Journal of Biological Chemistry
Volume 274, Issue 35, 27 August 1999, Pages 24485-24489
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PROTEIN CHEMISTRY AND STRUCTURE
The Carboxyl Terminus of the Bacteriophage T4 DNA Polymerase Contacts Its Sliding Clamp at the Subunit Interface*

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The location of the interaction of the COOH terminus of the bacteriophage T4 DNA polymerase with its trimeric, circular sliding clamp has been established. A peptide corresponding to the COOH terminus of the DNA polymerase was labeled with a fluorophore and fluorescence spectroscopy used to show that it forms a specific complex with the sliding clamp by virtue of its low KD value (7.1 ± 1.0 μm). The same peptide was labeled with a photoaffinity probe and cross-linked to the sliding clamp. Mass spectrometry of tryptic digests determined the sole linkage point to be Ala-159 on the sliding clamp, an amino acid that lies on the subunit interface. These results demonstrate that the COOH terminus of the DNA polymerase is inserted into the subunit interface of its sliding clamp, thereby conferring processivity to the DNA polymerase.

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*

This work was supported by National Institutes of Health Grants GM13306 (to S. J. B.) and GM19492 (to S. C. A.). The mass spectrometer was purchased in part with funds from National Institutes of Health Grant RR11318.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Laboratoire de Biochimie Physique et des Biopolymères, Université Catholique de Louvain, Bâtiment Lavoisier, 1/1B Place L. Pasteur, B-1348 Louvain-la-Neuve, Belgium.