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:

Crampton, D. J., Mukherjee, S., and Richardson, C. C. (2006) DNA-induced switch from independent to sequential dTTP hydrolysis in the bacteriophage T7 helicase.  Mol. Cell 21, 165-174.

Qimron, U., Lee, S., Hamdan, S. M., and Richardson, C. C. (2006) Primer initiation and extension by T7 DNA primase.  EMBO J. 25, 2199-2208.

Crampton, D. J., Ohi, M. O., Qimron, E., Walz, T., and Richardson, C. C,  (2006) Oligomeric states of the bacteriophage T7 primase/helicase.  J. Mol. Biol. 360, 667-677.

Marintcheva, B., Hamdan, S. M., Lee, S.,  and Richardson, C. C. (2006) Essential residues in the C-terminus of bacteriohage T7 gene 2.5 single-stranded DNA binding protein.  J. Biol. Chem. 281, 25831-25840.

Lee, S., Marintcheva, B., Hamdan, S. M.,  and Richardson, C. C. (2006) The C-terminal residues of bacteriophage T7 gene 4 helicas-primase coordinate helicase and DNA polymerase activities.  J. Biol. Chem. 281, 23841-25849.

Qimron, U., Tabor, S., Marintcheva, B., and Richardson, C. C. (2006) Genome-wide screens for Escherichia coli genes affecting Infection by T7 bacteriophage.   Proc. Natl. Acad. Sci. U.S.A. 103, 19039-19044.

Johnson, D., Takahashi, M., Hamdan, S. M., Lee, S. J., and Richardson, C. C. (2007) Exchange of DNA polymerases at the replication fork of bacteriophage T7.  Proc. Natl. Acad. Sci. U.S.A. 104, 5312-5317.

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.

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.