DNA-independent deoxynucleotidylation of the phi 29 terminal protein by the phi 29 DNA polymerase.
In this paper, we show that the phi 29 DNA polymerase, in the absence of DNA, is able to catalyze the formation of a covalent complex between the phi 29 terminal protein (TP) and 5'-dAMP. Like the reaction in the presence of phi 29 DNA, TP.dAMP complex formation is strongly dependent on activating Mn2+ ions and on the efficient formation of a TP/DNA polymerase heterodimer. The nature of the TP-dAMP linkage was shown to be identical (a O-5'-deoxyadenylyl-L-serine bond) to that found covalently linking TP to the DNA of bacteriophage phi 29, indicating that this DNA-independent reaction actually mimics that occurring as the initiation step of phi 29 DNA replication. Furthermore, as in normal TP-primed initiation on the phi 29 DNA template, this novel reaction showed the same specificity for TP Ser232 as the OH donor and the involvement of the YCDTD amino acid motif, highly conserved in alpha-like DNA polymerases. However, unlike the reaction in the presence of phi 29 DNA, the DNA-independent deoxynucleotidylation of TP by the phi 29 DNA polymerase did not show dATP specificity, being possible to obtain any of the four TP.dNMP complexes with a similar yield. This lack of specificity together with the poor efficiency of this reaction at low deoxynucleoside triphosphate (dNTP) concentration reflect a weak, but similar stability of the four dNTPs at the phi 29 DNA polymerase dNTP-binding site. Thus, the presence of a director DNA would mainly contribute to stabilizing a complementary nucleotide, giving base specificity to the protein-primed initiation reaction. According to all these data, the novel DNA polymerase reaction described in this paper could be considered as a "non-DNA-instructed" protein-primed deoxynucleotidylation.
Blanco
L
L
Centro de Biología Molecular, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Spain.
Bernad
A
A
Esteban
J A
JA
Salas
M
M
D001435:Bacteriophages / Q000201:enzymology* / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity
D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase