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We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By(More)
We show that the mechanisms of DNA-dependent and -independent dTTP hydrolysis by the gene 4 protein of bacteriophage T7 differ in the pathways by which these reactions are catalyzed. In the presence of dTTP, gene 4 protein monomers assemble as a ring that binds single-stranded DNA and couples the hydrolysis of dTTP to unidirectional translocation and the(More)
The DNA helicase encoded by gene 4 of bacteriophage T7 couples DNA unwinding to the hydrolysis of dTTP. The loss of coupling in the presence of orthovanadate (Vi) suggests that the gamma-phosphate of dTTP plays an important role in this mechanism. The crystal structure of the hexameric helicase shows Arg-522, located at the subunit interface, positioned to(More)
molecules decreases, there is a corresponding increase in the number of folded molecules (Fig. 2A), and eventually the distribution converges to its equilibrium form at the final concentration of denaturant. The positions of the peaks, however, remain constant , indicating that the average end-to-end distances of the molecules in the subpopula-tions do not(More)
Electron microscopic and crystallographic data have shown that the gene 4 primase/helicase encoded by bacteriophage T7 can form both hexamers and heptamers. After cross-linking with glutaraldehyde to stabilize the oligomeric protein, hexamers and heptamers can be distinguished either by negative stain electron microscopy or electrophoretic analysis using(More)
The multifunctional protein encoded by gene 4 of bacteriophage T7 (gp4) provides both helicase and primase activity at the replication fork. T7 DNA helicase preferentially utilizes dTTP to unwind duplex DNA in vitro but also hydrolyzes other nucleotides, some of which do not support helicase activity. Very little is known regarding the architecture of the(More)
An open reading frame from Arabidopsis thaliana, which is highly homologous to the human mitochondrial DNA helicase TWINKLE, was previously cloned, expressed, and shown to have DNA primase and DNA helicase activity. The level of DNA primase activity of this Arabidopsis Twinkle homolog (ATH) was low, perhaps due to an incomplete zinc binding domain (ZBD). In(More)
The ring-shaped helicase of bacteriophage T7 (gp4), the product of gene 4, has basic beta-hairpin loops lining its central core where they are postulated to be the major sites of DNA interaction. We have altered multiple residues within the beta-hairpin loop to determine their role during dTTPase-driven DNA unwinding. Residues His-465, Leu-466, and Asn-468(More)
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