Choreography of bacteriophage T7 DNA replication

Abstract

The replication system of phage T7 provides a model for DNA replication. Biochemical, structural, and single-molecule analyses together provide insight into replisome mechanics. A complex of polymerase, a processivity factor, and helicase mediates leading strand synthesis. Establishment of the complex requires an interaction of the C-terminal tail of the helicase with the polymerase. During synthesis the complex is stabilized by other interactions to provide for a processivity of 5 kilobase (kb). The C-terminal tail also interacts with a distinct region of the polymerase to captures dissociating polymerase to increase the processivity to >17kb. The lagging strand is synthesized discontinuously within a loop that forms and resolves during each cycle of Okazaki fragment synthesis. The synthesis of a primer as well as the termination of a fragment signal loop resolution.

Figure 1

Model of the T7 replisome. The hexameric helicase domain of gp4, encircling the lagging strand, unwinds the duplex DNA to provide two single-stranded DNA templates. Gp5/trx bound to the helicase catalyzes the polymerization of nucleotides on the leading strand. The ssDNA extruded by the translocating helicase forms a replication loop in which the primase domain of gp4 catalyzes the synthesis of a tetraribonucleotide (green) to serve as a primer for the lagging strand gp5/trx that is also bound to the helicase. Gp2.5 coats the exposed ssDNA.

Figure 2

Three modes of interactions between gp4 and gp5/trx during leading strand synthesis. (a) Gp5/trx is loaded onto the hexameric gp4 through an interaction of the acidic C-terminal tail of gp4 with a basic patch at the front side (Fbp) of gp5. (b) A high affinity complex between the two proteins coordinates their activities during DNA synthesis. (c) Gp5/trx temporarily dissociated from gp4 or present in solution is retained with gp4 through an interaction between two basic loops in the trx binding domain (TBDbp) of gp5 and the acidic C-terminal tail of gp4.

Figure 3

Two distinct modes involved in primer synthesis by the primase domain of gp4. Synthesis of tetraribonucleotides at the primase recognition sequence requires interactions between the zinc-binding domain (ZBD) and RNA polymerase domain (RPD) of the primase domain of gp4. (a) Diribonucleotide pppAC is synthesized from the 5’-GTC-3’ basic recognition sequence through interactions between the ZBD and RPD in the same subunit (cis mode). Extension of the diribonucleotide to the functional tetraribonucleotide is facilitated by shortening the linker between the ZBD and RPD. (b) In the hexameric gp4 structure, the ZBD in one subunit interacts with the RPD in an adjacent subunit to catalyze the synthesis of a primer (trans mode).