Physiological, Biochemical, and Molecular Genetic Events of Recognition and Resolution of RNA/DNA Hybrids

Robert J. Crouch, PhD, Head, Section on Formation of RNA
Susana M. Cerritelli, PhD, Staff Scientist
Hyongi Chon, PhD, Postdoctoral Fellow
Lina Gugliotti, PhD, Postdoctoral Fellow
Yutaka Suzuki, PhD, Postdoctoral Fellow
John Brad Holmes, BS, Predoctoral Fellow
Kiran Sakhuja, MS, MSc, Research Assistant
Mitsuru Haruki, PhD, Guest Researcher

The aim of our research is to understand processes involved in cellular DNA replication and the relationship of HIV replication to cellular DNA replication events and how to use the resultant information for therapeutic purposes. We are examining the formation and resolution of RNA/DNA hybrids that form during DNA replication or transcription. Ribonucleases H participate in the removal of the RNA of the RNA/DNA hybrids and are intimately related to DNA replication in cells and in HIV replication during conversion of the virus’s RNA genome to DNA. With similar protein architectures, RNases H of cells and HIV share common enzymatic mechanisms for cleaving RNA. Drugs that alter the levels of specific disease-related genes are in development to take advantage of RNases H within the cell. Regulated expression of RNases H could enhance the efficacy of the drugs. We employ molecular-genetic and biochemical tools and mouse animal models in our efforts.

Structure-function of ribonucleases H: RNase H1

Cerritelli, Crouch; in collaboration with Nowotny, Yang

RNA/DNA hybrids are essential intermediates in the replication of HIV’s RNA genome. In addition, the hybrids are believed to be necessary for mitochondrial DNA replication and important for switching immunoglobulin isotypes (e.g., from IgM to IgA). How these enzymes recognize and cleave the RNA is important in understanding the biology of these diverse events and for possible regulation of RNase H activity. Bacterial RNases HI are generally small proteins (150 amino acids) that share significant similarity with their eukaryotic counterparts in structure, interaction with their substrates, and the mechanism of cleavage. The protein structure of E. coli RNase HI unbound to a substrate is similar to that of the structure of human RNase H1 in complex with an RNA/DNA hybrid. The enzyme recognizes at least four hydroxyl groups of the ribose, with the DNA significantly distorted so that one phosphate is able to bind in a pocket on the enzyme (Nowotny et al., Cell 2005;121:1005). Both properties contribute to the specificities of the enzyme. The “basic protrusion” of the enzyme has extensive interactions with the hybrid, adding to the stability of the complex. Eukaroytic RNases H have an N-terminal domain (absent from the bacterial enzyme) that binds to RNA/DNA, conferring processivity to the enzyme (Gaidamakov et al., Nucleic Acids Res 2005;33:2166). This N-terminal domain (HBD) interacts with RNA/DNA through contacts with both the DNA and RNA strands and may provide the initial contact between the enzyme and hybrid.

The N- and C-terminal domains are connected by 65 amino acids in the human enzyme and by 64 in the mouse enzyme. The length and sequence of the connection domain are extremely variable in other species, suggesting that RNase H activity may not depend on any particular amino acid number or sequence. Our studies indicate that the connection domain is important for flexibility, allowing the protein to bind and cleave more effectively. Our current model of RNase H1 action posits that binding of the enzyme occurs mainly via the HBD and that the RNase H domain searches for an appropriate cleavage site and cleaves; following release of the RNase H domain from the hydrolyzed substrate, the HBD anchors the protein while the RNase H domain recognizes another site and cleaves; the process continues until either the HBD releases from the hybrid or no more RNase H cleavage sites are available to be attacked.

Nowotny M, Cerritelli SM, Ghirlando R, Gaidamakov SA, Crouch RJ, Yang W. Specific recognition of RNA/DNA hybrid and enhancement of human RNase H1 activity by HBD. EMBO J 2008;27:1172-1181.
Nowotny M, Gaidamakov SA, Ghirlando R, Cerritelli SM, Crouch RJ, Yang W. Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription. Mol Cell 2007;28:264-276.

Functions of RNase H1 in mitochondria and nuclei

Cerritelli, Sakhuja, Suzuki, Holmes, Crouch; in collaboration with Holt

One of the major challenges we face in understanding the roles of RNase H1 in cells is to determine how the translation of a single mRNA can produce both nuclear and mitochondrial forms of the enzyme. Knocking out the Rnaseh1 gene in mouse results in embryonic lethality at embryonic day 8.5 due to a failure to replicate mitochondrial DNA. The outcome indicates that (1) the enzyme is essential for the maintenance of mtDNA and (2) there is no need for newly synthesized RNase H1 for replication/repair of nuclear DNA. We have generated a conditional knockout that permits us to determine whether RNase H1 is necessary in adult animals either for mtDNA or nuclear DNA replication and repair. It may be that RNase H1 is important for mtDNA during embryogenesis only when, after implantation, rapid mtDNA synthesis occurs, at which time it is suddenly activated. In addition, we will be able to generate organ-/tissue-specific knockouts of the Rnaseh1 gene by using the Cre-lox system, including Tamoxifen®-inducible Cres for general ablation and others (e.g., heart-specific Cre expressers). However, the problem of inactivating the synthesis of both forms remains unresolved. Accordingly, we have generated transgenic mice that produce either both isoforms of RNase H1 or only the nuclear form in T- and B-cells. We are currently using the mice to examine effects on class switch recombination.

In collaboration with Ian Holt, we are searching for possible roles of RNase H1 in mitochondrial DNA replication by using, among other techniques, analysis of intermediates on two-dimensional gels. Our findings thus far indicate that elevated expression of RNase H1 in mitochondria alters mtDNA replication. In addition, data obtained in Holt’s laboratory by John Holmes supporting the existence of RNA/DNA hybrids as replication intermediates indicate a bi-directional mode of DNA replication for this organelle.

Structure-function of ribonucleases: RNase H2

Chon, Cerritelli, Crouch; in collaboration with Kanaya, Burgers

We previously found that RNase H2, the second type of RNase H, in Saccharomyces cerevisiae is composed of three subunits (Jeong et al., Nucleic Acids Res 2004;32:407), two of which we did not find in higher eukaryotes when using a BLAST search. We determined that there are three subunits of RNase H1 in human cells and were able to find similar RNases H2 in other mammals, in particular the mouse. A recent report in Nature Genetics demonstrated that mutations in any one of these proteins leads to the rare Aicardi-Goutières syndrome (AGS). Our studies on the human and yeast enzymes indicate a connection between RNase H2 and PCNA (proliferating cell nuclear antigen)—a clamp-loader protein involved in recruiting proteins to DNA for repair and replication. A PCNA-Interacting-Peptide (PIP) is present in the RNase H2B subunit of yeast, mouse, and human RNases H2. Using several types of analyses, we demonstrated that the PIP of the RNase H2B is indeed able to interact with PCNA, a finding whose biological significance we are continuing to explore. We also examined the effects of several of the AGS-causing mutations on recombinant RNase H2 activity. Only two of the mutant enzymes have shown decreased activity. The other altered proteins are most likely defective in vivo for complex formation and/or stability.

Rohman MS, Koga Y, Takano K, Chon H, Crouch RJ, Kanaya S. Effect of the disease-causing mutations identified in human RNase H2 on the activities and stabilities of yeast RNase H2 and archaeal RNase HII. FEBS J 2008;275:4836-4849.

Collaborators

Peter Burgers, PhD, Washington University, St. Louis, MO
Ian Holt, PhD, MRC-Dunn Nutrition Unit, Cambridge, UK
Shigenori Kanaya, PhD, Osaka University, Osaka, Japan
Marcin Nowotny, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Wei Yang, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD

For further information, contact crouchr@mail.nih.gov or visit http://sfr.nichd.nih.gov.