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European biophysics journal : EBJ

Is photocleavage of DNA by YOYO-1 using a synchrotron radiation light source sequence dependent?


PMID 21931957

Abstract

The photocleavage of double-stranded and single-stranded DNA by the fluorescent dye YOYO-1 was investigated in real time by using the synchrotron radiation light source ASTRID (ISA, Denmark) both to initiate the reaction and to monitor its progress using Couette flow linear dichroism (LD) throughout the irradiation period. The dependence of LD signals on DNA sequences and on time in the intense light beam was explored and quantified for single-stranded poly(dA), poly[(dA-dT)(2)], calf thymus DNA (ctDNA) and Micrococcus luteus DNA (mlDNA). The DNA and ligand regions of the spectrum showed different LD kinetic behaviors, and there was significant sequence dependence of the kinetics. However, in contrast to expectations from the literature, we found that poly(dA), mlDNA, low salt ctDNA and low salt poly[(dA-dT)(2)] all had significant populations of groove-bound YOYO. It seems that this mode was predominantly responsible for the catalysis of DNA cleavage. In homopolymeric DNAs, intercalated YOYO was unable to cleave DNA. In mixed-sequence DNAs the data suggest that YOYO in some but not all intercalated binding sites can cause cleavage. It is also likely that cleavage occurs at transient single-stranded regions. The reaction rates for a 100xa0mA beam current of 0.5-μW power varied from 0.6xa0h(-1) for single-stranded poly(dA) to essentially zero for low salt poly[(dG-dC)(2)] and high salt poly[(dA-dT)(2)]. At the conclusion of the experiments with each kind of DNA, uncleaved DNA with intercalated YOYO remained.