High Tech with a Human Touch
LEONARD, Alan C
Biological Sciences Department, College of Science
B.S. Baldwin-Wallace College 1975
Ph.D. State University of New York, Buffalo 1979
BIO 2010 Microbiology
BIO 5575 Cancer Biology
Before coming to Florida Tech in 1989, Dr. Leonard was an NIH postdoctoral fellow and later a faculty member in the Department of Experimental Biology at the Roswell Park Cancer Institute, in Buffalo, New York. While at Roswell Park he perfected the recombinant DNA technology required to isolate and clone the origin of replication from the Escherichia coli chromosome and constructed small plasmids, termed minichromosomes, 1/500th the size of the chromosome. Dr. Leonard has shown that minichromosomes replicate under the same control mechanism as the chromosome and are an excellent model system for the study of the regulation of bacterial chromosome replication. Recent studies have identified similarities among the DNA replication regulatory systems of all cells, further supporting the usefulness of the E. coli model system in dissecting the machinary of life.
Dr. Leonard's research is supported by the National Institutes of Health and his laboratory has been an active place for the training of MS and PhD students interested in the molecular biology of cell growth regulation.
We study the bacterium Escherichia coli because many of the important cellular components are known, and DNA synthesis must be triggered in these cells under a variety of different growth conditions. To examine the triggering mechanism, we have developed methods to probe DNA–protein interactions in living bacterial cells that are proceeding synchronously through the cell cycle. We find that in cycling E. coli cells, nucleoprotein complexes (orisomes) are assembled step-by-step at the chromosomal replication origin, oriC, to unwind the DNA. Orisomes comprise assemblies of initiator protein, DnaA, that are modulated by chromatin proteins that bend DNA. Recently we have identified instruction sets built into the oriC nucleotide sequence that direct the assembly of DnaA oligomers in an orderly fashion, and serve as a component of the timing mechanism for new rounds of DNA replication during the cell cycle. The novel arrangment of DnaA recognition sites produces several novel oligomeric protein assemblies that may be required to unwind the DNA as the first step in triggering new DNA synthesis. We are currently trying to determine how each step of orisome assembly is choreographed and what specific role each component plays. We believe that understanding the molecular mechanisms of orisome assembly is also important to design new inhibitors to block bacterial growth.
Rozgaja, T, Grimwade, J, Iqbal, M., Czerwonka, C., Vora. M., and Leonard, A. 2011. Two oppositely-oriented arrays of low affinity recognition sites in oriC guide progressive binding of DnaA during E. coli pre-RC assembly. Mol. Microbiol., 82: 475-488.
Leonard, A. and Grimwade, J. 2011. Regulation of DnaA assembly and activity: taking directions from the genome. Annual Review of Microbiology. 65:19-35.
Leonard, A.C., and Grimwade, J.E 2010. Regulating DnaA complex assembly: it's time to fill the gaps, Curr. Opin. Microbiol., 13:766-772.
Leonard, A.C., and Grimwade, J.E. 2010. “Chromosome replication and segregation”. In: Encyclopedia of Microbiology, 3d edition, .M. Schaechter, ed. pp. 493-506 Oxford: Elsevier
Leonard, A. C., and Grimwade, J.E. 2010. Chapter 4.4.1, Initiation of DNA Replication. In A. Böck, R. Curtiss III, J. B. Kaper, P. D. Karp, F. C. Neidhardt, T. Nyström, J. M. Slauch, C.L. Squires, and D. Ussery (ed.), EcoSal—Escherichia coli and Salmonella: Cellular and Molecular biology. http://www.ecosal.org. ASM Press, Washington, DC. doi: 10.1128/ecosal.4.4.1
Miller, D.T., Grimwade, J.E., Betteridge, T., Rozgaja, T., Torgue, J.J., and Leonard, A.C. 2009. Bacterial origin recognition complexes direct assembly of higher-order DnaA oligomeric structures. Proc. Natl. Acad. Sci. (USA). 106:18479-18484.
Leonard, A.C. and Grimwade, J.E. 2009. Initiating chromosome replication in E. coli: it makes sense to recycle. Genes and Development. 23:1145-50.
Grimwade, J.E., Torgue, J.J., McGarry, K.C., Rozgaja, T., Enloe, S.T., and Leonard, A.C. 2007. Mutational analysis reveals Escherichia coli oriC interacts with both DnaA-ATP and DnaA-ADP during pre-RC assembly. Mol. Microbiol.,66:428-39.
Nievera, C., Torgue, J., Grimwade, J.E., and A.C. Leonard. 2006. SeqA blocking of DnaA-oriC interactions ensures staged assembly of the E. coli pre-RC. Molecular Cell 24: 581-592.
Leonard, A.C. and J.E. Grimwade. 2005. Building a bacterial orisome: emergence of new regulatory features for replication origin unwinding. Mol Microbiol. 55: 978-985.
McGarry, K.C., V.T. Ryan, J.E. Grimwade and A.C. Leonard. 2004. Two discriminatory binding sites in the Escherichia coli replication origin are required for DNA strand opening by initiator DnaAATP. Proc. Natl. Acad. Sci (USA). 101: 2811-2816.
Ryan, V.T., J.E. Grimwade, J.E. Camara, E. Crooke and A.C. Leonard. 2004. Escherichia coli prereplication complex assembly is regulated by dynamic interplay among Fis, IHF and DnaA. Mol. Microbiol. 51:1347-1359.
Ryan, V.T., J.E. Grimwade, C.J. Nievera and A.C. Leonard. 2002. IHF and HU stimulate assembly of pre-replication complexes at Escherichia coli by two different mechanisms. Mol. Microbiol. 46: 113-124.
Grimwade, J.E., V.T. Ryan and A.C. Leonard. 2000. IHF redistributes bound initiator protein, DnaA, on supercoiled oriC of Escherichia coli. Mol. Microbiol. 35: 835-844.
Cassler, M.R., J..E. Grimwade and A.C. Leonard. 1995. Cell cycle-specific changes in nucleoprotein complexes at a chromosomal replication origin. EMBO J. 14: 5833-5841.
Leonard, A.C. and C.E. Helmstetter. 1986. Cell-Cycle Specific Replication of Escherichia coli minichromosomes. Proc. Natl. Acad. Sci. (USA). 83: 5101-5105.