During base excision repair, a transient single-stranded DNA (ssDNA) gap is produced at the apurinic/apyrimidinic (AP) site. Exonuclease III, capable of performing both AP endonuclease and exonuclease activity, are responsible for gap creation in bacteria. We used single-molecule fluorescence resonance energy transfer to examine the mechanism of gap creation. We found an AP site anchor-based mechanism by which the intrinsically distributive enzyme binds strongly to the AP site and becomes a processive enzyme, rapidly creating a gap and an associated transient ssDNA loop. The gap size is determined by the rigidity of the ssDNA loop and the duplex stability of the DNA and is limited to a few nucleotides to maintain genomic stability. When the 3′ end is released from the AP endonuclease, polymerase I quickly initiates DNA synthesis and fills the gap. Our work provides previously unidentified insights into how a signal of DNA damage changes the enzymatic functions.
region and so allows the helicase and polymerase to bind and begin unwinding and replication, respecively. After travelling all around the plasmid probably as a single protein complex, the proteins then reach the newly synthesised origin of replication, which provides the signal for termination. RepD then does a series of strand exchanges to close the two plasmid circles. We are using a combination of measurements with whole plasmids and oligonucleotide models to elucidate the series of events at each stage of the replication. In particular, by following individual processes in real time, we are able to describe the order of biochemical steps that enable this process to occur. of. SARS was epidemic in 2003 worldwide. SARS-CoV helicase plays critical roles in viral replication, and has been proposed to be a potential candidate for anti-SARS therapy. We use single molecule fluorescence resonance energy transfer to examine the unwinding and rewinding mechanism of nsP13 helicase on partial DNA duplexes as a function of protein, ATP concentration, and tail length. Our results reveal that the tail length of the substrates determines the total amount of DNA unwound by increasing the number of proteins loaded. In contrast, unwinding rate and step size increase as a function of the protein and ATP concentration for the partial duplex with a long tail (45nts long), but independent of protein concentration for the short tail (30nts long). We also observed a repetitive unwinding displaying multiple rounds of reunwinding and re-zipping events where re-unwinding becomes favorable at higher protein concentration. We also found that the relative extent of constitutive unwinding and repetitive fluctuation is defined by the modality of DNA-Protein complex in the presence or absence of ATP concentration. The ratio between them determines the processivity of the cooperative helicases in tandem. In general, our results identify the important cellular parameters, governing the cooperative unwinding and repetitive rewinding behavior of helicase. This is a new attempt to understand the complicate behavior of unwinding motor cohorts at the single molecule resolution.
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