Conditional Coupling of Leading-strand and Lagging-strand DNA Synthesis at Bacteriophage T4 Replication Forks

Farid A Kadyrov,John W Drake

doi:10.1074/jbc.m101310200

Abstract

Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution. If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed. Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis. A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis. Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA. It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase. Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner.

Highlights

Biochemical studies of the purified proteins and of DNA replication reconstituted in vitro have clarified many structural and mechanistic details of this complicated process
Because both polymerase and additional replication proteins are involved in laggingstrand synthesis in all analyzed replication systems, dilution experiments clarify whether these proteins, once loaded, remain bound within a replication complex or function distributively during repetitive cycles of Okazaki fragment synthesis
The synthesis of lagging strands depends strongly on the CTP and UTP used by the helicase-primase complex to synthesize pentaribonucleotide primers (Fig. 1B, lanes 7–9)

Summary

Introduction

Biochemical studies of the purified proteins and of DNA replication reconstituted in vitro have clarified many structural and mechanistic details of this complicated process. Dilution of pre-formed replication complexes is a powerful method for differentiating between coupled and uncoupled modes of DNA replication because in uncoupled synthesis, lagging-strand synthesis depends on the concentration of DNA polymerase and is sensitive to dilution Because both polymerase and additional replication proteins are involved in laggingstrand synthesis in all analyzed replication systems, dilution experiments clarify whether these proteins, once loaded, remain bound within a replication complex or function distributively (i.e. are recruited from solution for each cycle) during repetitive cycles of Okazaki fragment synthesis. In both the complicated Escherichia coli and the simpler phage T7 systems for replication in vitro, dilution experiments showed that leading-strand and lagging-strand DNA replica-. Direct interactions may occur between these two proteins within the replication fork, because a tryptic product of the gp helicase that lacks 17–20 amino acids from the COOH end has normal helicase activity but fails to function as a helicase in the T4 replication fork [21]

Methods

Results

Conclusion