Richard S. Myers

Lecturer of Biochemistry & Molecular Biology

Ph.D. (1989) University of Illinois, Urbana

Homologous Genetic Recombination, DNA exonuclease mechanisms

Tel: (305) 243-2056, Fax: (305) 243-3065

rmyers@molbio.med.miami.edu

Life depends on the faithful recreation of DNA sequences from generation to generation. DNA damage puts organisms at risk of losing or modifying important genetic information. DNA double strand breaks are frequently repaired by enzymes that splice in similar DNA sequences via homologous recombination. Although recombinational repair restores chromosomal integrity, DNA repair is often accompanied by rearrangement of genetic information. Such genetic exchange results in novel combinations of genes. The rate, position and extent of recombination are carefully controlled. Aberrant (unregulated) recombination events promote genome instability and are associated with genetic defects and cancer. The rate-limiting step in homologous recombination is formation of ssDNA from dsDNA.

Recombination hotspots correspond to regions of DNA that increase formation of ssDNA in their immediate vicinity. Our laboratory studies how enzymes that degrade, unwind and pair DNA collaborate to promote DNA repair and genetic exchange. Most of our studies are aimed at understanding regulation of recombination enzymes at hotspots. In bacteria, as in other organisms, DNA exonucleases process broken DNA to initiate search for homology, provoking repair of DNA damage with coincident genetic exchange. These exonucleases processively degrade DNA and, when untamed, inhibit recombination by destroying the substrate. In E. coli, two strategies are employed to regulate DNA digestion at recombination hotspots: (1) Chi ( χ ) is a DNA sequence that converts RecBCD enzyme from an anti recombinagenic nuclease to a recombinase; (2) the exonuclease of phage χ digests DNA until assimilation of a complementary DNA strand attenuates further digestion. We are dissecting the regulatory mechanisms operating at χ and at DNA double-strand breaks in E. coli.

All The World's A Phage – Roger Hendrix

Molecular Biology arose as a multidisciplinary effort to determine the underlying principles of Life. Bacterial viruses (phage) were selected as the simplest systems for dissecting mechanisms of genetic inheritance. Fifty years later, phage studies continue to inform our understanding of fundamental biological processes, including homologous genetic recombination. Recombination generates genetic diversity, is required for the accurate segregation of chromosomes during meiosis and for repair of various types of DNA damage, particularly DNA strand breaks. Aberrant (unregulated) recombination events promote genome instability (1, 2), genetic defects and cancer. Understanding the molecular mechanisms of recombination is central to our knowledge of genetic diseases. Our long-term goal is to determine how recombination is regulated to effect DNA damage repair without causing collateral genetic damage.

In prokaryotes and eukaryotes, free-living creatures and obligate intracellular visitors, recombination is initiated at double-strand DNA (dsDNA) breaks by 5’ to 3’ dsDNA exonucleases that expose 3’ single-strand DNA (ssDNA) termini. In prokaryotes and eukaryotes, free-living creatures and obligate intracellular visitors, recombination is initiated at double-strand DNA (dsDNA) breaks by 5’ to 3’ dsDNA exonucleases that expose 3’ single-strand DNA (ssDNA) termini. The nascent ssDNA is bound by homologous pairing proteins (Synaptases) to form nucleoprotein filaments that search the genome for homologous DNA with which to pair. This homologous DNA is used either as a patch to fill the “hole” in the broken DNA or as a template to effect repair of the dsDNA break by priming DNA synthesis.

Based on my previous studies of E. coli recombination and the results of other studies of phage and fungi, I hypothesize that genetic recombination is regulated by a negative feedback loop in which DNA pairing (synapsis) inhibits additional DNA processing and limits recombination to a single act of exchange (3). This general hypothesis provides the scaffold for about half the work in the lab. We are dissecting the mechanisms that regulate recombination in two model systems, the Red recombination system of bacteriophage λ and the RecBCD recombination system of E. coli (4, 5, A. Larrea and R. Myers, unpublished)..

The phage Lambda ( λ ) Red system is a two-component recombinase consisting of a 5’ to 3’ dsDNA exonuclease and a Synaptase. In collaboration with Kenn Rudd (UM), homologs of λ exonuclease were found in the genomes of over 80 viruses that infect bacteria, plants or animals (6); many of these genomes contain Synaptase genes, too. We studied the activities of some of the homologs and have determined that they behave like λ’s recombinase. For example, we identified a novel Herpes recombinase that acts like the λ Red system. In collaboration with the Weller lab (U. Conn.), we determined that the Herpes recombinase catalyzes homologous strand exchange in vitro (7) and is required for viral development when DNA replication is inhibited by a common antiviral drug (N. Reuven, R. Martinez, R. Myers and S. Weller, unpublished). This last observation indicates that the Herpes recombinase is a dandy target for drug development, especially because the two-component recombinases are restricted to viruses (R. Myers, unpublished). We isolated λ mutants that illuminate the conserved mechanism operating in this large group of recombinases (8), isolated and characterized a novel phage nuclease that shares mechanistic features with the λ exonuclease (9), and have isolated and partially characterized another novel recombination nuclease from a plant (G. Tolun and R. Myers, unpublished). We are currently hunting the synaptase components of the plant nuclease in collaboration with the van Etten lab (U. Neb.) and helped discover the synaptase for an insect virus nuclease (with the Rohrman lab, OSU). We are also reconstituting the Herpes and phage SPP1 recombinases in E. coli, to study their genetic properties using λ as a model. This work comprises the other half of the lab’s activities.

While the formal mechanism of dsDNA break repair recombination is conserved throughout life,

the two-component recombination mechanism is restricted to viruses that require genome concatemerization for DNA maturation and packaging. This is a blessing and a curse. The detailed mechanism of the viral recombinases will be of limited utility in understanding cellular recombinases. But the unique structural and enzymatic properties of these enzymes make them attractive therapeutic targets as they may be inhibited with few side effects to the patient. While the formal mechanism of dsDNA break repair recombination is conserved Furthermore, two-component viral recombinases are great tools for Recombineering (in vivo genetic engineering). While recombineering studies have been restricted to bacteria, the ubiquity of these enzymes in Nature suggests that they will be useful for genetic engineering in plants and animals as well.

All our work combines genetics, biochemistry, bioinformatics, and a host of physical techniques (notably fluorescence spectroscopy, 10) to tease apart these complex machines. We employ simple model organisms, primarily bacteria and phage, because these systems are so well understood and experimentally accessible. Our multidisciplinary approach is designed to create a broad training environment and to allow associates to develop independent projects based on complementary techniques with non-overlapping limitations.

References:

1. Boulton, A., Myers, R.S., Redfield, R.J. (1997) Proc Natl Acad Sci USA 94:8058-8063.

2. Stahl F., Bowers R. Jr, Mooney D., Myers R., Stahl M., Thomason L. (2001) Mol Gen Genet 264:716-723.

3. Myers, R.S., Stahl, M.M., and Stahl, F.W. (1995) Genetics 141:805-812.

4. Jockovich, M.E., and Myers, R.S. (2001) Mol Microbiol 41:949-962.

5. Jockovich, M.E., Larrea, A., and Myers, R.S. (under revision)

6. Myers, R.S., and Rudd, K.E. (1998) Proc Miami Nature Biotech Winter Symp 9:49-50.

7. Reuven, N.B., Staire, A.E., Myers, R.S., and Weller, S.K. (2003) J Virol 77:7425-7433.

8. Subramanian, K., Ruttvisutinunt, W., Scott, W., and Myers R.S. (2003) Nucleic Acids Res 31:1585-1596.

9. Vellani, T.S., and Myers, R.S. (2003) J Bacteriol 185:2465-2474.

10. Tolun, G., and Myers, R.S. (2003) Nucleic Acids Res 131:1-6.

Representative Publications

1.    Thomason, L.C., Myers R.S., Oppenheim A.B., Costantino N., Sawitzke J.A., Datta S., Bubunenko M., Court D.L. Recombineering in Prokaryotes. In: Phages: Their Role in Bacterial Pathogenesis and Biotechnology; Ed. M.W. Waldor, D.I. Friedman, S.L. Adhaya, ASM Press; p. 383-399. 2005

2.    Tolun, G., Myers, R.S., A Realtime DNase Assay (ReDA) Based on PicoGreen® Fluorescence. Nucleic Acids Res. 31e111:1-6, 2003.

3.    Reuven N.B., Staire A.E., Myers R.S., Weller S.K., The Herpes Simplex Virus-1 Alkaline Nuclease and Single-strand DNA binding Protein Mediate Strand Exchange in vitro. J. Virol. 77:7425-7433, 2003.

4.    Vellani T.S., Myers R.S., Bacteriophage SPP1 Chu is an alkaline exonuclease in the Red family of viral two-component recombinases. J. Bacteriol. 185:2465-2474, 2003.

5.   Subramanian K., Rutvisuttinunt W., Scott W., Myers R.S., The enzymatic basis of processivity in lambda exonuclease. Nucleic Acids Res. 31:1585-1596, 2003.

Honors and Professional Activities:

Genetics Society of America

American Society for Biochemistry & Molecular Biology

American Society for Microbiology