个人简介

B.S. Baldwin-Wallace College 1975 Ph.D. State University of New York, Buffalo 1979 Before coming to Florida Tech in 1989, Dr. Leonard was an NIH postdoctoral fellow and later a research scientist 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.

研究领域

My research is directed toward understanding the complex molecular machinery that regulates cell growth. Members of my lab are focused on studying the DNA–protein interactions and molecular assemblies that must take place during the cell cycle to ensure efficient duplication of the cell’s genome. Our fascination with these cellular machines is based on the remarkable temporal precision with which they are assembled and disassembled. Since many of the fundamental molecular cell growth mechanisms found in E. coli are shared by all cells, we hope that what we learn help to advance the areas of human cell growth and cancer.

We study the bacterium Escherichia coli because many of the important cellular components are known, and exquisitely timed 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 and using purified cell components. 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 copies of the initiator protein, Dna and we have identified assembly instruction encoded into the oriC nucleotide sequence that direct the assembly of DnaA oligomers in an orderly fashion. The novel arrangment of DnaA recognition sites also produces several distinctive sub-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 stage of orisome assembly is choreographed in E. coli and what specific role each component plays. However, the arrangement of DnaA recognition sites is highly variable in the replication origins of different bacterial types. To address this issue, sudies are underway to transplant different replication origins into E. coli to more easily study the variety of orisome assembly. We believe that understanding the diversity of orisome assembly mechanisms and identifying shared attributes among bacterial types will be a critical step in designing new inhibitors that block bacterial growth.

近期论文

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Leonard, A.C., and Grimwade, J.E. (2015) The orisome: structure and function. Front. Microbiol. 6:545. doi: 10.3389/fmicb.2015.00545 Kaur, G., Vora, M.P., Czerwonka, C.A., Rozgaja, T.A., Grimwade, J.E., and Leonard, A.C. (2014) Building the bacterial orisome: high-affinity DnaA recognition plays a role in setting the conformation of oriC DNA, Mol. Microbiol. 91:1148-1163. Leonard, A.C., and Mechali, M. (2013) DNA Replication Origins. In S.D. Bell, M. Mechali, and M. DePamphilis, (eds.), DNA Replication, Cold Spring Harbor Laboratory Press 5(10) a010116. doi: 10.1101/cshperspect.a010116. 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.