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

Aberrations in the intimate role of RNA associated with DNA create genomic damage and, therefore formation and resolution of RNA-DNA interaction is critical.  Among the several human disorders resulting from persistent RNA-DNA associations are Amyotrophic Lateral Sclerosis (ALS), Human Immune Deficiency Virus (HIV-AIDS), mitochondrial disease (MtDis), and Aicardi-Goutières Syndrome (AGS). Each of these debilitating disorders is associated with RNA/DNA hybrids; a complex of an RNA complementary in sequence to DNA. In HIV-AIDS, the RNA genome of the virus is copied as DNA forming a simple RNA/DNA hybrid. In contrast, ALS, MtDis and AGS have R-loops in which RNA/DNA duplexes are formed during RNA synthesis with one of the duplex strand is looped out (single stranded DNA). HIV-AIDS, MtDis and AGS are all affected by ribonucleases H (RNases H), enzymes that degrade the RNA strand of RNA/DNA hybrids including R-loops.

Our laboratory interested in understanding how RNA/DNA hybrids are resolved and what role RNases H play in their elimination. We know a great deal about the recognition of RNA/DNA hybrids and the enzymatic mechanism of hydrolysis of the RNA. We have played a central role in defining the types and structural properties of the two classes of RNases H present in most organisms.

Our studies have shown that mice deleted for the Rnaseh1 gene arrest embryonic development at day 10 due to a failure to amplify mitochondrial DNA. Others have found that Aicardi-Goutières Syndrome (AGS), a severe neurological disorder with symptoms appearing at or soon after birth, can be caused by defective human RNase H2. We employ molecular-genetic and biochemical tools and yeast and mouse models in our research.

It is known that during RNA synthesis R-loops can form and that aberrant R-loop formation can result in chromosome breakage. However, R-loop formation has been observed in the normal recombination process of switching (recombination) from one form of immunoglobulin to another resulting in different isoforms of antibodies.

Structure-function of ribonucleases H: RNase H1

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 for our understanding of 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. Eukaryotic 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.

Functions of RNase H1 in mitochondria and nuclei

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 both 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.

Translation of the single Rnaseh1 mRNA initiates at two distinct start codons, with the resulting proteins being targeted to either the mitochondria or the nucleus. Our current studies suggest that the level of each protein is affected by a short upstream open reading frame. Thus, the amount of protein(s) is (are) kept at low levels by differential translation. This finding suggests that the very low amount of Rnaseh1 mRNA observed in most cells is more than sufficient to generate the quantity of RNase H1 that is optimal and that relatively poor translation is used to limit the amount of enzyme produced.

We have generated transgenic mice that express elevated levels of RNase H1 in B-cells; the increase results in no obvious change in mitochondria. We are examining effects of even higher levels of the nuclear form of RNase H1 on B cells, where recombination of the immunoglobulin locus results first in joining of the VDJ regions and then later in B-cell development when isotype switching occurs. These events involve numerous proteins used during repair of damaged 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 synthesis 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.

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

We previously found that, in Saccharomyces cerevisiae, RNase H2, the second type of RNase H, 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 paper in Nature Genetics reported 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.

A complete list of Dr Crouch's publications can be found on My NCBI Collections.

Our studies will provide information related to Ribonucleases H which are exceedingly important to maintain genome stability. Formation of RNA/DNA hybrids occurs during transcription, DNA replication, several other cellular processes and also during replication of the HIV-AIDS virus. We have described the basic structure of both RNase H1 and H2 and are now aiming to study their impact in vivo. Ribonucleases H are enzymes that recognize RNA/DNA hybrids and digest the RNA. Two types of cellular RNases H are known, mutations in type 1 lead to arrest of embryonic development in mice due to a failure to amplify mitochondrial DNA while mutations in type 2 enzymes can lead to a serious neurological syndrome. Replication of the HIV-AIDS virus requires a virally encode RNase H that is absolute necessary for production of infectious viral particles, making it a useful target for drugs to block replication of the virus. Type 1 and the HIV-AIDS enzymes are similar in structure and mechanism of action making imperative to examine effects of any potential drugs on the cellular type 1 RNase H. Aicardi Goutieres Syndrome is a severe human disorder caused by mutations in five genes, three of which encode subunits of RNase H2. Understanding how this RNase H is related to AGS is one of our major efforts.

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