The (I/Y)XGG motif of adenovirus DNA polymerase affects template DNA binding and the transition from initiation to elongation

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-direc...

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Bibliographic Details
Published in:The Journal of biological chemistry Vol. 276; no. 32; p. 29846
Main Authors: Brenkman, A B, Heideman, M R, Truniger, V, Salas, M, van der Vliet, P C
Format: Journal Article
Language:English
Published: United States 10.08.2001
Subjects:
Adenoviridae - enzymology
Amino Acid Motifs
Amino Acid Sequence
Amino Acids - chemistry
Binding Sites
Catalysis
DNA - metabolism
DNA Mutational Analysis
DNA-Binding Proteins - metabolism
DNA-Directed DNA Polymerase - chemistry
Dose-Response Relationship, Drug
Glycerol - pharmacology
Kinetics
Molecular Sequence Data
Mutagenesis, Site-Directed
Mutation
Protein Binding
ISSN:0021-9258
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Summary: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 K(m)(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.
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ISSN:0021-9258
DOI:10.1074/jbc.M103159200