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Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/38486
Title: An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal
Authors: Dufour, Emmanuelle; Méndez, Juan; Lázaro, José M.; Vega, Miguel de; Blanco, Luis; Salas, Margarita
Keywords: Linear DNA replication
Terminal protein
Issue Date: 1-Dec-2000
Publisher: Elsevier
Citation: Journal of Molecular Biology 304(3): 289-300 (2000)
Abstract: A multiple sequence alignment of eukaryotic-type DNA polymerases led to the identification of two regions of amino acid residues that are only present in the group of DNA polymerases that make use of terminal proteins. (TPs) as primers to initiate DNA replication of linear genomes. These amino acid regions (named terminal region (TPR protein-1 and TPR-2) are inserted between the generally conserved motifs Dx2SLYP and Kx3NSxYG (TPR-1) and motifs Kx3NSxYG and YxDTDS (TPR-2) of the eukaryotic-type family of DNA polymerases. We carried out site-directed mutagenesis in two of the most conserved residues of φ29 DNA polymerase TPR-1 to study the possible role of this specific region. Two mutant DNA polymerases, in conserved residues AsP332 and Leu342, were purified and subjected to a detailed biochemical analysis of their enzymatic activities. Both mutant DNA polymerases were essentially normal when assayed for synthetic activities in DNA-primed reactions. However, mutant D332Y was drastically affected in φ29 TP-DNA replication as a consequence of a large reduction in the catalytic efficiency of the protein-primed reactions. The molecular basis of this defect is a non-functional interaction with TP that strongly reduces the activity of the DNA polymerase/TP heterodimer.
Publisher version (URL): http://dx.doi.org/10.1006/jmbi.2000.4216
URI: http://hdl.handle.net/10261/38486
DOI: 10.1006/jmbi.2000.4216
ISSN: 0022-2836
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