Modular architecture of the bacteriophage T7 primase couples RNA primer synthesis to DNA synthesis.
DNA primases are template-dependent RNA polymerases that synthesize oligoribonucleotide primers that can be extended by DNA polymerase. The bacterial primases consist of zinc binding and RNA polymerase domains that polymerize ribonucleotides at templating sequences of single-stranded DNA. We report a crystal structure of bacteriophage T7 primase that reveals its two domains and the presence of two Mg(2+) ions bound to the active site. NMR and biochemical data show that the two domains remain separated until the primase binds to DNA and nucleotide. The zinc binding domain alone can stimulate primer extension by T7 DNA polymerase. These findings suggest that the zinc binding domain couples primer synthesis with primer utilization by securing the DNA template in the primase active site and then delivering the primed DNA template to DNA polymerase. The modular architecture of the primase and a similar mechanism of priming DNA synthesis are likely to apply broadly to prokaryotic primases.
Kato
Masato
M
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Ito
Takuhiro
T
Wagner
Gerhard
G
Richardson
Charles C
CC
Ellenberger
Tom
T
D000595:Amino Acid Sequence / Q000235:genetics; D017123:Bacteriophage T7 / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis* / Q000235:genetics; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics; D012313:RNA / Q000096:biosynthesis* / Q000235:genetics; D015032:Zinc / Q000378:metabolism
C034477:RNA primers; D012313:RNA; D004247:DNA; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase; D008274:Magnesium; D015032:Zinc