A fluorescence photobleaching study of the microsecond reorientational motions of DNA - PubMed (original) (raw)

A fluorescence photobleaching study of the microsecond reorientational motions of DNA

B A Scalettar et al. Biophys J. 1988 Feb.

Abstract

We have conducted a polarized fluorescence photobleaching recovery (FPR) study of the rotational dynamics of ethidium azide labeled DNA. Polarized photobleaching experiments provide data on microsecond and millisecond molecular reorientation that complement the information available from nanosecond fluorescence depolarization studies. In polarized FPR experiments an anisotropic angular concentration of fluorophore is created by bleaching dye molecules in a preferred orientation with a short, intense pulse of polarized light. The sample is then weakly illuminated, and the temporal variation in the emitted fluorescence is monitored. The fluorescence signal will systematically change as molecules undergo post-bleach reorientation and the angular distribution of dye tends toward isotropy. We have observed that the time dependence of our microsecond FPR curves is also determined in part by nonrotational phenomena. To isolate the reorientational recovery we conduct our FPR experiments in two modes (called parallel and perpendicular) that differ only in the polarization of the bleaching light. A quotient function, R(t), is constructed from the data obtained in these two modes; the variation with time of this new quantity is governed solely by processes that are sensitive to the polarization of the incident light (e.g., molecular rotation). It is found experimentally that R(t) remains constant, as expected, for rotationally restricted DNA systems despite a temporal recovery in the parallel and perpendicular FPR curves. We also follow the dynamics of solutions of phage lambda DNA as revealed in the temporal dependence of R(t). This DNA system rotationally relaxes after approximately 100 microseconds and the dye/DNA complex reorients substantially during the 10-microseconds bleach period. Our FPR data are interpreted in terms of dynamic models of DNA motion.

PubMed Disclaimer

Similar articles

Cited by

References

    1. Biopolymers. 1974 Jan;13(1):29-61 - PubMed
    1. Biopolymers. 1973;12(7):1543-64 - PubMed
    1. Biochim Biophys Acta. 1978 Dec 21;521(2):502-9 - PubMed
    1. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4165-9 - PubMed
    1. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6341-5 - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources