Tus prevents overreplication of oriC plasmid DNA.

H Hiasa,K J Marians

doi:10.1016/s0021-9258(18)47112-8

Abstract

Minichromosome plasmid DNA templates containing oriC and two Ter sites, oriented as they are on the Escherichia coli chromosome, have been used to study the role of Tus in termination of bidirectional replication. In replication reactions reconstituted with purified proteins where it could be demonstrated that each active template was replicating bidirectionally, Tus was required to prevent extensive overreplication. In the presence of Tus, both replication forks terminated DNA synthesis at one or the other Ter site in an apparent stepwise manner. First, the progress of one replication fork was arrested by a properly oriented Tus-Ter complex. Then, either because of steric hindrance resulting from the stalled replication machinery of the first fork or because of the formation of a branched DNA structure, the progression of the second opposing fork was halted at the same site on the DNA template. In the absence of Tus, overreplication required DNA ligase and arose via a template strand-switching mechanism. Thus, the role of Tus in E. coli is more likely to prevent overreplication rather than to ensure accurate termination.

Highlights

Minichromosome Template DNAs-Four oriC plasmids, all 6 kb in length, thadtiffer in the orientationof oriC and two 7krB sequences have been used in this study (Fig. 1).One pair of plasmids, which c a n y “535”in their designation, have TtheerB sites oriented to exclude the passage of replication forks between them
In the type I plasmids, oriC was oriented versus the terminus region as it ison the E. coli chromosome
In the type I1plasmids, this orientation waresversed. Both orientations of oriC were used to ensure that any results observed on termination of replication did not resultfrom interference with replication fork progression because of plasmid sequences

Summary

MATERIALS AND METHODS

Rently, six!I'er sites have beenidentified on the E. coli chromo- Plasmid DNAs-Fourtypes of minichromosomes (see Fig. 1)were some (9-12). Gel ElectrophoreticAnalysis-A portion of the reaction mixtures was mixed with 0.2 volume of a dye mixture containing 50 mM EDTA, 12.5% glycerol, 2%Sarkosyl, 0.05% xylene cyanol,and 0.05% bromphenol blue and fractionated by electrophoresis through vertical 0.8% agarose gels (Sea Kem ME, 14 x 10 x 0.3 cm) at 2 V/cm for 16 h in electrophoresis buffer containing 50 mM Tris-HC1(pH 7.9 at 23 “C), mM NaOAc, and 1m EDTA (referred to as a neutralgel).Gels weredried under vacuum onto filter paper (Whatman No.3M) and autoradiographed with Hyperfilm-MP film(Amersham Corp.). Sodium hydroxideand sucrose were added to the reaction mixtures to final concentrations of 80 m and 4%, respectively,and the replication products were resolvedby electrophoresisthrough horizontal 0.7%agarose gels(13 x 15 x 0.4 cm)at 1.5 V/cmfor 14h in electrophoresisbuffer containing 30 mM NaOH and 2 mM EDTA. Nascent leading strands were analyzed by electrophoresisthrough vertical 1.2%alkaline agarose gels (14 x 13 x 0.3 cm) at 2 V/cm for 18 h (as described above)

RESULTS

Experlmenr 1 Experimenr2

DISCUSSION