Interaction of adjacent primase domains within the hexameric gene 4 helicase-primase of bacteriophage T7

Abstract

The interaction of primase monomers within the hexameric gene 4 helicase-primase of bacteriophage T7 has been examined by using two genetically distinct gene 4 proteins. The T7 56-kDa gene 4 protein differs from the full-length 63-kDa protein in that it lacks the N-terminal zinc motif essential for the recognition of primase recognition sites. A second gene 4 protein, gp4-K122A, is unable to catalyze the synthesis of phosphodiester bonds as the result of an amino acid change in the catalytic site. Although each protein alone is inactive, the two together catalyze the synthesis of RNA primers. Reconstitution of activity depends on hexamer formation. We propose that the zinc motif of one subunit in the hexamer interacts with the catalytic sites of adjacent subunits.

Zinc-binding Domain of the Bacteriophage T7 DNA Primase Modulates Binding to the DNA Template

“…The loss of ability to catalyze the template-directed synthesis of oligoribonucleotides could result from defective function of either the ZBD or RPD (10). However, all the hybrid DNA primases, like the 56-kDa gene 4 protein, catalyze the templateindependent synthesis of random diribonucleotides (supplemental Fig.…”

Heterohexamer of 56- and 63-kDa Gene 4 Helicase-Primase of Bacteriophage T7 in DNA Replication

“…M13 ssDNA, DNA, and T4 polynucleotide kinase were purchased from New England Biolabs. The 63-kDa gp4, 56-kDa gp4, 63-kDa gp4-D237A, 56-kDa gp4-D237A, gp5/ trx, and gp2.5 were overproduced and purified as described (2,14,20,24,25). Minicircular DNA was prepared as described (26).…”

“…Finally, the 56-kDa gp4 cannot catalyze oligoribonucleotide synthesis due to the absence of the ZBD. An altered 63-kDa gp4 in which lysine-122 is replaced with alanine in the active site of the RPD (gp4-K122A) cannot catalyze the formation of phosphodiester bonds and thus also is unable to synthesize oligoribonucleotides (20). However, mixing the two proteins results in the template-directed synthesis of oligoribonucleotides (20).…”

“…An altered 63-kDa gp4 in which lysine-122 is replaced with alanine in the active site of the RPD (gp4-K122A) cannot catalyze the formation of phosphodiester bonds and thus also is unable to synthesize oligoribonucleotides (20). However, mixing the two proteins results in the template-directed synthesis of oligoribonucleotides (20). In this "trans" synthesis of oligoribonucleotides the ZBD of the gp4-K122A must contact the RPD of an adjacent 56-kDa gp4 subunit, indicating the formation of heterohexamer between the 63-and 56-kDa gp4.…”

The Roles of Tryptophans in Primer Synthesis by the DNA Primase of Bacteriophage T7

“…A shallow cleft is present between these two subdomains, a feature that is also found in E. coli DnaG primase (6,7). The flexible linker allows the ZBD of one subunit in a hexameric gp4 to interact with the RPD of an adjacent subunit to reconstitute a functional primase and catalyze the primer synthesis in a trans mode (8,9).…”