Kenneth J. Marians

Coupling of a Replicative Polymerase and Helicase: A τ–DnaB Interaction Mediates Rapid Replication Fork Movement

The E. coli replication fork synthesizes DNA at the rate of nearly 1000 nt/s. We show here that an interaction between the tau subunit of the replicative polymerase (the DNA polymerase III holoenzyme) and the replication fork DNA helicase (DnaB) is required to mediate this high rate of replication fork movement. In the absence of this interaction, the polymerase follows behind the helicase at a rate equal to the slow (approximately 35 nt/s) unwinding rate of the helicase alone, whereas upon establishing a tau-DnaB contact, DnaB becomes a more effective helicase, increasing its translocation rate by more than 10-fold. This finding establishes the existence of both a physical and communications link between the two major replication machines in the replisome: the DNA polymerase and the primosome.

DNA gyrase and topoisomerase IV: Biochemical activities, physiological roles during chromosome replication, and drug sensitivities

DNA gyrase and topoisomerase IV are the two type II topoisomerases present in bacteria. Though clearly related, based on amino acid sequence similarity, they each play crucial, but distinct, roles in the cell. Gyrase is involved primarily in supporting nascent chain elongation during replication of the chromosome, whereas topoisomerase IV separates the topologically linked daughter chromosomes during the terminal stage of DNA replication. These different roles can be attributed to differences in the biochemical properties of the two enzymes. The biochemical activities, physiological roles, and drug sensitivities of the enzymes are reviewed.

The Ordered Assembly of the ϕX174-type Primosome I. ISOLATION AND IDENTIFICATION OF INTERMEDIATE PROTEIN-DNA COMPLEXES

The φX-type primosome was discovered during the resolution and reconstitution in vitro of the complementary strand DNA replication step of the φX174 viral life cycle. This multienzyme bidirectional helicase-primase complex can provide the DNA unwinding and Okazaki fragment-priming functions at the replication fork and has been implicated in cellular DNA replication, repair, and recombination. We have used gel mobility shift assays and enhanced chemiluminescence Western analysis to isolate and identify the pathway of primosome assembly at a primosome assembly site (PAS) on a 300-nucleotide-long single-stranded DNA fragment. The first three steps do not require ATP and are as follows: (i) PriA recognition and binding to the PAS, (ii) stabilization of the PriA-PAS complex by the addition of PriB, and (iii) formation of a PriA-PriB-DnaT-PAS complex. Subsequent formation of the preprimosome involves the ATP-dependent transfer of DnaB from a DnaB-DnaC complex to the PriA-PriB-DnaT-PAS complex. The final preprimosomal complex contains PriA, PriB, DnaT, and DnaB but not DnaC. A transient interaction between the preprimosome and DnaG generates the five-protein primosome. As described in an accompanying article (Ng, J. Y., and Marians, K. J. (1996) J. Biol. Chem. 271, 15649-15655), when assembled on intact φX174 phage DNA, the primosome also contains PriC.

A general method for inserting specific DNA sequences into cloning vehicles

A general method has been developed to introduce any double-stranded DNA molecule into cloning vehicles at different restriction endonuclease sites. In this method a chemically synthesized decadeoxyribonucleotide duplex, containing a specific restriction endonuclease sequence, is joinlex DNA is cut by the same restriction endonuclease to generate the cohesive ends. It is then inserted into the restriction endonuclease cleavage site of the cloning vehicle. To demonstrate the feasibility of this new method, we have inserted separately the synthetic lac operator DNA at the Bam I and HindIII cleavage sites of the plasmid pMB9 DNA.

Temporal Regulation of Topoisomerase IV Activity in E. coli

We isolated a mutant allele of dnaX, encoding the tau and gamma subunits of the DNA polymerase III holoenzyme, that causes extreme cell filamentation but does not affect either cell growth or DNA replication. This phenotype results from a defect in daughter chromosome decatenation during rapid growth. In these cells, ParC, one subunit of topoisomerase IV, no longer associated with the replication factory, as occurs in wild-type cells, and was instead distributed uniformly on the nucleoid; the distribution of ParE, the other subunit of topoisomerase IV, was unaffected. In addition, the majority of topoisomerase IV activity in synchronized cell populations was restricted to late in the cell cycle, when replication was essentially complete. These observations suggest that topoisomerase IV activity in vivo might be dependent on release of ParC from the replication factory.

Recruitment to stalled replication forks of the PriA DNA helicase and replisome-loading activities is essential for survival

PriA, a 3-->5 superfamily 2 DNA helicase, acts to remodel stalled replication forks and as a specificity factor for origin-independent assembly of a new replisome at the stalled fork. The ability of PriA to initiate replication at stalled forked structures ensures complete genome replication and helps to protect the cell from illegitimate recombination events. This review focuses on the activities of PriA and its role in replication fork assembly and maintaining genomic integrity.

The Ordered Assembly of the ϕX174-type Primosome III. PriB FACILITATES COMPLEX FORMATION BETWEEN PriA AND DnaT

The properties of two mutant PriA proteins, PriA C439Y and PriA C445Y have been used to reveal the role of PriB during assembly of the φX174-type primosome. The replication defects of both mutant PriA proteins could be rescued by high concentrations of DnaT. Analysis of the formation of intermediate complexes in primosome assembly and the effect of PriB on PriA binding to DNA demonstrated that the mutant PriA proteins could not form a PriA-PriB complex on DNA carrying a primosome assembly site. Consequently, the mutant proteins also could not form PriA-PriB-DnaT complexes at concentrations of DnaT sufficient to form such a complex with wild-type PriA. In addition, PriB was found to stabilize wild-type but not mutant PriA proteins on DNA. At high concentrations of DnaT, both mutant and wild-type PriA proteins could form a PriA-DnaT complex and support PriB-independent φX174 complementary strand DNA replication. Thus, during primosome assembly, PriB facilitates complex formation between PriA and DnaT.

Role of the Core DNA Polymerase III Subunits at the Replication Fork α IS THE ONLY SUBUNIT REQUIRED FOR PROCESSIVE REPLICATION

The DNA polymerase III holoenzyme is composed of 10 subunits. The core of the polymerase contains the catalytic polymerase subunit, α, the proofreading 3′ → 5′ exonuclease, ε, and a subunit of unknown function, θ. The availability of the holoenzyme subunits in purified form has allowed us to investigate their roles at the replication fork. We show here that of the three subunits in the core polymerase, only α is required to form processive replication forks that move at high rates and that exhibit coupled leading- and lagging-strand synthesis in vitro. Taken together with previous data this suggests that the primary determinant of replication fork processivity is the interaction between another holoenzyme subunit, τ, and the replication fork helicase, DnaB.

Actin Homolog MreB Affects Chromosome Segregation by Regulating Topoisomerase IV in Escherichia coli

In Escherichia coli, topoisomerase IV, a type II topoisomerase, mediates the resolution of topological linkages between replicated daughter chromosomes and is essential for chromosome segregation. Topo IV activity is restricted to only a short interval late in the cell cycle. However, the mechanism that confers this temporal regulation is unknown. Here we report that the bacterial actin homolog MreB participates in the temporal oscillation of Topo IV activity. We show that mreB mutant strains are deficient in Topo IV activity. In addition, we demonstrate that, depending upon whether it is in a monomeric or polymerized state, MreB affects Topo IV activity differentially. In addition, MreB physically interacts with the ParC subunit of Topo IV. Together, these results may explain how dynamics of the bacterial cytoskeleton are coordinated with the timing of chromosome segregation.

Studies on the lactose operon. III. Visualization and physical mapping of the lactose repressor-operator complex.

A physical mapping of the location of the lactose operator in the hybrid phage γh80dlacUV5 was accomplished by electron microscopy after mounting the lac repressor-lac DNA complex to a cytochrome c film followed by high-resolution shadowing. The location of the lac repressor peaked within a 0·15 μm wide zone at 3·2 μm from one end of the 15·2 μm long DNA molecule. The lac gene is thus located at 21% from one end of the DNA length.