PMID:11279245

Citation	Kato, M, Frick, DN, Lee, J, Tabor, S, Richardson, CC and Ellenberger, T (2001) A complex of the bacteriophage T7 primase-helicase and DNA polymerase directs primer utilization. J. Biol. Chem. 276:21809-20
Abstract	The lagging strand of the replication fork is initially copied as short Okazaki fragments produced by the coupled activities of two template-dependent enzymes, a primase that synthesizes RNA primers and a DNA polymerase that elongates them. Gene 4 of bacteriophage T7 encodes a bifunctional primase-helicase that assembles into a ring-shaped hexamer with both DNA unwinding and primer synthesis activities. The primase is also required for the utilization of RNA primers by T7 DNA polymerase. It is not known how many subunits of the primase-helicase hexamer participate directly in the priming of DNA synthesis. In order to determine the minimal requirements for RNA primer utilization by T7 DNA polymerase, we created an altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase.
Links	PubMed Online version:10.1074/jbc.M101470200
Keywords	Amino Acid Sequence; Bacteriophage T7/enzymology; Bacteriophage T7/genetics; Base Pair Mismatch; Base Sequence; DNA Primase/chemistry; DNA Primase/metabolism; DNA Primers; DNA Replication; DNA, Viral/biosynthesis; DNA-Directed DNA Polymerase/chemistry; DNA-Directed DNA Polymerase/metabolism; Kinetics; Models, Molecular; Molecular Sequence Data; Peptide Fragments/chemistry; Protein Structure, Secondary; Templates, Genetic
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
9CAUD:A0A0K2CZV8		GO:0003896: DNA primase activity	ECO:0000247:	PMID:11279245 UniProtKB:P03692	F		"An altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity was created. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase."	complete CACAO 11467
BPT7:PRIM		GO:0003896: DNA primase activity	ECO:0000314:		F		Figs. 5, 6. "An altered gene 4 protein that does not form functional hexamers and consequently lacks detectable DNA unwinding activity was created. Remarkably, this monomeric primase readily primes DNA synthesis by T7 DNA polymerase on single-stranded templates. The monomeric gene 4 protein forms a specific and stable complex with T7 DNA polymerase and thereby delivers the RNA primer to the polymerase for the onset of DNA synthesis. These results show that a single subunit of the primase-helicase hexamer contains all of the residues required for primer synthesis and for utilization of primers by T7 DNA polymerase."	complete CACAO 11625

References

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