Functional characterization of highly processive protein-primed DNA polymerases from phages Nf and GA-1, endowed with a potent strand displacement capacity.

Abstract:

This paper shows that the protein-primed DNA polymerases encoded by bacteriophages Nf and GA-1, unlike other DNA polymerases, do not require unwinding or processivity factors for efficient synthesis of full-length terminal protein (TP)-DNA. Analysis of their polymerization activity shows that both DNA polymerases base their replication efficiency on a high processivity and on the capacity to couple polymerization to strand displacement. Both enzymes are endowed with a proofreading activity that acts coordinately with the polymerization one to edit polymerization errors. Additionally, Nf double-stranded DNA binding protein (DBP) greatly stimulated the in vitro formation of the TP-dAMP initiation complex by decreasing the K(m) value for dATP of the Nf DNA polymerase by >20-fold. Whereas Nf DNA polymerase, as the phi29 enzyme, is able to use its homologous TP as well as DNA as primer, GA-1 DNA polymerase appears to have evolved to use its corresponding TP as the only primer of DNA synthesis. Such exceptional behaviour is discussed in the light of the recently solved structure of the DNA polymerase/TP complex of the related bacteriophage phi29.

Polymerases:

Topics:

Source / Purification, Kinetic Parameters, Nucleotide Incorporation, Exonuclease Activity, Fidelity, Structure and Structure/Function

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Phi29 Longás E2006 3-5' Exonuclease (proofreading) Yes
Phi29 Longás E2006 Cloned or native Cloned in E. coli
Phi29 Longás E2006 Full length or truncated Full length
Phi29 Longás E2006 Vmax 2280 /minute Reaction: Nucleotide incorporation; Substrate: dNTPs
GA-1 Longás E2006 3-5' Exonuclease (proofreading) Yes
GA-1 Longás E2006 Cloned or native Native organism
GA-1 Longás E2006 Full length or truncated Full length
GA-1 Longás E2006 kcat 2260 /minute Reaction: Nucleotide incorporation; Substrate: dNTPs
Nf Longás E2006 3-5' Exonuclease (proofreading) Yes
Nf Longás E2006 Cloned or native Native organism
Nf Longás E2006 Full length or truncated Full length
Nf Longás E2006 KM 0.6uM Reaction: Nucleotide incorporation; Substrate: dNTPs
Nf Longás E2006 Vmax 2400 /minute Reaction: Nucleotide incorporation; Substrate: dNTPs
Nf Longás E2006 Kd 1.5nM Reaction: Nucleotide incorporation; Substrate: dNTPs

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