Acidic residues in the nucleotide-binding site of the bacteriophage T7 DNA primase.
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.
Lee
Seung-Joo
SJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Richardson
Charles C
CC
D019943:Amino Acid Substitution; D024342:Amino Acids, Acidic; D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D002846:Chromatography, Affinity; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism; D009711:Nucleotides / Q000378:metabolism*; D009843:Oligoribonucleotides / Q000096:biosynthesis; D020349:Surface Plasmon Resonance
D024342:Amino Acids, Acidic; D009711:Nucleotides; D009843:Oligoribonucleotides; D019915:DNA Primase