Charles C. Richardson, M.D.

Edward S. Wood Prof. of Biological Chemistry and Molecular Pharmacology

Harvard Medical School
C2-219a
240 Longwood Ave
Boston, MA 02115
Tel: (617) 432-1864
Fax:(617) 432-3362
e-mail:ccr@hms.harvard.edu
9 postdoctoral fellows, 1 graduate students
website:http://richardson.med.harvard.edu

The replication of the chromosome of bacteriophage T7 provides a model system to study DNA replication. Emphasis is on the protein-protein and protein-DNA interactions responsible for the coordination of events at the replication fork. The crystal structures of all of the T7 replication proteins are available. T7 DNA polymerase forms a 1:1 complex with thioredoxin of E. coli, an interaction that confers processivity on polymerization. The T7 gene 4 protein contains both a helicase and primase domain. The hexameric helicase translocates 5' to 3' on DNA and catalyzes the unwinding of duplex DNA. Studies are designed to examine the mechanism by which the energy of hydrolysis of a NTP fuels its unidirectional movement. The primase domain catalyzes the synthesis of RNA primers at specific sites for lagging strand synthesis. The recognition event involves a zinc motif in the protein. The gene 2.5 protein, a DNA binding protein, is essential for T7 DNA replication. It modulates the activities of the polymerase, helicase, and primase and facilitates the cycling of the lagging strand DNA polymerase. Using a mini-circle primer-template the reconstituted replisome mediates coordinated leading and lagging strand DNA synthesis in which a replication loop containing a nascent Okazaki fragment is present.  Additional DNA polymerases are electrostatically bound to the helicase thus providing an immediate source of polymerase for the multiple priming events on the lagging strand and for the rapid replacement of the leading strand polymerase in the event it dissociates from the primer template.

References:

Marintcheva, B., Marintchev, A.,  Wagner, G.,  and Richardson, C. C. (2008) The acidic C-terminal tail of the ssDNA-binding protein of bacteriophage T7 functions as an electrostatic shield.  Proc. Natl. Acad. Sci. U.S.A. 105, 1855-1860.

Lee, S. J., Qimron, U., and Richardson, C. C. (2008) Communication between subunits critical to DNA binding by hexameric helicase of bacteriophage T7.  Proc. Natl. Acad. Sci. U.S.A. 105, 8908-8913.

Tran, N., Rezende, L. F., Richardson, C. C., Tabor, S., (2008) The role of bacteriophage T7 gene 1.7 in dideoxyribonucleotide sensitivity.  Proc. Natl. Acad. Sci. U.S.A. 105, 9373-9378.

Hamdan, S. M., Loparo, J. J., Takahashi, M., Richardson, C. C., and van Oijen,,  A. M. (2009) Dynamics of DNA replication loops reveal temporal control of lagging-strand synthesis. Nature 457, 336-339.

Akabayov, B.,  Lee, S. J., Akabayov,  S.,  Rekhi, S., Zhu, B., and Richardson, C. C. (2009) DNA Recognition by the DNA Primase of Bacteriophage T7: A Structure-Function Study of the Zinc Binding Domain." ? Biochemistry 48, 1763–1773.

Marintcheva, B., and Richardson, C. C. (2009) Mutations in the gene 5 DNA polymerase of bacteriophage T7 suppress the dominant lethal phenotype of gene 2.5 ssDNA binding protein lacking the C-terminal phenylalanine.  Molecular Microbiology 72, 869-880.

Hamdan, S. M., Johnson, D. E., Tanner,  N. A.,  Lee, J. B., Qimron, U., Tabor, S., van Oijen, A. M., and Richardson, C. C.  (2007) Dynamic DNA helicase-DNA polymerase interactions assure processive replication fork movement.  Mol. Cell  27, 539-549.

Qimron, U., Kulczyk, A. W., Hamdan, S. M., Tabor, S., and Richardson, C. C.  (2007) Inadequate inhibition of host RNA polymerase restricts T7 bacteriophage on hosts overexpressing udk.   Molecular Microbiology 67, 448-457.