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DNA extension under varying forces was measured by video microscopy.
DNA extension is only observed in reactions where dATP is included.
DNA extension mediated by Pol η was also observed by Li et al. [21], however, our data indicate that at the reported salt concentration the DNA extension is PCNA-independent (Fig. 4C), in accordance with previous analyses of human Pol η activity using synthetic D-loop substrate [41].
DNA extension was measured with varying concentrations of ATP (left panel) and ATPγS (right panel) on 12 nt oh HP to determine the lengths of primer synthesized as a function of NTPs.
The quality of the DNA was checked on a 0.8% agarose 1X TAE gel alongside a 1 kb DNA Extension Ladder (Invitrogen, Carlsbad, CA), and the concentration was measured on a NanoDrop spectrophotometer.
Recently, Ichikawa et al. [4] have presented a novel DNA extension technique via laser heating.
It was based on cyclic enzymatic signal amplification (CESA) and template-free DNA extension reaction.
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Telomere proteins from ciliated protozoa bind to the single-stranded G-rich DNA extensions at the ends of macronuclear chromosomes.
In this study we have used nucleosomes with DNA extensions of 240 1200 bp together with single molecule AFM and high resolution PAGE.
These observations allowed the authors to separate the contributions from the unwinding and wrapping of the DNA around the enzyme during RPo formation to the DNA extensions.
The authors observed four different DNA extensions, which were assigned to (1) the initial state, (2) RPo, (3) RNAP promoter initial transcribing complex, and (4) RNAP DNA elongation complex.
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