Coordinated DNA Replication by the Bacteriophage T4 Replisome

Abstract

The T4 bacteriophage encodes eight proteins, which are sufficient to carry out coordinated leading and lagging strand DNA synthesis. These purified proteins have been used to reconstitute DNA synthesis in vitro and are a well-characterized model system. Recent work on the T4 replisome has yielded more detailed insight into the dynamics and coordination of proteins at the replication fork. Since the leading and lagging strands are synthesized in opposite directions, coordination of DNA synthesis as well as priming and unwinding is accomplished by several protein complexes. These protein complexes serve to link catalytic activities and physically tether proteins to the replication fork. Essential to both leading and lagging strand synthesis is the formation of a holoenzyme complex composed of the polymerase and a processivity clamp. The two holoenzymes form a dimer allowing the lagging strand polymerase to be retained within the replisome after completion of each Okazaki fragment. The helicase and primase also form a complex known as the primosome, which unwinds the duplex DNA while also synthesizing primers on the lagging strand. Future studies will likely focus on defining the orientations and architecture of protein complexes at the replication fork.

Keywords: DNA replication; T4 bacteriophage; replisome.

Figure 1

A model of the T4 bacteriophage DNA replisome. Replication of T4 genomic DNA is accomplished by a replication complex composed of eight proteins. The helicase (gp41) and primase (gp61) interact to form the primosome with the assistance of the helicase loader (gp59). The primosome complex encircles the lagging strand DNA, unwinding duplex DNA while synthesizing RNA primers for use by the lagging strand polymerase (gp43). DNA synthesis on both strands is catalyzed by a holoenzyme complex formed by a polymerase (gp43) and a trimeric processivity clamp (gp45). The clamp is loaded onto the DNA by the clamp loader complex (gp44/62). The leading and lagging strand holoenzymes interact to form a dimer. Single-stranded DNA formed by the helicase is coated with single-stranded DNA-binding protein (gp32).

Figure 2

The two models of primosome activity used by T4 to initiate lagging strand synthesis. The helicase (gp41) and primase (gp61) interact as stacked rings encircling the lagging strand. This complex unwinds duplex DNA while synthesizing pentaribonucleotide RNA primers for use by the lagging strand polymerase (gp43). Primer synthesis occurs while the helicase continues to unwind DNA in the opposite direction. Two models have been proposed to accommodate these coupled activities. In the primosome disassembly model (shown left), one of the primase subunits dissociates from the primosome complex and remains with the newly synthesized primer. In the DNA looping model (shown right), the excess DNA unwound by the helicase during primer synthesis loops out allowing the primase to stay intact. In both models, the clamp loader (gp44/62) loads a clamp (gp45) onto the newly synthesized primer. The lagging strand polymerase is then signaled to release and recycle to the new primer.