An aspartic acid residue in TPR-1, a specific region of protein-priming DNA polymerases, is required for the functional interaction with primer terminal protein.
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 Dx(2)SLYP and Kx(3)NSxYG (TPR-1) and motifs Kx(3)NSxYG 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 phi29 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 phi29 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.
Dufour
E
E
Centro de Biologia Molecular "Severo Ochoa", Universidad Autonoma, Cantoblanco, Madrid, 28049, Spain.
Méndez
J
J
Lázaro
J M
JM
de Vega
M
M
Blanco
L
L
Salas
M
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D017103:Bacillus Phages / Q000201:enzymology* / Q000235:genetics; D017104:Bacteriophage M13 / Q000235:genetics; D001665:Binding Sites; D017124:Conserved Sequence / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D019281:Dimerization; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013698:Templates, Genetic; D013816:Thermodynamics; D014764:Viral Proteins / Q000378:metabolism*
D004279:DNA, Viral; D003838:Deoxyadenine Nucleotides; D011994:Recombinant Proteins; D014764:Viral Proteins; D001224:Aspartic Acid; D004259:DNA-Directed DNA Polymerase; C026600:2'-deoxyadenosine triphosphate