Mechanism of sequence-specific template binding by the DNA primase of bacteriophage T7.
DNA primases catalyze the synthesis of the oligoribonucleotides required for the initiation of lagging strand DNA synthesis. Biochemical studies have elucidated the mechanism for the sequence-specific synthesis of primers. However, the physical interactions of the primase with the DNA template to explain the basis of specificity have not been demonstrated. Using a combination of surface plasmon resonance and biochemical assays, we show that T7 DNA primase has only a slightly higher affinity for DNA containing the primase recognition sequence (5'-TGGTC-3') than for DNA lacking the recognition site. However, this binding is drastically enhanced by the presence of the cognate Nucleoside triphosphates (NTPs), Adenosine triphosphate (ATP) and Cytosine triphosphate (CTP) that are incorporated into the primer, pppACCA. Formation of the dimer, pppAC, the initial step of sequence-specific primer synthesis, is not sufficient for the stable binding. Preformed primers exhibit significantly less selective binding than that observed with ATP and CTP. Alterations in subdomains of the primase result in loss of selective DNA binding. We present a model in which conformational changes induced during primer synthesis facilitate contact between the zinc-binding domain and the polymerase domain.
Lee
Seung-Joo
SJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA.
Zhu
Bin
B
Hamdan
Samir M
SM
Richardson
Charles C
CC
D000255:Adenosine Triphosphate / Q000378:metabolism; D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D001665:Binding Sites; D003570:Cytidine Triphosphate / Q000378:metabolism; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D009843:Oligoribonucleotides / Q000096:biosynthesis; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009843:Oligoribonucleotides; D003570:Cytidine Triphosphate; D000255:Adenosine Triphosphate; D019915:DNA Primase