Results: 4

1.
Fig. 2.3

Fig. 2.3. From: Experimental and computational analysis of DNA unwinding and polymerization kinetics.

Fluorescence-base stopped-flow assay for DNA unwinding. a) DNA unwinding fork substrate design with fluorescein in the lower strand and GGG at the 3′ end in the top strand. T7 gp4 moves in the 5′ to 3′ direction to unwind the dsDNA substrate. b) Representative kinetic trace showing the unwinding of the fork DNA by the time-dependent increase in fluorescence. The kinetics is fit to the gfit unwinding model (unwinding.m), which provides kf and s, from which the average rate of unwinding (kf × s) was calculated. A good fit for the 40ds DNA unwinding data in the presence of top ssDNA trap was obtained at N = 2 and hence gave parameters for the two phases; A1 = 0.862, A2 = 0.137, kf1 = 4.488, kf2 = 1.413, minD = 0, s = 5.395 and F0 = 0.995 and hence an average rate of unwinding for the fast population is ~ 24.2 bp/s and for the slow population is ~7.6 bp/s.

Manjula Pandey, et al. Methods Mol Biol. 2010;587:57-83.
2.
Fig. 2.2

Fig. 2.2. From: Experimental and computational analysis of DNA unwinding and polymerization kinetics.

Gel-based radiometric assay for DNA unwinding. a) The DNA unwinding fork substrate design with radiolabeled top strand. T7 gp4 assembles on the top strand and moves in the 5′ to 3′ direction to unwind the dsDNA substrate. b) Representative native gel showing the ds and ss DNA resolved as a function of reaction time. Time points here are 0, 0.05, 0.08, 0.1, 0.15, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.2, 1.5, 1.7, 2, 2.5, 3, 4, 6, 8, 15, 30 s (for 40ds duplex). c) Kinetics of ds40 unwinding at 18°C in reactions containing T7 gp4, dTTP, MgCl2 and SSB as the trap. The kinetics is fit to the gfit unwinding model (unwinding.m), which provides kf and s, from which the average rate of unwinding (kf × s) was determined. A good fit for the 40ds DNA unwinding data here gave parameters; A = 0.829, kf = 9.89, minD = 0, s = 6.34 and F0 = 0.002 and hence an average rate of unwinding ~ 62.7 bp/s.

Manjula Pandey, et al. Methods Mol Biol. 2010;587:57-83.
3.
Fig. 2.4

Fig. 2.4. From: Experimental and computational analysis of DNA unwinding and polymerization kinetics.

Strand displacement DNA synthesis by helicase-polymerase replisome. a) T7 DNA polymerase and T7 gp4 are assembled on the replication fork substrate with a radiolabeled DNA primer. T7 gp4 on top strand moves to unwind the dsDNA and DNA polymerase extends the primer in the 5′ to 3′ direction by duplicating the bottom strand. Sequencing gel shows progressive strand displacement DNA synthesis activity of the replisome. Time points here are 0, 0.004, 0.006, 0.008, 0.01, 0.015, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.1, 0.12, 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.6, 0.75, 1, 1.5, 2, 3, 4 s (for 41 nt base extensions). The 24 mer primer is elongated to 65 nt runoff product. b) Rate constants of individual nucleotide addition plotted against nucleotide added (rates of first three nucleotide addition are not plotted here as they are always high probably due to premelting of duplex with helicase binding). Rate constants are estimated by fitting the kinetics of individual DNA product synthesis to the polymerization model of gfit (polymerase_ni.m). Errors are calculated from the global fits to the polymerization model.

Manjula Pandey, et al. Methods Mol Biol. 2010;587:57-83.
4.
Fig. 2.1

Fig. 2.1. From: Experimental and computational analysis of DNA unwinding and polymerization kinetics.

Instrumental designs for the rapid kinetic studies. a) Chemical quenched-flow RQF-3 (www.kintek-corp.com, figure kindly provided by Prof. K.A. Jhonson). Sample A and Sample B are loaded in sample loops from the load ports via a three way valve. Upon firing the instrument, solution A and B are forced through the delay line by water from the drive syringes A and B, and then reactants are mixed in the valve to start the reaction. The reaction mix flows through the selected delay line and mixed with the quench solution from syringe C after predetermined time intervals. The quenched sample is collected into a tube from the exit line. Different length delay lines are selected through an eight – way valve for reaction times in the range of 2–100 msec by selecting different reaction loops. b) Stopped-flow instrument. The KinTek stopped flow has a stable light source and sensitive detection system that can measure absorbance and fluorescence simultaneously. There are two channels for fluorescence detection, and three drive syringes, but in normal mode of operation two syringes are connected (those shown) and used to drive mixing of two reactants A and B into the observation cell. Reaction time is under computer control allowing times from few msec to several minutes. The instrument dead-time is determined as outlined in www.kintek-corp.com.

Manjula Pandey, et al. Methods Mol Biol. 2010;587:57-83.

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