Charles Richardson

Department of Biological Chemistry and Molecular Pharmacology
Harvard Medical School
Building C, Room 219
240 Longwood Avenue
Boston, MA 02115
Tel: (617) 432-1864
Fax: (617) 432-3362
Email: ccr@bcmp.med.harvard.edu
Web Page: The Richardson Lab Page

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.

Rotation Projects:

1. Characterization of mutationally altered helicase and primase proteins of bacteriophage T7: We have identified a number of amino acid changes in the helicase and primase that affect their activities in vivo. The proteins are purified from cells overexpressing the genes and characterized biochemically. The altered proteins can be characterized for their ability to interact with the other T7 replication proteins.

2. Structure-function studies on T7 DNA polymerase: T7 DNA polymerase physically interacts with a number of replication proteins to achieve processivity (thioredoxin), to replicate duplex DNA (helicase), and to initiate lagging strand DNA synthesis (primase). Mutational analysis of the DNA polymerase will be carried out to dissect these interactions and to identify the domains on their three-dimensional crystal structures.

3. The ability of T7 DNA polymerase in solution to exchange with the replicating DNA polymerase without affecting processivity will be investigated by identifying the mechanism by which the helicase serves as a reservoir for additional DNA polymerases. The role of this exchange in lagging strand synthesis will be examined.

References:

• 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.