The role of the zinc motif in sequence recognition by DNA primases.
The DNA primase of bacteriophage T7 has a zinc-binding motif that is essential for the recognition of the sequence 3'-CTG-5'. The T7 primase also catalyzes helicase activity, a reaction coupled to nucleotide hydrolysis. We have replaced the zinc motif of the T7 primase with those found in the gene 61 primase of phage T4 and the DnaG primase of Escherichia coli. The T4 and E. coli primases recognize the sequences 3'-T(C/T)G-5' and 3'-GTC-5', respectively. Both chimeric proteins can partially replace T7 primase in vivo. The two chimeric primases catalyze the synthesis of oligoribonucleotides albeit at a reduced rate and DNA dependent dTTPase activity is reduced by 3-10-fold. Both chimeric proteins recognize 3'-(A/G)CG-5' sites on single-stranded DNA, sites that differ from those recognized by the T7, T4, or E. coli primases, indicating that the zinc motif is only one determinant in site-specific recognition.
Kusakabe
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Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, Boston, Massachusetts 02115, USA.
Richardson
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CC
D000595:Amino Acid Sequence; D017122:Bacteriophage T4 / Q000201:enzymology; D001483:Base Sequence; D019915:DNA Primase; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005816:Genetic Complementation Test; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012316:RNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013942:Thymine Nucleotides / Q000378:metabolism; D015032:Zinc / Q000378:metabolism*
D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D013942:Thymine Nucleotides; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; C034970:bacteriophage T7 induced DNA polymerase; D004259:DNA-Directed DNA Polymerase; D015032:Zinc; C024157:thymidine 5'-triphosphate