Gene 4 DNA primase of bacteriophage T7 mediates the annealing and extension of ribo-oligonucleotide at primase recognition sites

Takahiro Kusakabe, Charles C. Richardson

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

The 63-kDa gene 4 primase of bacteriophage T7 recognizes a core trinucleotide sequence, 5'-GTC-3', on single-stranded DNA at which it catalyzes the synthesis of the ribodinucleotide pppAC. The dinucleotide is extended to a tetranucleotide primer at the sites 5'-(G/T)GGTC-3' and 5'- GTGTC-3'. In the presence of T7 primase, T7 DNA polymerase extends the synthetic ribotetranucleotide pACCA (1 μM), but not pCACA, on M13 DNA templates. The reaction is specific for T7 DNA polymerase and depends on dTTP and translocation of the gene 4 protein, T7 primase extends the dinucleotide AC and trinucleotide ACC to ACCC in the presence of CTP and an appropriate template, whereas other dinucleotides are extended less efficiently; the deoxyribodinucleotide dAC is not extended. The Cys4 zinc motif of the primase is essential for extension of the dinucleotides. The 5'-cryptic cytidine of the recognition sequence is essential for extension of the dinucleotide AC to tri- and tetranucleotides. At a preformed replication fork, the dinucleotide AC provides for primer synthesis on the lagging strand. The synthesis of all Okazaki fragments is initiated by primers arising from the recognition sequence 5'-GGGTC-3'; none arise at an adjacent 5'-GGGTT-3' sequence. If ADP or AMP replaces ATP in the primase reaction, primers terminating in di- or monophosphate, respectively, are synthesized.

Original languageEnglish
Pages (from-to)12446-12453
Number of pages8
JournalJournal of Biological Chemistry
Volume272
Issue number19
DOIs
Publication statusPublished - May 9 1997
Externally publishedYes

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this