Abstract
DNA primases catalyze the synthesis of oligoribonucleotides to initiate lagging strand DNA synthesis during DNA replication. Like other prokaryotic homologs, the primase domain of the gene 4 helicase-primase of bacteriophage T7 contains a zinc motif and a catalytic core. Upon recognition of the sequence, 5'-GTC-3' by the zinc motif, the catalytic site condenses the cognate nucleotides to produce a primer. The TOPRIM domain in the catalytic site contains several charged residues presumably involved in catalysis. Each of eight acidic residues in this region was replaced with alanine, and the properties of the altered primases were examined. Six of the eight residues (Glu-157, Glu-159, Asp-161, Asp-207, Asp-209, and Asp-237) are essential in that altered gene 4 proteins containing these mutations cannot complement T7 phage lacking gene 4 for T7 growth. These six altered gene 4 proteins can neither synthesize primers de novo nor extend an oligoribonucleotide. Despite the inability to catalyze phosphodiester bond formation, the altered proteins recognize the sequence 5'-GTC-3' in the template and deliver preformed primer to T7 DNA polymerase. The alterations in the TOPRIM domain result in the loss of binding affinity for ATP as measured by surface plasmon resonance assay together with ATP-agarose affinity chromatography.
Highlights
DNA primase is an essential component of most replisomes in that it catalyzes the synthesis of oligoribonucleotides that serve as primers for the lagging strand DNA polymerase [1]
An iterative profile search combined with structural modeling proposed that motifs IV–VI form a unique fold, the TOPRIM fold, found in topoisomerases [15]
The crystal structures of T7 DNA primase and E. coli DnaG primase confirmed that motifs IV and V are Acidic Residues in the Catalytic Site of T7 Primase
Summary
Materials—Oligonucleotides were obtained from the Biopolymer Laboratory at Harvard Medical School. All assays (templatedirected oligoribonucleotide synthesis, template-directed oligoribonucleotide extension, template-independent diribonucleotide synthesis, and RNA-primed DNA synthesis by DNA polymerase) used a reaction buffer containing 40 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM DTT, and 50 mM potassium glutamate plus the additional components described in each assay. Template-directed Oligoribonucleotide Synthesis—The ability of gene 4 protein to catalyze de novo synthesis of oligoribonucleotides was determined by measuring the incorporation of [␣-32P]CMP into oligoribonucleotides using a synthetic DNA template containing a primase recognition site. Determination of Binding Ability of Gene 4 Protein to Nucleotide Resin—A small volume (0.4 ml) of pre-swollen nucleotide-coupled agarose resin was packed into a column and washed with buffer A (20 mM potassium phosphate, pH 6.8, 10 mM MgCl2, 1 mM DTT, and 10% glycerol). The signal obtained when the proteins were flowed over the control lane was subtracted from the signal measured when proteins were flowed over the biotinylated NTP lane
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