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.