Lagging-strand Polymerase Research Articles

Inversion/dimerization of plasmids mediated by inverted repeats

In contrast with earlier studies on the lambda and Escherichia coli genomes, recombination between inverted repeats on plasmids is highly efficient and shown to be recA-independent. In addition, the recombination product is exclusively a head-to-head inverted dimer. Here, we show that this recombination/rearrangement event can occur on different plasmid replicons and is not specific to the particular sequence within the inverted repeats. Transcription readthrough into the inverted repeats has little effect on this event. Genetic analysis has also indicated that most known recombination enzymes are not involved in this process. Specifically, single or double mutants defective in Holliday junction resolution systems (RuvABC and/or RecG/RusA) do not abolish this recombination/rearrangement event. This result does not support the previous models (i.e. the reciprocal-strand-switching and the cruciform-dumbbell models) in which intermediates containing Holliday junctions are proposed. Further analysis has demonstrated that the recombination/rearrangement frequency is dramatically (over 1000-fold) reduced if mismatches (2.8%) are present within the inverted repeats. Mutations in dam, mutH and mutL genes partially or completely restored the recombination/rearrangement frequency to the level exhibited by the perfect inverted repeats, suggesting the formation of heteroduplexes during recombination/rearrangement. Sequencing analysis of the recombination/rearrangement products have indicated that the majority of the products do not involve crossing-over. We discuss a possible mechanism in which blockage of the lagging strand polymerase by a hairpin triggers recombination/rearrangement mediated by inverted repeats.

Coordinated Leading and Lagging Strand DNA Synthesis on a Minicircular Template

The coordinated synthesis of both leading and lagging DNA strands is thought to involve a dimeric DNA polymerase and a looping of the lagging strand so that both strands can be synthesized in the same direction. We have constructed a minicircle with a replication fork that permits an assessment of the stoichiometry of the proteins and a measurement of the synthesis of each strand. The replisome consisting of bacteriophage T7 DNA polymerase, helicase, primase, and single-stranded DNA-binding protein mediates coordinated replication. The criteria for coordination are fulfilled: (1) a replication loop is formed, (2) leading and lagging strand synthesis are coupled, (3) the lagging strand polymerase recycles from one Okazaki fragment to another, and (4) the length of Okazaki fragments is regulated. T7 single-stranded DNA-binding protein is essential for coordination.

τCouples the Leading- and Lagging-strand Polymerases at the Escherichia coli DNA Replication Fork

Synthesis of an Okazaki fragment occurs once every 1 or 2 s at the Escherichia coli replication fork. To account for the rapid recycling required of the lagging-strand polymerase, it has been proposed that it is held at the replication fork by protein-protein interactions with the leading-strand polymerase as part of a dimeric polymerase assembly. Solution studies showed that the replicative polymerase, the DNA polymerase III holoenzyme, was indeed a dimer with two catalytic cores held together by the tau subunit. However, the functionality of this arrangement at the replication fork has never been demonstrated. We showed previously that the lagging-strand polymerase acted processively during multiple rounds of Okazaki fragment synthesis, i.e. the same polymerase core assembly synthesized each and every fragment made by the fork. Using extreme dilution of active replication forks and the isolation of protein-DNA complexes capable of supporting coupled leading- and lagging-strand synthesis, we demonstrate here that this coupling of leading- and lagging-strand synthesis is, in fact, mediated by the tau subunit of the holoenzyme acting as a physical bridge between the core assemblies synthesizing the leading and lagging strands.

Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin

Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components. DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex. A model for polymerase switching during initiation of DNA replication is presented.

The asymmetric dimeric polymerase hypothesis: A progress report

In 1983, my laboratory first proposed that the DNA polymerase III holoenzyme is an asymmetric dimer with distinguishable leading and lagging strand polymerases. Here, I review progress by my laboratory and others in testing this hypothesis. To date, the hypothesis is supported by our demonstration of (i) an asymmetry in function of two populations of holoenzyme in solution in their ability to use the ATP analog, ATPγS, to support initiation complex formation, (ii) the stabilization of a dimeric polymerase structure by the τ subunit, (iii) allosteric communication between polymerase halves and (iv) the coexistence of γ and the τ, subunits which share common sequences, within the same holoenzyme assemblies. This latter observation may provide a structural basis for holoenzyme asymmetry. I discuss the implications of the asymmetric dimer hypothesis to the solution of problems encountered by polymerases at the replication fork and delineate further tests required before the hypothesis can be firmly established.