Abstract
DNA polymerase III holoenzyme was assembled from pure proteins onto a primer template scaffold. The assembly process could be divided into two stages. In the time-consuming first stage, beta subunit and gamma.delta subunit complex were required in forming a tightly bound ATP-activated "preinitiation complex" with a single-stranded DNA bacteriophage circle uniquely primed with a synthetic pentadecadeoxyribonucleotide. This finding substantiates an earlier study using crude protein preparations in a homopolymer system lacking Escherichia coli single-stranded DNA binding protein (Wickner, S. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 3511-3515). In the second stage, the polymerase III core and the tau subunit rapidly seek out and bind the preinitiation complex to form DNA polymerase III holoenzyme capable of rapid and entirely processive replication of the circular DNA. ATP is not required beyond formation of the preinitiation complex. It is remarkable that the fully assembled DNA polymerase III holoenzyme is so stably bound to the primed DNA circle (4-min half-time of dissociation), yet upon completing a round of synthesis the polymerase cycles within 10 s to a new preinitiation complex on a challenge primed DNA circle. Efficient polymerase cycling only occurred when challenge primed DNA was endowed with a preinitiation complex implying that cycling is mediated by a polymerase subassembly which dissociates from its accessory proteins and associates with a new preinitiation complex. These subunit dynamics suggest mechanisms for polymerase cycling on the lagging strand of replication forks in a growing chromosome.
Highlights
I11 holoenzyme is so stably bound to the primedDNA fork.Rapidly growing E. coli cells dividing within 30 min circle (4-min half-timeof dissociation), yet upon com- initiate new rounds of replication before the parental chropleting a round of synthesisthepolymerase cycles mosome is completed [1].This “dichotomous” mode of repliwithin 10 s to a new preinitiation complex on a chal- cation generates six replication forks in one cell; two forks lenge primedDNA circle
Efficient polymerase cycling are initiated at the unique bidirectional origin of replication only occurred when challenge primed DNA was en- on the parentaclhromosome, and four other forks resulftrom dowed with a preinitiation complex implying that cy- bidirectionalfiring of the two daughter originsbefore full cling is mediated by a polymerase subassembly which duplication of the parental chromosome [1]
The directioofnreplication on thelagging strand is opposite that of fork movement, and synthesis is DNA polymerase I11 holoenzyme, a multiprotein complex, repeatedly primed with RNA primers [1].The lagging strand is the replicase of Escherichia coli [1, 2]. pol I11 holoenzyme’ fragments(Okazakifragments) produced uponextending is composed of at least seven differentsubunits; four are accessory proteins (p, y, 6, T ), and three others are tightly bound in a particle called pol I11 core: a! (DNA polymerase), t (3’,5’-exonuclease), and 8 [2, 3]
Summary
BounAd TP-activate"dpreinitiatiocnomplexw" ith the p and 7 . 6 Form aPreinitiation Complex withPrimed primed ssDNA circle. Preinitiation complexes on primed synthesis (Fig. 1).The lag is convertedto a burst in synthesis ssDNA circles serve as an efficient substrate for the pol I11 upon a 2-min preincubation of the subunitswithprimed core and promote rapid cyclinofgpolymerase from completed ssDNA (Fig. 1,circles). The simultaneous presence of y .6, p, ATP, and primed ssDNA wasessentialduringpreincubation to elicit burst synthesis upon addition of the rest of the subunits (Fig. 1) This result may be interpreted as rate-limiting formation of an ATP-activated preinitiation complex of one or a combination of y, 6, and subunitswith the primed ssDNA. Primed DNAs-&X and M13mp18viral ssDNA were singly primed by annealing aunique synthetic DNA oligonucleotide (15-mers 1and 6, respectively [10]).Hybridization was in 10 mM Tris-C1 (pH 8.0),
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