-16 |
| Chan, King C; Budihas, Scott R; Le Grice, Stuart F J et al. (2004) A capillary electrophoretic assay for ribonuclease H activity. Anal Biochem 331:296-302 |
| Jeong, Ho-Sang; Backlund, Peter S; Chen, Hao-Chia et al. (2004) RNase H2 of Saccharomyces cerevisiae is a complex of three proteins. Nucleic Acids Res 32:407-14 |
| Broccoli, Sonia; Rallu, Fabien; Sanscartier, Patrick et al. (2004) Effects of RNA polymerase modifications on transcription-induced negative supercoiling and associated R-loop formation. Mol Microbiol 52:1769-79 |
| Backer, Marina V; Gaynutdinov, Timur I; Gorshkova, Inna I et al. (2003) Humanized docking system for assembly of targeting drug delivery complexes. J Control Release 89:499-511 |
| Pileur, Frederic; Andreola, Marie-Line; Dausse, Eric et al. (2003) Selective inhibitory DNA aptamers of the human RNase H1. Nucleic Acids Res 31:5776-88 |
| Cerritelli, Susana M; Frolova, Ella G; Feng, Chiguang et al. (2003) Failure to produce mitochondrial DNA results in embryonic lethality in Rnaseh1 null mice. Mol Cell 11:807-15 |
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