@article{cb42739515fe4839bba7339578a402b5,
title = "Single strand breakage and repair in eukaryotic DNA as assayed by S1 nuclease",
abstract = "A sensitive new approach for measuring the repair of single strand breaks 1n DNA induced by low doses of gamma irradiation was tested 1n cultured fibroblasts from Chinese hamster lung, human afflicted with ataxia telangiectasia or Fanconi's anemia and 1n normal cells of early and late passages. The assay 1s based on the increasing rate of strand separation of DNA duplexes in alkali for molecules with increasing numbers of single strand scissions. DNA strand separation 1s shown to follow the relation, 1n F + -(1/Mn. const) .tβ where F 1s the proportion of double-stranded DNA, detected as S1 nuclease resistant, after alkaline denaturation time, t. Mn 1s the number-average molecular weight of DNA between single strand breaks. β<1 is an empirically determined constant. The results suggest an increase 1n the number-average molecular weight between breaks, Mn, with increasing times for repair. The final level attained corresponds to the Mn of control DNA in unirradiated cells. As few as one break introduced into 109 daltons of single-stranded control cell DNA can be detected. The kinetics, requirements and sensitivities of this assay are described.",
author = "Sheridan, {R. B.} and Huang, {P. C.}",
note = "Funding Information: the number of alkali labile sites introduced into the DNA by the acidic condition. Less than 0.4 breaks per 10 daltons DNA resulted from such a treatment. Thus the incubation mixture does not seriously degrade the proportion of DNA which remains double-stranded . Variations within replicates in the proportion of double-stranded DNA remaining after treatment, may be a consequence of the geometry of denaturation. The kinetics of unwinding are different depending on whether the cells are denatured as a suspension or attached to a surface. Cells treated in monolayer show a slower rate of strand separation which may be a consequence of the high local viscosity resulting from the release of DNA from the surface. Inspection of such a monolayer during denaturation reveals what appears to be a gel overlaying the surface. Variations 1n the viscosity of this gel could drastically affect the rate of unwinding. Unfortunately, repair should only be measured in cells which have been attached to a growth surface for at least one generation time; otherwise, it is difficult to interpret the results. Due to the simplicity and sensitivity of this technique, its applicability should be wide ranging. It should be possible to monitor changes in the number-average molecular weight of DNA 1n many systems in which denaturation of DNA can be effectuated by alkali or other means. The single strand specific nuclease, S,, is a hardy enzyme and should quite easily adapt to many other reaction conditions. By adopting a similar technique, it should be possible to follow many processes Involving breakage and re-joinino of DNA such as excision of UV-induced dimers and recombination. This study shows that repair processes can be monitored in eukaryotic cells given nonlethal doses of gamma radiation. Thus this technique should be useful in the elucidation of molecular mechanisms for several mutations affecting DNA repair processes. ACKNOWLEDGMENTS He wish to thank Drs. L. Grossman and L. Hamilton for their critical reading of this manuscript and S.Y. Wang for the use of the gamma source. This study was supported by a Kun1tz-Worthington Scholarship (R.B.S.) and The National Foundation/March of Dimes Grant CRBS300. A preliminary report was represented before the FASEB meetings, San Francisco (Fed. Proc. 35, 1490) 1976.",
year = "1977",
month = feb,
doi = "10.1093/nar/4.2.299",
language = "English (US)",
volume = "4",
pages = "299--318",
journal = "Nucleic acids research",
issn = "0305-1048",
publisher = "Oxford University Press",
number = "2",
}