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Originally published In Press as doi:10.1074/jbc.M103159200 on June 4, 2001

J. Biol. Chem., Vol. 276, Issue 32, 29846-29853, August 10, 2001
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The (I/Y)XGG Motif of Adenovirus DNA Polymerase Affects Template DNA Binding and the Transition from Initiation to Elongation*

Arjan B. BrenkmanDagger , Marinus R. HeidemanDagger , Veronica Truniger§, Margarita Salas§, and Peter C. van der VlietDagger

From the Dagger  University Medical Center, Department of Physiological Chemistry and Center for Biomedical Genetics, Utrecht, The Netherlands and § Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain

Adenovirus DNA polymerase (Ad pol) is a eukaryotic-type DNA polymerase involved in the catalysis of protein-primed initiation as well as DNA polymerization. The functional significance of the (I/Y)XGG motif, highly conserved among eukaryotic-type DNA polymerases, was analyzed in Ad pol by site-directed mutagenesis of four conserved amino acids. All mutant polymerases could bind primer-template DNA efficiently but were impaired in binding duplex DNA. Three mutant polymerases required higher nucleotide concentrations for effective polymerization and showed higher exonuclease activity on double-stranded DNA. These observations suggest a local destabilization of DNA substrate at the polymerase active site. In agreement with this, the mutant polymerases showed reduced initiation activity and increased Km(app) for the initiating nucleotide, dCMP. Interestingly, one mutant polymerase, while capable of elongating on the primer-template DNA, failed to elongate after protein priming. Further investigation of this mutant polymerase showed that polymerization activity decreased after each polymerization step and ceased completely after formation of the precursor terminal protein-trinucleotide (pTP-CAT) initiation intermediate. Our results suggest that residues in the conserved motif (I/Y)XGG in Ad pol are involved in binding the template strand in the polymerase active site and play an important role in the transition from initiation to elongation.

* This work was supported in part by the Netherlands Organization for scientific research (to P. C. v. d. V.), by National Institutes of Health Grant 2R01 GM27242-21 and Dirección General de Investigación Científica y Técnica grant PB98-0645 (to M. S.), by an institutional grant from the Fundación Ramón Areces (to the Centro de Biología Molecular "Severo Ochoa"), and by European Union Contract FMRX-CT97-0125 (to P. C. v. d. V. and M. S.).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.

To whom correspondence should be addressed: Universiteitsweg 100, P.O. Box 85060, 3508 AB, Utrecht, The Netherlands. Tel.: 31-302538989; Fax: 31-302539035; E-mail: p.c.vandervliet@med.uu.nl.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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