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PHYSIOLOGICAL, BIOCHEMICAL, AND MOLECULAR
GENETIC EVENTS OF RECOGNITION AND
RESOLUTION OF RNA-DNA HYBRIDS
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Robert J. Crouch, PhD, Head, Section
on Formation of RNA Susana M. Cerritelli, PhD, Staff
Scientist Inna Gorshkova, PhD, Senior Research Fellow Sergei A. Gaidamakov, PhD, Postdoctoral
Fellow Ho-Sang Joeng, PhD, Postdoctoral
Fellow Nicole Follmer, BS, Postbaccalaureate Fellow Robert Wirka, BS, Postbaccalaureate Fellow Yelena Skripchenko, MS, Research
Assistant |
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Our
research is directed toward understanding the processes involved in cellular
DNA replication, the relationship of HIV replication to these cellular events,
and how to use the resultant information for therapeutic purposes. We are
examining the formation and resolution of RNA-DNA hybrids formed during DNA
replication or transcription. Ribonucleases H are important enzymes
participating in removal of the RNA of the RNA-DNA hybrids and are intimately
related to DNA replication in cells as well as in HIV replication during the
conversion of the RNA genome of HIV to DNA. RNases H of cells and HIV share
common enzymatic mechanisms of cleavage of RNA in similar protein
architectures. Drugs to 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 the drugs. We use
molecular genetic, biochemical, and mouse animal models in these efforts. Formation
of R-loop in RNA-DNA hybrids Cerritelli, Crouch; in
collaboration with Drolet Transcribing
DNA usually produces an RNA molecule that, in prokaryotic cells, is almost
immediately recognized by the protein-synthetic machinery and translated into
the appropriate polypeptide. In eukaryotic cells, the nascent mRNA is
recognized by a set of pre–mRNA proteins, also resulting in the
stabilization of single-stranded RNA. Together with the Drolet laboratory, we
reported that inhibition of protein synthesis leads to extensive R-loop
formation whereby the nascent RNA anneals to the DNA after exiting the RNA
polymerase. R-loop formation is most noticeable when topoisomerase, the
enzyme responsible for rewinding the DNA, is defective. Others have obtained
similar results in S. cerevisiae when the nascent RNA-binding proteins
are defective. Tying transcription to R-loop formation aids in our
understanding of the role of RNases H in resolution of R-loops and suggests
that these R-loops may occur at special sites and times during the life of
the cell, providing means of initiating DNA repair and/or replication. Broccoli S, Rallu F, Sanscartier P, Cerritelli SM, Crouch RJ,
Drolet M. Mechanisms of transcription-induced hypernegative supercoiling in topA
null mutants of Escherichia coli. Mol Microbiol
2004;52:1769-1779. Mechanism
of action of mouse and human RNases H1 Gorshkova, Gaidamkov,
Cerritelli, Crouch The
observations described below support a role for eukaryotic RNases H1 in
recognition and cleavage of rather long RNA-DNA hybrids. Our previous results
have shown that RNase H1 is required for mitochondrial DNA replication during
which rather extensive RNA-DNA hybrids may form. It has been suggested that
the occurrence of such long RNA-DNA hybrids aids in the generation of the
various isotypes of antibody molecules. Others have postulated a role for
RNase H in the immune system but have not proven such a role. A few years
ago, we discovered that eukaryotic RNases H1 have an RNase H domain similar
in size, amino acid sequence, and structure to that we reported for E.
coli RNase HI. Eukaryotic RNases H1 have an additional domain connected
to the N-terminus of the RNase H domain that can bind to duplex RNAs even in
the absence of the RNase H domain. We have now shown that this
“extra” dsRNA-binding domain aids in dimerization of the
full-length protein when an RNA-DNA substrate is present. Dimerization is
greatly reduced when a conserved amino acid residue or two adjacent lysines
are changed. The simultaneous mutation of all three amino acids results in a
dramatic reduction in binding to RNA-DNA hybrids. Pileur F, Andreola M-L, Dausse E, Michel J, Moreau S, Yamada H,
Gaidamakov SA, Crouch RJ, Toulmé J-J, Cazenave C. Selective inhibitory DNA
aptamers of the human RNase H1. Nucleic Acids Res 2003;31:5776-5788. Drugs
targeted to the RNase H of HIV reverse transcriptase Gaidamakov, Gorshkova,
Crouch; in collaboration with Beutler, Le Grice, Parniak The
genome of retroviruses is RNA that is copied into DNA by the virally encoded
reverse transcriptase (RT). During the course of DNA synthesis, an RNA-DNA
hybrid is produced that is processed in several ways by the RNase H of RT to
yield the final duplex DNA that integrates into chromosomal DNA. RNase H is
absolutely required for making infectious HIV and thus is an excellent target
for therapeutic drugs. Our finding that RNase H1 (a cellular RNase H with
sequence, structural, and mechanistic properties similar to those of the HIV
RNase H) is required for completion of embryogenesis suggested that potential
HIV RNase H drugs should be specific for the viral enzyme. Of more than
200,000 chemicals screened, about 50 were sufficiently active as inhibitors
of the HIV enzyme to be selected for further screening. We tested these
compounds against a variety of RNases H, including the human protein, and
divided them into several classes depending on their relative inhibition of
the various RNases H. We examined some of the compounds in our laboratory for
interaction with RNA-DNA duplexes to distinguish between direct inhibition of
the enzyme and binding to the substrate and thus alteration of duplex
structure. Most of the inhibitors seem to interact with the enzyme or the
enzyme-substrate complex. Some of the most interesting compounds are those
that are more effective on the cellular enzyme. They will provide us with
tools to examine RNase H1 function in cells. Chan KC, Budihas SR, Le Grice SFJ, Parniak MA, Crouch RJ,
Gaidamakov SA, Isaaq HJ, Wamiru A, McMahon JB, Beutler JA. A capillary
electrophoretic assay for ribonuclease H activity. Anal Biochem
2004;331:296-302. Eukaryotic
RNase H2 activation Jeong, Crouch Defining
the composition of enzymes is sometimes a straightforward task and at other
times somewhat convoluted. Bacterial and archael RNases HII are active as a
single polypeptide chain, with several key residues part of the active site.
The eukaryotic RNase H2 enzyme is considerably larger than RNases HII, yet
the orthologous eukaryotic polypeptide is similar in size and sequence to the
smaller RNases HII. We expressed the RNase H2 polypeptide encoded by human,
mouse, Caenorhabditis elegans, and Saccharomyces cerevisiae in E.
coli and uniformly found the protein to have no RNase H enzymatic
activity. It seems that eukaryotic RNases H2 require some modification and/or
that more than one subunit is necessary for RNase H2 activity. From S.
cerevisiae cells, we affinity-purified the RNase H2 polypeptide of S.
cerevisiae and found two proteins copurifying in a complex. We determined
the identities of these proteins by using mass spectrometry. RNase H2
activity in S. cerevisiae strains deleted for either of these two
genes is absent, suggesting that both proteins may be components of the
active enzyme. Co-expression in E. coli of the three proteins
identified as components of RNase H2 generates an active RNase H2. Thus, the
three proteins are necessary and sufficient for forming the three-subunit
RNase H2. Several studies in S. cerevisiae indicate a possible
interaction of one or more of the subunits of RNase H2 that might interact
with a variety of other proteins, suggesting a complex between RNase H2 and
other parts of the cell’s replication/repair machinery. We tested the
effects on steady-state levels of RNase H2 in strains deleted for the
hypothetical interactants and found no change in RNase H2 levels. Jeong H-S, Backlund PS, Chen H-C, Karavanov AA, Crouch RJ.
Ribonuclease H2 of Saccharomyces cerevisiae is a complex of three
proteins. Nucleic Acids Res 2004;32:407-414. COLLABORATORS John Beutler, PhD, Molecular Targets
Program, Center for Cancer Research, NCI, Marc Drolet, PhD, Stuart Le Grice, PhD, Resistance Mechanisms
Laboratory, HIV Drug Resistance Program, NCI, Frederick, MD Michael Parniak, PhD, Peter Schuck, PhD, Protein Biophysics
Resource, ORS, NIH,
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