DNA replication is a fundamental cellular process. However, the structure and dynamics of the eukaryotic DNA replication machinery remain poorly understood. A soluble extract system prepared from Xenopus eggs recapitulates eukaryotic DNA replication outside of a cell on a variety of DNA templates. This system has been used to reveal many aspects of DNA replication using a variety of ensemble biochemical techniques. Single-molecule fluorescence imaging is a powerful tool to dissect biochemical mechanisms. By immobilizing or confining a substrate, its interaction with individual, soluble, fluorescently-labeled reactants can be imaged over time and without the need for synchrony. These molecular movies reveal binding parameters of the reactant and any population heterogeneity. Moreover, if the experiments are imaged in wide-field format, the location or motion of the labeled species along the substrate can be followed with nanometer accuracy. This dissertation describes the use and development of novel single-molecule fluorescence imaging techniques to study eukaryotic DNA replication. A biophysical characterization of a replication fork protein, PCNA, revealed both helical and non-helical sliding modes along DNA. Previous experiments demonstrate that the egg extracts efficiently replicate surface-immobilized linear DNA. This finding suggested replication of DNA could be followed as motion of the replication fork along the extended DNA. However, individual proteins bound at the replication fork could not be visualized in the wide-field due to the background from the high concentration of the fluorescent protein needed to compete with the extract’s endogenous protein. To overcome this concentration barrier, I have developed a wide-field technique that enables sensitive detection of single molecules at micromolar concentrations of the labeled protein of interest. The acronym for this method, PhADE, denotes three essential steps: (1) Localized PhotoActivation of fluorescence at the immobilized substrate, (2) Diffusion of unbound fluorescent molecules to reduce the background and (3) Excitation and imaging of the substrate-bound molecules. PhADE imaging of flap endonuclease I (Fen1) during replication revealed the time-evolved pattern of replication initiation, elongation and termination and the kinetics of Fen1 exchange during Okazaki fragment maturation. In the future, PhADE will enable the elucidation of the dynamic events at the eukaryotic DNA replication fork. PhADE will also be broadly applicable to the investigation of other complex biochemical process and low affinity interactions. It will be especially useful to those researchers wishing to correlate motion with binding events.

Each cell division, the nuclear DNA must be replicated efficiently and with high accuracy to avoid mutations which can have an effect on cell function. There are three replicative DNA polymerases essential for the synthesis of DNA during replication in eukaryotic cells. DNA polymerase α (Pol α) synthesize short primers required for DNA polymerase δ (Pol δ) and DNA polymerase ε (Pol ε) to carry out the bulk synthesis. The role of Pol δ and Pol ε at the replication fork has been unclear. The aim of this thesis was to examine what role Pol ε has at the replication fork, compare the biochemical properties of Pol δ and Pol ε, and to study the function of the second largest and essential subunit of Pol ε, Dpb2. To identify where Pol ε replicates DNA in vivo, a strategy was taken where the active site of Pol ε was altered to create a mutator polymerase leaving a unique error-signature. A series of mutant pol ε proteins were purified and analyzed for enzyme activity and fidelity of DNA synthesis. Two mutants, M644F and M644G, exhibited an increased mutation rate and close to normal polymerase activity. One of these, the M644G gave rise to a specific increase of mismatch mutations resulting from T-dTMP mis-pairing during DNA synthesis in vitro. The M644G mutant was introduced in yeast strains carrying a reporter gene, URA3, on either side of an origin in different orientations. Mutations which inactivated the URA3 gene in the M644G mutant strains were analyzed. A strand specific signature was found demonstrating that Pol ε participates in the synthesis of the leading strand. Pol δ and Pol ε are both stimulated by the processivity clamp, PCNA, in in vitro replication assays. To clarify any differences they were challenged side by side in biochemical assays. Pol ε was found to require that single-stranded template (ssDNA) was entirely coated with RPA, whereas Pol δ was much less sensitive to uncoated ssDNA. The processivity of Pol δ was stimulated to a much higher degree by PCNA than of Pol ε. In presence of PCNA the processivity of Pol δ and Pol ε was comparable. In contrast, Pol ε was approximately four times slower than Pol δ when replicating a single-primed circular template in the presence of all accessory proteins and an excess of polymerase. The biochemical characterization of the system suggests that Pol ε and Pol δ are loaded onto the PCNA-primer-ternary complex by separate mechanisms. A model is proposed where the loading of Pol ε onto the leading strand is independent of the PCNA interaction motif which is required by enzymes acting on the lagging strand. The essential gene DPB2 encodes for the second largest subunit of Pol ε. We carried out a genetic screen in S.cerevisiae and isolated a lethal mutant allele of dpb2 (dpb2-200). When over-expressed together with the remaining three subunits of Polε, Pol2, Dpb3 and Dpb4, the dpb2-201 did not copurify. The biochemical property of Pol2/Dpb3/Dpb4 complex was compared with wild-type four-subunit Pol ε (Pol2/Dpb2/Dpb3/Dpb4) and a Pol2/Dpb2 complex in replication assays. The absence of Dpb2 in the complex did not significantly affect the specific activity or the processivity, but gave a slightly reduced efficiency in holoenzyme assays when compared to wild-type four-subunit Pol ε. We propose that Dpb2 is not essential for the enzyme activity of Pol ε.

In eukaryotes there are three DNA polymerases which are essential for the replication of chromosomal DNA: DNA polymerase alpha (Pol alpha), DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon). In vitro studies of viral DNA replication showed that Pol alpha and Pol delta are sufficient for DNA replication on both leading and lagging DNA strands, thus leaving the function of Pol epsilon unknown. The low abundance and the reported protease sensitivity of Pol epsilon were holding back biochemical studies of the enzyme. The aim of this study was to characterize the structural and functional properties of eukaryotic Pol epsilon. We first developed a protocol for over-expression and purification of Pol epsilon from the yeast Saccharomyces cerevisiae. Pol epsilon consists of four subunits: Pol2 (catalytic subunit), Dpb2, Dpb3 and Dpb4. This four-subunit complex was purified to homogeneity by conventional chromatography and the subunit stoichiometry of purified Pol epsilon was estimated from colloidal coomassie-stained gels to be 1:1:1:1. The quaternary structure was determined by sedimentation velocity and gel filtration experiments. Molecular mass (371 kDa) was calculated from the experimentally determined Stokes radius (74.5 Å) and sedimentation coefficient (11.9 S) and was in good agreement with a theoretical molecular mass calculated for a heterotetramer (379 kDa). Analytical sedimentation equilibrium ultracentrifugation experiments supported the proposed heterotetrameric structure of Pol epsilon. By cryo-electron microscopy and single-particle image analysis we determined the structure of Saccharomyces cerevisiae Pol epsilon to 20-Å resolution. The four-subunit complex was found to consist of a globular domain, comprising the Pol2 subunit, flexibly connected to an elongated domain, including Dpb2, Dpb3 and Dpb4 subunits. We found that Pol epsilon requires a minimal length of 40 base pairs of primer-template duplex to be processive. This length corresponds to the dimensions of the elongated domain. To characterize the fidelity by which Pol epsilon synthesizes DNA, we purified wild type and exonuclease-deficient Pol epsilon. Wild type Pol epsilon synthesizes DNA with a very high accuracy. Analysis of the exonuclease-deficient Pol epsilon showed that Pol epsilon proofreads more than 90% of the errors made by its polymerase activity. Exonuclease-deficient Pol epsilon was shown to have a specific spectrum of errors not seen in other DNA polymerases: a high proportion of transversions resulting from T-dTTP, T-dCTP and C-dTTP mispairs. This unique error specificity and amino acid sequence alignment suggest that the structure of the polymerase active site of Pol epsilon differs from those of other members of B family DNA polymerases. With recombinant proteins and circular single-stranded DNA templates, we partially reconstituted DNA replication in vitro, in which we challenged Pol epsilon and Pol delta in side-by-side comparisons regarding functional assays for polymerase activity and processivity, as well as physical interactions with nucleic acids and PCNA. We found that Pol epsilon activity and “on-DNA” PCNA interactions are dependent on RPA-coated template DNA. By the surface plasmon resonance technique, we showed that Pol epsilon has a high affinity for DNA and low affinity for immobilized PCNA. By contrast, Pol delta was found to have low affinity for DNA and high affinity for PCNA. We suggest that a possible function of RPA is to regulate down the DNA synthesis through Pol epsilon, and that the mechanism by which Pol epsilon and Pol delta load onto the template is different due to different properties of the interaction with DNA and PCNA.