Characterization of a novel DNA primase from the Salmonella typhimurium bacteriophage SP6.
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.
Tseng
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Frick
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Richardson
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D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004265:DNA Helicases / Q000737:chemistry / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009841:Oligonucleotides / Q000737:chemistry; D009843:Oligoribonucleotides / Q000096:biosynthesis / Q000378:metabolism; D012483:Salmonella Phages / Q000201:enzymology* / Q000235:genetics; D012486:Salmonella typhimurium / Q000821:virology*; D017422:Sequence Analysis, DNA
D017931:DNA Primers; D004279:DNA, Viral; D009841:Oligonucleotides; D009843:Oligoribonucleotides; C031656:guanosine triphosphate cytidine monophosphate; D019915:DNA Primase; D004265:DNA Helicases