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Updated: Jul 13, 2026
Kinetics of Lagging-strand DNA Synthesis In Vitro by the Bacteriophage T7 Replication Proteins
Published on: February 25, 2017
Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6
T Y Tseng1, D N Frick, C C Richardson
1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Abstract:
The gene for the DNA primase encoded by Salmonella typhimurium bacteriophage SP6 has been cloned and expressed in Escherichia coli and its 74-kDa protein product purified to homogeneity. The SP6 primase is a DNA-dependent RNA polymerase that synthesizes short oligoribonucleotides containing each of the four canonical ribonucleotides. GTP and CTP are both required for the initiation of oligoribonucleotide synthesis. In reactions containing only GTP and CTP, SP6 primase incorporates GTP at the 5'-end of oligoribonucleotides and CMP at the second position. On synthetic DNA templates, pppGpC dinucleotides are synthesized most rapidly in the presence of the sequence 5'-GCA-3'. This trinucleotide sequence, containing a cryptic dA at the 3'-end, differs from other known bacterial and phage primase recognition sites. SP6 primase shares some properties with the well-characterized E. colibacteriophage T7 primase. The T7 DNA polymerase can use oligoribonucleotides synthesized by SP6 primase as primers for DNA synthesis. However, oligoribonucleotide synthesis by SP6 primase is not stimulated by either the E. coli- or the T7-encoded ssDNA binding protein. An amino acid sequence alignment of the SP6 and T7 primases, which share only 22.4% amino acid identity, indicates amino acids likely critical for oligoribonucleotide synthesis as well as a putative Cys(3)His zinc finger motif that may be involved in DNA binding.
Insights
The SP6 bacteriophage primase, a DNA-dependent RNA polymerase, synthesizes short RNA primers. Its unique recognition sequence differs from other known primases, though it shares properties with T7 primase.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- The DNA primase enzyme is essential for initiating DNA replication by synthesizing RNA primers.
- Bacteriophage SP6 encodes a DNA primase with unique characteristics.
- Understanding primase function is crucial for comprehending DNA replication mechanisms.
Purpose of the Study:
- To clone, express, and purify the SP6 bacteriophage DNA primase.
- To characterize the enzymatic activity and substrate specificity of SP6 primase.
- To compare SP6 primase with other known primases, such as T7 primase.
Main Methods:
- Gene cloning and expression in Escherichia coli.
- Protein purification to homogeneity.
- Enzymatic assays to determine RNA synthesis activity and template dependence.
- Amino acid sequence alignment with related enzymes.
Main Results:
- The 74-kDa SP6 primase protein was successfully purified.
- SP6 primase functions as a DNA-dependent RNA polymerase, synthesizing oligoribonucleotides.
- Guanosine triphosphate (GTP) and cytidine triphosphate (CTP) are required for initiation, with GTP incorporated at the 5'-end and CTP at the second position.
- The enzyme shows optimal synthesis of pppGpC dinucleotides on templates with a 5'-GCA-3' sequence, a novel recognition site.
- SP6 primase shares functional similarities with T7 primase, and its products can prime T7 DNA polymerase.
- Unlike E. coli or T7 primase, SP6 primase activity is not stimulated by single-stranded DNA binding proteins.
- Sequence alignment reveals low overall identity (22.4%) with T7 primase but highlights conserved regions and a potential zinc finger motif.
Conclusions:
- SP6 bacteriophage primase is a distinct DNA-dependent RNA polymerase with a unique DNA recognition site.
- The enzyme's properties provide insights into the diversity of primase mechanisms in bacteriophages.
- Structural analysis suggests potential DNA-binding domains critical for its function.
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