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.

Figure 1

Organization of T7 gene 4 protein. Gene 4 protein consists of a primase and a helicase domain, connected by a linker region. The primase domain contains two subdomains: a zinc motif and a catalytic core. The gene 4 proteins used in this study are schematically presented. A 63-kDa protein; 56-kDa protein, a truncated gene 4 protein that lacks the N-terminal 63-aa residue containing the zinc motif; gp4-K122A, a gene 4 protein in which K122 at the catalytic site is replaced by alanine; PF, a primase fragment that contains only the primase domain from amino acid 1 to 271; and PF-K122A, a primase fragment in which K122 is replaced by alanine. Signs next to the abbreviated protein names denote the ability to catalyze the synthesis of primers.

Figure 2

Oligoribonucleotide synthesis catalyzed by mixtures of gene 4 proteins. A fixed amount (50 ng) of the indicated gene 4 protein was mixed with increasing amount (0, 50, 100, or 200 ng) of the indicated other gene 4 protein. The mixture was incubated with 10 μM DNA template 5′-GGGTCA₁₀-3′ in the standard reaction for 30 min at 37°C. Note that dTTP was omitted. Reaction products were separated and analyzed as described in Materials and Methods. In the autoradiograph of the gel, the major product of the reaction, tetraribonucleotide ACCC, is denoted on the left.

Figure 3

Effect of dTTP and its analog β,γ-methylene dTTP on primase activity. (A) The reconstituted primases consisting of 0.15 μM each of the 56-kDa protein and gp4-K122A were incubated with 25 μM template 5′-GGGTCA₁₀-3′ in the standard reaction with various concentrations of either dTTP or its nonhydrolyzable analog β,γ-methylene dTTP for 20 min at 37°C. Reaction products were separated and analyzed as described in Materials and Methods. Incorporation of [α-³²P]CMP into the product was plotted against the concentration of the dNTPs. (B) Inhibition of primase activity by preincubation with β,γ-methylene dTTP. The indicated gene 4 protein (0.08 μM) was incubated with 1 mM β,γ-methylene dTTP for the indicated period at room temperature before the addition of an equal amount of the other gene 4 protein. After an additional 5-min incubation, primase reactions were initiated by the addition of 10 μM template 5′-GGGTCA₁₀-3′, and rNTPs, and carried out for 20 min at 37°C. Incorporation of [α-³²P]CMP into the product was plotted against the preincubation time with the dTTP analog.

Figure 4

Two modes of primer synthesis catalyzed by gene 4 protein. (A) Effect of 3′-flanking sequence on the primase activity of gene 4 protein. Both the 6-nt (5′-GGGTCA-3′) and 15-nt (5′-GGGTCA₁₀-3′) templates have the same primase recognition site but differ in the 3′-side flanking length. The indicated gene 4 proteins were incubated with the template in the standard reaction with or without 0.1 mM dTTP for 20 min at 37°C. The protein concentration of either the 63-kDa protein or the primase fragment was 0.1 μM, and total concentration of equimolar mixture of the 56-kDa protein and gp4-K122A was 0.3 μM. Incorporation efficiency of [α-³²P]CMP into the product is presented as height of bars in graphs. The presence or absence of dTTP is denoted. (B) Effect of defective gene 4 proteins on the primase activity of 63-kDa gene 4 protein. On the two different sets of templates described above, increasing concentrations of the 63-kDa protein alone were incubated in the standard reaction at 37°C for 20 min (▵). Either the 56-kDa protein (○) or gp4-K122A (●) was added to the 63-kDa protein to yield a total 0.3 μM protein. The mixtures of proteins used for the 15-nt template were incubated for 10 min at 37°C before the primer synthesis reaction. Incorporation of [α-³²P]CMP into the product is plotted against the 63-kDa protein concentration. (C) Effect of mixing ratio on primer synthesis catalyzed by the 56-kDa protein and g-K122A. Reaction condition was the same as the one used for the 15-mer template in B.

Figure 5

Reconstituted primase synthesizes functional primers for T7 DNA polymerase. The ability of gene 4 protein to prime DNA synthesis catalyzed by T7 DNA polymerase was determined by measuring the incorporations of dNTP into DNA product (15). The reaction contained 9.8 nM M13 single-stranded DNA, 0.3 mM of all four dNTPs, 0.1 μCi [α-³²P]dCTP, 0.1 mM each of ATP and CTP, the increasing amounts of the indicated gene 4 protein, and 20 nM T7 DNA polymerase. After incubation for 10 min at 37°C, radioactivity incorporated into DNA products was measured and plotted against the concentration of gene 4 proteins used.

Figure 6

Model for primer synthesis catalyzed by hexamer composed of 56- and 63-kDa gene 4 proteins.