Roles of the helicase and primase domain of the gene 4 protein of bacteriophage T7 in accessing the primase recognition site.
The 63 kDa gene 4 protein of bacteriophage T7 provides both helicase and primase activities. The C-terminal helicase domain of the gene 4 protein is responsible for DNA-dependent NTP hydrolysis and for hexamer formation, whereas the N-terminal primase domain contains the zinc motif that is, in part, responsible for template-directed oligoribonucleotide synthesis. In the presence of beta, gamma-methylene dTTP, the protein forms a hexamer that surrounds and binds tightly to single-stranded DNA and consequently is unable to translocate to primase recognition sites, 5'-GTC-3', or to dissociate from the molecule to which it is bound. Nonetheless, in the presence of beta,gamma-methylene dTTP, it catalyzes the synthesis of pppAC dimers at primase sites on M13 DNA. When bound to single-stranded DNA in the presence of beta,gamma-methylene dTTP, the primase can function at recognition sites on the same molecule to which it is bound provided that a sufficient distance exists between the recognition site and the site to which it is bound. Furthermore, the primase bound to one DNA strand can function at a primase site located on a second DNA strand. The results indicate that the primase domain resides on the outside of the hexameric ring, a location that enables it to access sites distal to its site of binding.
Kusakabe
T
T
Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, 240 Longwood Avenue, Boston, MA 02115, USA.
Baradaran
K
K
Lee
J
J
Richardson
C C
CC
D017123:Bacteriophage T7 / Q000201:enzymology*; D001483:Base Sequence; D004265:DNA Helicases / Q000378:metabolism*; D019915:DNA Primase / Q000378:metabolism*; D017931:DNA Primers / Q000096:biosynthesis; D004270:DNA, Circular / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D008969:Molecular Sequence Data; D009843:Oligoribonucleotides / Q000096:biosynthesis; D011485:Protein Binding; D013942:Thymine Nucleotides
D017931:DNA Primers; D004270:DNA, Circular; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D009843:Oligoribonucleotides; D013942:Thymine Nucleotides; C041083:beta, gamma-methylenedeoxythymidine 5'-triphosphate; D019915:DNA Primase; D004265:DNA Helicases