A molecular handoff between bacteriophage T7 DNA primase and T7 DNA polymerase initiates DNA synthesis.
The T7 DNA primase synthesizes tetraribonucleotides that prime DNA synthesis by T7 DNA polymerase but only on the condition that the primase stabilizes the primed DNA template in the polymerase active site. We used NMR experiments and alanine scanning mutagenesis to identify residues in the zinc binding domain of T7 primase that engage the primed DNA template to initiate DNA synthesis by T7 DNA polymerase. These residues cover one face of the zinc binding domain and include a number of aromatic amino acids that are conserved in bacteriophage primases. The phage T7 single-stranded DNA-binding protein gp2.5 specifically interfered with the utilization of tetraribonucleotide primers by interacting with T7 DNA polymerase and preventing a productive interaction with the primed template. We propose that the opposing effects of gp2.5 and T7 primase on the initiation of DNA synthesis reflect a sequence of mutually exclusive interactions that occur during the recycling of the polymerase on the lagging strand of the replication fork.
Kato
Masato
M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Ito
Takuhiro
T
Wagner
Gerhard
G
Ellenberger
Tom
T
D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000096:biosynthesis* / Q000737:chemistry; D019915:DNA Primase / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics; D015032:Zinc / Q000737:chemistry
D017931:DNA Primers; D004268:DNA-Binding Proteins; C022262:Okazaki fragments; D014764:Viral Proteins; C085913:gene 2.5 protein, Enterobacteria phage T7; D004247:DNA; D019915:DNA Primase; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D015032:Zinc; D000409:Alanine