Robert Crouch, who leads the Section on Formation of RNA, studies the relationship between formation and resolution of RNA-DNA interactions seen in normal and disease states. From the earliest studies, the research has focused on RNases H, enzymes that degrade RNA in RNA/DNA hybrids. Recent exploration of the biological roles of RNases H, as well as their relationships to human diseases, has been greatly aided by the lab’s contributions defining the composition and structural interactions with their substrates of the two classes of these enzymes in prokaryotes and eukaryotes. Type I RNase H is required for mammalian development with a critical role in mitochondrial DNA replication and is structurally and functionally related to an essential RNase H of the HIV-AIDS virus. Thus, how Type I enzymes are related to important maintenance of cells or how they can be protected for HIV-induced damage are major areas of ongoing studies in the lab. Type II RNase H is important for (i) embryonic development and (ii) because mutations in human RNase H2 cause Aicardi-Goutières syndrome (AGS), a severe neurological disorder with a majority of patients having inability to communicate and poor or non-existent mobility. It is currently thought that self-nucleic acids mimic those of viruses and patient’s cells respond by inducing an innate immune response as if they were continuously being infected.
Mouse models based on RNase H1 or H2 are being used to further our knowledge of the roles of RNases H. Conditional knockout (KO) mice allow us to delete the Rnaseh1 gene in any tissue. As adaptive immunity is critical to fight off infections yet is not required for viability in a germ-free environment, studies on mouse B-cell development provide us a unique system for defining the role(s) of RNase H1 in a well-defined pathway without causing lethality. RNase H1 is directed to mitochondria and nuclei presenting a major challenge for any study with Rnaseh1-KO mice because of simultaneous loss of function in the two cellular compartments. Meeting this challenge in B-cell studies will provide not only what role RNase H1 plays in the progress of development but also provide a means of specifically eliminating RNase H1 in other tissues. Mice deleted of Rnaseh2a, the catalytic subunit of the heterotrimeric mouse RNase H2, are embryonic lethal, but in AGS patients mutations result in RNase H2 with lower enzymatic activity. Using the mouse version of the AGS-RNase H2 mutation, Rnaseh2aG37S, we are examining how mice develop and how their physical properties are related to that seen with AGS patients. In addition, we have developed another mouse model that is based on our structure-function studies of RNase H2. The mutant mouse, RNase H2-RED (RNA Excision Defective), is capable of hydrolysis of RNA/DNA hybrids but is defective in excising ribonucleotides incorporated during DNA replication. We anticipate studies with each of these mouse models will take us into a wide range of biological areas including innate immunity, neurological studies and many more.
Chon, H., Sparks, J.L., Rychlik, M., Nowotny, M., Burgers, P.M., Crouch, R.J., and Cerritelli, S.M. RNase H2 roles in genome integrity revealed by unlinking its activities. Nucleic Acids Res. 41:3130-3143, 2013. PMID: 23355612
Suzuki,Y., Holmes, J.B., Cerritelli, S.M., Sakhuja K., Minczuk, M., Holt, I.J. and Crouch, R.J., An uORF and context of the two AUG codons affect the abundance of mitochondrial and nuclear RNase H1, Mol. Cell. Biol., 30, 5123-5134, 2010. PMID: 20823270
- Rychlik MP, Chon H, Cerritelli SM, Klimek P, Crouch RJ, Nowotny M. Crystal structures of RNase H2 in complex with nucleic acid reveal the mechanism of RNA-DNA junction recognition and cleavage. Mol Cell. 2010 Nov 24;40(4):658-70. PMID: 21095591
- Suzuki Y, Holmes JB, Cerritelli SM, Sakhuja K, Minczuk M, Holt IJ, Crouch RJ.
An upstream open reading frame and the context of the two AUG codons affect the abundance of mitochondrial and nuclear RNase H1. Mol Cell Biol. 2010 Nov;30(21):5123-34. Epub 2010 Sep 7.PMID: 20823270
- Pohjoismäki JL, Holmes JB, Wood SR, Yang MY, Yasukawa T, Reyes A, Bailey LJ, Cluett TJ, Goffart S, Willcox S, Rigby RE, Jackson AP, Spelbrink JN, Griffith JD, Crouch RJ, Jacobs HT, Holt IJ. Mammalian mitochondrial DNA replication intermediates are essentially duplex but contain extensive tracts of RNA/DNA hybrid. J Mol Biol. 2010 Apr 16;397(5):1144-55. Epub 2010 Feb 23.PMID: 20184890
- Cerritelli SM, Crouch RJ. Ribonuclease H: the enzymes in eukaryotes. FEBS J. 2009 Mar;276(6):1494-505. Epub 2008 Feb 18. Review.PMID: 19228196
- Chon H, Vassilev A, DePamphilis ML, Zhao Y, Zhang J, Burgers PM, Crouch RJ, Cerritelli SM. Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex. Nucleic Acids Res. 2009 Jan;37(1):96-110. Epub 2008 Nov 16.PMID: 19015152
Nowotny, M., Gaidamakov, S.A., Crouch, R.J., and Yang, W. Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis, Cell,121, 1005-1016, 2005. PMID: 15989951
Jeong, H.-S. Backlund, P.S., Chen, H.-C., Karavanov, A.A., and Crouch, R.J., Ribonuclease H2 of Saccharomyces cerevisiae is a complex of three proteins, Nucleic Acids Res., 32, 407-414, 2004. PMID: 14734815