PHYSIOLOGICAL, BIOCHEMICAL, AND MOLECULAR-GENETIC EVENTS OF RECOGNITION AND RESOLUTION OF RNA/DNA HYBRIDS
, Head, Section on Formation of RNA
Susana M. Cerritelli, PhD, Staff Scientist
Sergei A. Gaidamakov, PhD, Research Fellow
Lina Gugliotti, PhD, Postdoctoral Fellow
Hyongi Chon, PhD, Visiting Fellow
Yutaka Suzuki, PhD, Visiting Fellow
John Brad Holmes, BS, Predoctoral Fellow
Kiran Sakhuja,MS, MSC, Research Assistant
We focus on understanding the processes involved in cellular DNA replication, elucidating the relationship between HIV replication and cellular DNA replication events, and determining how best to use the resultant information for therapeutic purposes. We are examining the formation and resolution of RNA/DNA hybrids associated with DNA replication or transcription. Ribonucleases H are important enzymes that participate in removal of the RNA of RNA/DNA hybrids; they are also intimately related to DNA replication in cells and to HIV replication—during the conversion of the RNA genome of HIV to DNA. With similar protein architectures, RNases H of cells and HIV share common enzymatic mechanisms of RNA cleavage. Drugs that alter levels of specific disease-related genes are under development to take advantage of RNases H within the cell. Regulated expression of RNases H could enhance the efficacy of these drugs.
Structure-function of ribonucleases H: RNase H1
RNA/DNA hybrids are essential intermediates in the replication of the RNA genome of HIV and are necessary for mitochondrial DNA replication. It has been suggested that the hybrids are important in 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 replication events and for possible regulation of RNase H activity. Bacterial RNases HI are generally small (150–amino acid) proteins that share significant similarity with their eukaryotic counterparts with respect to their 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 can bind in a pocket on the enzyme. 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. Eukaryotic RNases H have an N-terminal domain (absent from the bacterial enzyme) that binds to RNA/DNA, conferring processivity to the enzyme. The N-terminal domain (dsRHbd) interacts with RNA/DNA through contacts to both the DNA and RNA strands and may provide the initial contact between the enzyme and hybrid. In the human enzyme, 65 amino acids connect the N- and C-terminal domains; in the mouse, 64 do so. In other species, the length and sequence of the connection domain vary considerably, suggesting that a particular amino acid number or sequence is not required for RNase H activity. We have shortened the connection domain of mouse RNase H1 and found that the ability of the dsRHbd to bind to duplex RNA and the ability of the RNase H domain to cleave hybrid substrates are both greatly diminished. Employing a new internally labeled substrate that permits us to demonstrate processivity on a defined RNA sequence, we found that mouse RNase H1 proteins with short connection domains remain processive and cleave at the same sites as the full-length enzyme. Our observations indicate that the connection domain is important for providing 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 dsRHbd 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 dsRHbd anchors the protein while the RNase H domain recognizes another site and cleaves; the process continues until either the dsRHbd releases from the hybrid or no more RNase H cleavage sites are available to be attacked. Our current substrate is of such a length as to allow the enzyme to be anchored via the dsRHbd to one end; even with our shortest connection domain, the RNase H domain can cleave anywhere on the RNA. Using a longer substrate, we should be able to estimate the “reach” of the enzyme provided by the connection domain (“reach” is defined as the maximum distance—from the attachment site—at which the enzyme can cleave once it is attached to the substrate via the dsRHbd). The ability of RNase H1 to cleave long RNA/DNA hybrids suggests that the enzyme may play a role in resolving long RNA/DNA hybrids generated in vivo such as those thought to be involved in switching immunoglobulin isotypes and other instances when nascent RNA forms long R-loop structures.
Gaidamakov SA, Gorshkova II, Schuck P, Steinbach PJ, Yamada H, Crouch RJ, Cerritelli SM. Eukaryotic RNases H1 act processively by interactions through the duplex RNA-binding domain. Nucleic Acids Res 2005;33:2166-75.
Nowotny M, Gaidamakov SA, Cerritelli SM, Ghrilando R, Crouch RJ, Yang W. Structure of human RNase H1 complexed with an RNA/DNA hybrid: insight into HIV reverse transcription. Mol Cell 2007;28:264-76.
Nowotny M, Gaidamakov SA, Crouch RJ, Yang W. Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis. Cell 2005;121:1005-16.
Sorting out the functions of RNase H1 in mitochondria and nuclei
One of the major challenges in understanding the role of RNase H1 in cells relates to the translation of a single mRNA to 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 owing to a failure of mitochondrial DNA replication. This embryonic lethality reveals that the enzyme is essential for the maintenance of mtDNA and that there is no need for newly synthesized RNase H1 for replication/repair of nuclear DNA. We have generated a conditional knockout (KO) that permits us to determine if 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 only when rapid mtDNA synthesis occurs during embryogenesis, whereby it is suddenly activated after implantation. In addition, we will generate organ-/tissue-specific KOs of the Rnaseh1 gene by using the Cre-lox system, including tamoxifen-inducible Cres for general and specific (e.g., heart-specific Cre expressers) ablation. However, the problem of inactivation of synthesis of both forms remains. Accordingly, we have generated transgenic mice that produce either both isoforms of RNase H1 or only the nuclear form in T and B cells. Moreover, we will generate an additional KO in which the Met codon, which is used for initiation of the nuclear isoform, is mutated, resulting in little or no nuclear isoform. In collaboration with Ian Holt, we are searching for possible roles of RNase H1 in mitochondrial DNA replication by using, among other techniques, two-dimensional gel analysis of intermediates.
Structure-function of ribonucleases: RNase H2
We previously found that, in Saccharomyces cerevisiae, RNase H2, the second type of RNase H, is composed of three subunits, two of which were not found with a BLAST search in higher eukaryotes. We determined that human cells contain three subunits of RNase H1 and found similar RNases H2 in other mammals, in particular in the mouse. Last year, a report in Nature Genetics demonstrated that mutations in any one of these proteins compromises RNase H2 activity and leads to the rare Aicardi-Goutières syndrome. Our studies on the yeast enzyme indicate a possible 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 the yeast enzyme as well as in mouse and human RNases H2. Using several types of analysis, we demonstrated, in a finding whose biological significance we are continuing to pursue, that the PIP of the RNase H2B is indeed able to interact with PCNA.
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, contactrobert_crouch@nih.gov.
