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Journal of Virology, December 2000, p. 11681-11689, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Identification of Conserved Residues Contributing to the Activities of Adenovirus DNA Polymerase

Huanting Liu, James H. Naismith, and Ronald T. Hay*

Centre for Biomolecular Science, The University of St. Andrews, North Haugh, St. Andrews KY16 9ST, United Kingdom

Received 18 July 2000/Accepted 26 September 2000

Adenovirus codes for a DNA polymerase that is a member of the DNA polymerase alpha  family and uses a protein primer for initiation of DNA synthesis. It contains motifs characteristic of a proofreading 3'-5'-exonuclease domain located in the N-terminal region and several polymerase motifs located in the C-terminal region. To determine the role of adenovirus DNA polymerase in DNA replication, 22 site-directed mutations were introduced into the conserved DNA polymerase motifs in the C-terminal region of adenovirus DNA polymerase and the mutant forms were expressed in insect cells using a baculovirus expression system. Each mutant enzyme was tested for DNA binding activity, the ability to interact with pTP, DNA polymerase catalytic activity, and the ability to participate in the initiation of adenovirus DNA replication. The mutant phenotypes identify functional domains within the adenovirus DNA polymerase and allow discrimination between the roles of conserved residues in the various activities carried out by the protein. Using the functional data in this study and the previously published structure of the bacteriophage RB69 DNA polymerase (J. Wang et al., Cell 89:1087-1099, 1997), it is possible to envisage how the conserved domains in the adenovirus DNA polymerase function.

* Corresponding author. Mailing address: Centre for Biomolecular Science, Biomolecular Science Building, The University of St. Andrews, North Haugh, St. Andrews KY16 9ST, United Kingdom. Phone: (44) 1334 463396. Fax: (44) 1334 462595. E-mail: rth{at}st-andrews.ac.uk.

Journal of Virology, December 2000, p. 11681-11689, Vol. 74, No. 24
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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