PubMed · 1497322
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K J Marians. 1992. Prokaryotic DNA replication.. https://doi.org/10.1146/annurev.bi.61.070192.003325
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Capillary gel electrophoresis has proven to be a powerful tool in biomedical research. We report our investigation of some of the critical parameters affecting separations of single-stranded DNA fragments as monitored by ultraviolet (UV) absorbance detection. Although not as sensitive as laser-induced fluorescence (LIF), UV absorbance detection allows one to calculate quite accurately, and inexpensively, the molarity of each separated DNA fragment and, moreover, the signal "fading" effect normally observed with LIF detection can be, in many cases, substituted for fluorescence to detect the many different single-stranded DNAs, as well as for detection of sequencing reactions.
The nascent DNA synthesized after UV irradiation contained discontinuity, i.e., daughter-strand gaps. The sizes of these gaps produced in the leading and lagging strands of UV-irradiated Escherichia coli cells were determined by using strand-specific DNA probes. The DNA isolated from irradiated uvrA delta(lac-pro) cells was hybridized with the 32P-labeled single-stranded DNA probes. After digestion with S1 nuclease, the sizes of the bound radioactive DNA fragments were determined by electrophoresis in an alkaline agarose gel. It was found that the average size of gaps produced in the leading strand was about 0.12 kb, whereas those produced in the lagging strand was slightly smaller than 0.12 kb. No gaps larger than 0.5 kb were detected.
The induction of mutations by ultraviolet light and delayed photoreversal in bacteria defective for SOS mutagenesis is discussed in terms of two models: the two-step misincorporation and bypass model, and the model involving simple deamination of cytosine-containing dimers. In phage S13 the latter appears to be the predominant mechanism. In Escherichia coli there is little evidence that the simple deamination mechanism is of any significance except in ung strains lacking uracil glycosylase where uracils left after photoreversal are not removed. Deamination might, however, occur during the operation of translesion synthesis via the two-step model and if it did, subsequent photoreversal would lead to the "mutation" being extended from one to both strands by uracil glycosylase repair rather than being removed.