Robert J. Crouch, PhD, Head, Section on Formation of RNA
Susana M. Cerritelli, PhD, Staff Scientist
Sergei A. Gaidamakov, PhD, Research Fellow
Hyongi Chon, PhD, Postdoctoral Fellow
Lina Gugliotti, PhD, Postdoctoral Fellow
Yutaka Suzuki, PhD, _Postdoctoral Fellow-
John Brad Holmes, BS, Predoctoral Fellow 1
Kiran Sakhuja, MS, MSC, Research Assistant
Our research is directed toward understanding the processes involved in cellular DNA replicatio and the relationship of HIV replication to these cellular events in order to use the information gained for therapeutic purposes. We are examining the formation and resolution of RNA/DNA hybrids during DNA replication or transcription. Ribonucleases H are important enzymes that participate in removal of the RNA of RNA/DNA hybrids. These enzymes are 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 cleavage of RNA. Drugs that alter levels of specific disease-related genes are undergoing development to take advantage of RNases H within the cell. Regulated expression of RNases H could enhance the efficacy of the drugs.
Structure-function of ribonucleases H: RNase H1
Cerritelli, Gaidamakov, Crouch; in collaboration with Gorshkova, Nowotny, Schuck, Yang
We have made considerable progress in understanding how RNase H1 binds to and cleaves an RNA/DNA hybrid substrate. Solving the crystal structure of RNase H1 domains from different sources has expedited our recent advances. Last year, we published the structure of the RNase domain of Bacillus halodurans in complex with an RNA/DNA; that domain differs in several ways from the more common form of Type 1 RNases H. Recently, we determined the structure of the RNase H domain of the human enzyme in complex with an RNA/DNA hybrid. The data reveal that the human enzyme is almost superimposable on the substrate-free structure of E. coli RNase HI that we described several years ago, indicating that binding to substrate does not alter the protein conformation in any significant way. We have also solved the co-crystal structure of the double-stranded RNA/hybrid binding domain (dsRHbd) of human RNase H1 in complex with a 12 bp RNA/DNA hybrid. The structure not only reveals the binding of this part of the enzyme to the deoxyribose residues in the DNA strand and two ribose moieties of the RNA strand but also demonstrates that three of these short regions can bind independently and in basically the same manner to the 12 bp RNA/DNA. Using surface plasmon resonance, we observed two moles of RNase H1 bound to a short 12 bp RNA/DNA hybrid. Careful enzymatic studies on catalysis clearly demonstrate that the enzyme acts as a monomer. The response curve of increasing enzyme concentration was linear (not sigmoidal), and the addition of catalytically inactive mutant protein to the assay mixture neither stimulated nor inhibited the rate of reaction. Given that the enzyme is first attracted to the RNA/DNA substrate via the dsRHbd, it seems that the 2:1 binding is attributable to the dsRHbd in a manner that is to some extent related to the small size of the dsRHbd. These results and our data indicating that eukaryotic RNases H1 act in a processive manner led to a tentative model in which the enzyme binds to the substrate via the dsRHbd with the RNase H domain contacting and cleaving the RNA of the hybrid. After cleavage, the RNase H domain disengages from the hybrid but, because the dsRHbd has high affinity for the substrate and remains bound, the RNase H domain is able to search for a nearby hybrid, resulting in several (processive) cleavages in one binding event. The "reach" of the enzyme could then be related to the length of the domain that connects the dsRHbd and the RNase H domain. In one extreme, the enzyme might be able to hydrolyze RNA of RNA/DNA when two very distant regions are located in close proximity to one another, such as during class switch recombination.
Structure-function of ribonucleases H: RNase H2
Chon, Crouch; in collaboration with DePamphilis, Vassilev
We previously found that RNase H2, the second type of RNase H, of Saccharomyces cerevisiae is composed of three subunits, two of which were not found in other organisms as revealed by a Blast search. Employing the same strategy that we used to determine the subunit composition of the yeast enzyme, we found that two additional proteins purify with the readily identifiable catalytic subunit of human RNase H2. A recent report in Nature Genetics demonstrated that mutations in either one of these proteins compromise RNase H2 activity and lead to the rare human Aicardi-Goutieres syndrome (AGS). Our studies on the yeast enzyme indicate a possible connection between RNase H2 and at least one protein involved in DNA replication/repair. We are in the process of determining if the human or mouse RNase H2 interacts with similar proteins.
Sorting out the functions of RNase H1 in mitochondria and nuclei
Cerritelli, Sakhuja, Chon, Holmes, Crouch
One of the major challenges we face in understanding the roles of RNase H1 in cells is related to the translation of a single mRNA to produce both a nuclear and mitochondrial form of the enzyme. Knocking out the Rnaseh1 gene in mouse results in lethality at embryonic day 8.5 due to a failure to replicate mitochondrial DNA. This outcome tells us that (1) the enzyme is essential for the maintenance of mtDNA and that (2) there is no need for newly synthesized RNase H1 for replication/repair of nuclear DNA. We have generated a conditional knockout that will permit us to determine if RNase H1 is necessary in adult animals for either mtDNA or nuclear DNA replication and repair. It may be that RNase H1 is important for mtDNA replication only when rapid mtDNA synthesis occurs during embryogenesis when it is suddenly activated post-implantation. In addition, we will be able to generate organ-/tissue-specific knockout of the Rnaseh1 gene by using the Cre-lox system, including tamoxifen-inducible Cres for general ablation and other (e.g., heart-specific) Cre expressers. However, the problem of inactivation of synthesis of both forms of RNase H1 remains. Accordingly, we 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 knockout in which the Met codon used for initiation of the nuclear isoform is mutated, resulting in little or no nuclear isoform. Establishing these mice has been a time-consuming effort, which will bear fruit in the near future.
We also developed RNA interference and are examining a series of chemical inhibitors of RNase H1. Developing cell lines from our various transgenic mice will be extremely rewarding. We already have two cell lines that will be helpful. One is a rho-zero cell in which mtDNA has been eliminated, and the second is a mouse cell line that contains mtDNA but, due to lack of cytochrome c, is unable to carry out oxidative phosphorylation. Because cytochrome c is involved in the major apoptotic pathway, these cells are less prone to undergo programmed cell death and should provide us with a means of inhibiting mtDNA without too many side effects.
1 Cambridge University
COLLABORATORS
Melvin DePamphilis, PhD, Laboratory of Molecular Growth Regulation, NICHD, Bethesda, MD
Inna Gorshkova, PhD, Protein Biophysics Resource, OD, NIH, Bethesda, MD
Marcin Nowotny, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
Peter Schuck, PhD, Protein Biophysics Resource, OD, NIH, Bethesda, MD
Alex Vassilev, PhD, Laboratory of Molecular Growth Regulation, NICHD, Bethesda, MD
Wei Yang, PhD, Laboratory of Molecular Biology, NIDDK, Bethesda, MD
For further information, contact robert_crouch@nih.gov.