RNase H (Ribonuclease H) is conserved in species ranging from bacteria to humans. It degrades the RNA strand in an RNADNA hybrid and removes RNA primers during DNA replication. Knock-out of rnh1 gene in mice results in embryonic lethality due to failure of mitochondrial DNA replication. HIV encodes its own viral RNase H activity, which is essential for converting genomic RNA to dsDNA before integration into human genome. In addition to removal of RNA primers during both (-) and (+) strand synthesis, HIV RNase H degrades viral genomic RNA after reverse transcription of the (-) strand and generates a primer for the (+) strand synthesis. As one of only four enzymes encoded by HIV and essential for its infection, the viral RNase H is an obvious target for developing anti-HIV drugs. A number of RNase H structures from cellular and viral origins have been determined by X-ray crystallography or NMR in the past 17 years, but an enzyme-substrate complex revealing how an RNA/DNA hybrid is recognized by RNase H remained elusive until two years ago. With the IATAP funding and in collaboration with Dr. Robert Crouchs group in NICHD, my group determined the first crystal structure of RNase H (from B. haloduran) complexed with an RNA/DNA hybrid substrate and elucidated the substrate specificity and catalytic mechanism in 2005. Our results were published in Cell. Since then, we have determined the crystal structures of B. haloduran RNase H bound to cleavage intermdiate analog and product, thereby resolving a long standing mystery about the requirement for two metal ions for nucleic acid synthesis and degradation. These results have been published in EMBO J. and Molecular Cell in 2005-2006. In the last fiscal year, we determined the crystal structure of human RNase H catalytic domain (RNase HC) complexed with RNA/DNA substrate to reveal the difference between cellular and viral enzymes. They share a conserved active site, but differ in substrate binding and cleavage specificity. Modeling of HIV reverse transcriptase (RT), which contains both the polymerase and RNase H activity, suggests that an RNA/DNA substrate cannot simultaneously occupy the polymerase and RNase H active sites and must undergo a conformational change to toggle between the two catalytic centers. The RT region that accommodates this conformational change offers a new target to develop HIV-specific inhibitors. The results have been written up and are in press at Molecular Cell. In this fiscal year, we have determined the N-terminal substrate binding domain of human RNase H in complex with RNA/DNA hybrid and characterized its binding preference of RNA/DNA hybrid over dsRNA and dsDNA. The results has been published in EMBO J. To capture a crystal structure of viral RNase H complexed with its substrate, we have produced the full-length HIV reverse transcriptase and RNase H catalytic mutant. Special RNA/DNA substrates are being designed to target the RNase active site for crystallization trials with HIV RT. In collaboration with Dr. Stuart Le Grice's group at NCI, we have started a broad screen of HIV RT variants and RNA/DNA hybrids for co-crystals with RNA/DNA substrate at the RNase H active site. References Nowotny, M., Gaidamakov, S. A., Ghirlando, R., Cerritelli, S. M., Crouch, R. J. and Yang, W. (2007). Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription. Mol. Cell, 28, 264-276.

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Tian, Lan; Kim, Min-Sung; Li, Hongzhi et al. (2018) Structure of HIV-1 reverse transcriptase cleaving RNA in an RNA/DNA hybrid. Proc Natl Acad Sci U S A 115:507-512
Samara, Nadine L; Yang, Wei (2018) Cation trafficking propels RNA hydrolysis. Nat Struct Mol Biol 25:715-721
Samara, Nadine L; Gao, Yang; Wu, Jinjun et al. (2017) Detection of Reaction Intermediates in Mg2+-Dependent DNA Synthesis and RNA Degradation by Time-Resolved X-Ray Crystallography. Methods Enzymol 592:283-327
Cheng, Xiaoming; Xia, Yuchen; Serti, Elisavet et al. (2017) Hepatitis B virus evades innate immunity of hepatocytes but activates cytokine production by macrophages. Hepatology 66:1779-1793
Rosta, Edina; Yang, Wei; Hummer, Gerhard (2014) Calcium inhibition of ribonuclease H1 two-metal ion catalysis. J Am Chem Soc 136:3137-44
Lapkouski, Mikalai; Tian, Lan; Miller, Jennifer T et al. (2013) Complexes of HIV-1 RT, NNRTI and RNA/DNA hybrid reveal a structure compatible with RNA degradation. Nat Struct Mol Biol 20:230-236
Lapkouski, Mikalai; Tian, Lan; Miller, Jennifer T et al. (2013) Reply to ""Structural requirements for RNA degradation by HIV-1 reverse transcriptase"". Nat Struct Mol Biol 20:1342-3
Yang, Wei (2011) Nucleases: diversity of structure, function and mechanism. Q Rev Biophys 44:1-93
Rosta, Edina; Nowotny, Marcin; Yang, Wei et al. (2011) Catalytic mechanism of RNA backbone cleavage by ribonuclease H from quantum mechanics/molecular mechanics simulations. J Am Chem Soc 133:8934-41
Nowotny, Marcin; Yang, Wei (2009) Structural and functional modules in RNA interference. Curr Opin Struct Biol 19:286-93