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DNA replication requires the coordinated activity of a large number of enzymes at the replication fork. Understanding the mechanisms controlling this organization requires a direct probing of the dynamics of fully functional replisomes during replication. Observations at the single-molecule level provide the most direct way to visualize the complex biochemistry of the replisome and to quantify the many transient intermediates essential to replication. We present a novel assay that combines the observation of individual fluorescently labeled proteins with the mechanical manipulation of DNA. Surface-tethered DNAs labeled with quantum dots are hydrodynamically stretched and imaged with a TIRF microscope. Activity of the replisome is observed as a change in the DNA length due to the differing force-dependent extension of single- and double-stranded DNA at low pico-Newton forces. We employ a two-color imaging scheme to monitor DNA length in real-time and to stroboscopically image fluorescently labeled single proteins interacting with DNA. Observation of labeled proteins in an ongoing replication reaction allows us to pose structural questions about the stoichiometry and exchange of proteins at the prokaryotic replication fork. We will discuss preliminary results on primer extension by the T7 DNA polymerase and strand-displacement synthesis by the coupled activity of the T7 helicase and polymerase.

The bacteriophage T7 DNA polymerase consists of a tight, 1:1 complex of T7 gp5, encoded by the phage, and thioredoxin, produced by the E. coli host. In the absence of thioredoxin, gp5 is capable of adding only a few nucleotides to the 3′ end of a primer before dissociating from the primer-template. But when complexed with thioredoxin, gp5 becomes highly processive, capable of polymerizing thousands of nucleotides complementary to the template strand. The mechanism by which thioredoxin acts as a processivity factor to gp5 is not fully understood. To understand the role of the thioredoxin in stabilizing polymerase-DNA interactions, we use a single-molecule imaging approach to observe individual, fluorescently labeled T7 DNA polymerase complexes diffusing along double-stranded DNA. Our results show that the average diffusion coefficient of T7 DNA polymerase complexes is insensitive to ionic strength and does not exceed the theoretical diffusion limit for a protein that tracks the helical pitch and rotates as it diffuses along the DNA helix. These results suggest that the T7 DNA polymerase slides along the DNA, remaining tightly bound to the DNA and tracking the helical pitch. However, the mean diffusion coefficients for fluorescently labeled T7 gp5 in the absence of thioredoxin increase with salt concentration, and exceed the theoretical limit for a protein tracking the DNA helix. Upon addition of unlabeled thioredoxin, the mean diffusion coefficient is restored to the value observed for the labeled T7 DNA polymerase, and becomes salt independent. These observations indicate that, in the absence of thioredoxin, T7 gp5 intermittently dissociates from the DNA as it diffuses, and that thioredoxin binding suppresses microscopic hopping on and off the DNA.

In all organisms, the protein machinery responsible for the replication of DNA, the replisome, is faced with a directionality problem. The antiparallel nature of duplex DNA permits the leading-strand polymerase to advance in a continuous fashion, but forces the lagging-strand polymerase to synthesize in the opposite direction. By extending RNA primers, the lagging-strand polymerase restarts at short intervals and produces Okazaki fragments1,2. At least in prokaryotic systems, this directionality problem is solved by the formation of a loop in the lagging strand of the replication fork to reorient the lagging-strand DNA polymerase so that it advances in parallel with the leading-strand polymerase. The replication loop grows and shrinks during each cycle of Okazaki-fragment synthesis3. Here, we employ single-molecule techniques to visualize, in real time, the formation and release of replication loops by individual replisomes of bacteriophage T7 supporting coordinated DNA replication. Analysis of the distributions of loop sizes and lag times between loops reveals that initiation of primer synthesis and the completion of an Okazaki fragment each serve as a trigger for loop release. The presence of two triggers may represent a fail-safe mechanism ensuring the timely reset of the replisome after the synthesis of every Okazaki fragment.