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This thesis investigates ways in which a nanoscale production line may be built from synthetic DNA components. One property of a production line is motion, the coordinated movement of components, in this case strands of DNA, between specific locations. Another property is the ability to assemble a product, where smaller molecular building blocks are attached to D A and react when brought together by the DNA assembly line. An important fea- ture of either task is the ability of the mechanism to proceed with minimum user interaction: it is preferable that the assembly line be autonomous. The challenges and design principles of molecular machines working in nano- scale environments are first considered. Previous studies demonstrating the use of synthetic DNA not only as a self-assembling material to build nano- structures, but also to coordinate motion, are summarized. All DNA nano- machines that operate through the exchange of DNA strands are coordinated by toeholds. A 'split toehold', one that combines two smaller toeholds on distal sections of DNA held in proximity, is proposed as a way to allow a single cargo strand to interact with many different components. A molecular motor is then developed that transports a cargo between track locations. The fuel strands are hairpins, that carry instructions directing the cargo to the next anchorage. The switching of cargo direction in response to the chemical environment is also investigated. Two mechanisms that may allow the autonomous assembly of components are investigated, one of which is demonstrated using DNA-linked cleavable molecular building blocks. Further extensions to the mechanism are investi- gated, for example, the ability to use the DNA mechanism itself as a barcode containing information on the order of assembled ingredients.

Deoxyribonucleoside triphosphate (dNTP) concentrations measured in cells are not symmetric. dGTP almost always represents only 5-10% of the total dNTP pools in cells. In an in vitro replication system involving semiconservative replication from an SV 40 origin, the mutation frequency of an M13 phagemid replicated by human cell extracts in reaction mixtures containing "biologically biased" dNTP pools estimated from HeLa cell nuclei is not significantly different from that seen when replication is done with equimolar dNTP concentrations. Significant reduction of dGTP pool while keeping other dNTPs at "biologically biased" dNTP concentrations during replication reaction also did not increase mutation frequency. In contrast, in vitro replication with dNTP concentrations calculated from normal diploid fibroblast cells, which are three- to four-fold lower in concentrations, showed a marked reduction of the observed mutation frequency, showing the importance of overall dNTP levels during replication on mutation frequency in vitro. When whole-cell dNTP concentrations in HeLa cells were measured during S-phase, dNTP levels underwent a transient decrease in the middle of S-phase. Average HeLa cells' dNTP levels were also found to correlate with average DNA accumulation rates during S-phase, although no detailed relationship can yet be deducted from the available data. No significant changes in the ratio of the four dNTP concentrations were found during S-phase. Mutation rates of green fluorescent protein (GFP) inserted in either middle or late-replicating region of a chromosome in HeLa cells also correspond to average DNA accumulation rates and dNTP levels during middle and late S-phase. The late-replicating GFP-HeLa cells have a higher mutation rate than the middle-replicating GFP-HeLa cells, as the average DNA accumulation rates and dNTP pool levels were also lower in the middle compared to late S-phase. Taken together, these observations indicate that dNTP levels could play a role in determining the S-phase DNA replication rate and also the replication fidelity in mammalian cells. / Graduation date: 2002

The work in this thesis has provided conclusive genetic evidence that "panhandle" intermediates form during adenovirus replication. Adenovirus chromosomes lacking 51 by from their left -hand termini are infectious and capable of regenerating the missing origin sequence. Yet if an entire inverted terminal repeat is removed, the adenovirus chromosome is no longer viable. This first suggested, but did not prove, that "panhandles" formed during adenovirus replication. Homologous recombination or postreplicative overlap recombination could generate the same outcome. Analysis of the segregation of markers in the inverted repeats of adenovirus minichromosomes shows that homologous recombination does not mediate end repair. A special case was also found where postreplicative overlap recombination failed to transfer sequences between the inverted repeats, but similar molecules could exchange sequence information during "panhandle" formation. The exchange of information between inverted repeats is referred to as sequence conversion. A number of length and/or orientation constraints on sequence conversion during adenovirus DNA replication were identified. A length- and orientation-dependent constraint was found for gap filling close to "panhandle" loops. Polymerization towards the loop could occur even when the gap was only 6 by away. In contrast, polymerization away from the "panhandle" loop at a gap at 6 bp, did not take place. This steric constraint could reflect an asymmetry in the action of adenovirus DNA polymerase. A similar length and/or orientation dependent constraint was found for the removal of bulges (3 by and 4 by mismatches). Incision in the bulge of the 5' inverted repeat caused a block to the completion of sequence conversion at that site. When the bulge was in the 3' inverted repeat, a length requirement for successful removal was demonstrated. When 6 by or 39 by separated the bulge from the "panhandle" loop, removal of the bulge was not detected. When the distance was 79 bp, 184 bp, or 217 bp, bulges were successfully removed. The molecular basis for this obstruction remains to be determined. Moreover, incision in bulges located in the 3' inverted repeat triggers directional coconversion. Finally, small loops placed close to the site of polymerization did not cause the same length and orientation dependent constraints as did the "panhandle" loop. / Graduation date: 1991

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The bacteriophage T4 utilizes at least three modes of initiation of replication, termed primary, secondary and tertiary (Mosig, 1983; Kreuzer and Alberts, 1985). Two origins of replication have been isolated that utilize the tertiary mode of initiation. The DNA sequence requirements of the two tertiary origins have been characterized at the nucleotide level. Maximal replication of each origin-containing plasmid required both an intact gpmotA-dependent middle-mode promoter sequence and approximately 50 basepairs of the downstream region. In contrast, gpmotA-dependent transcription from the origin promoter was found to be independent of the downstream region. The requirement for a promoter element within the tertiary origins is striking, particularly since the replication of tertiary origin-containing plasmids is resistant to the RNA polymerase inhibitor rifampicin.