A CELL-FREE ASSAY USING CYTOPLASMIC CELL-EXTRACTS TO STUDY REJOINING OF RADIATION-INDUCED DNA DOUBLE-STRAND BREAKS IN HUMAN CELL-NUCLEI

We describe a cell-free assay that can be employed to study rejoining of radiation-induced DNA double-strand breaks (dsbs) under in vitro co nditions. The assay uses nuclei prepared from irradiated, agarose-embe dded human A549 cells as substrate and cytoplasmic cell extracts prepa red from exponentially growing HeLa cells as the source of enzymes. We demonstrate that rejoining of dsbs is absolutely dependent on cell ex tract and that, under optimal reaction conditions, it proceeds to an e xtent similar to that observed in intact cells, albeit with about six times longer half time. Dsb rejoining in this assay requires Mg2+ and is inhibited by high concentrations of either K+ or Na+. The assay sho uld provide means for the biochemical characterization of the enzymolo gy of eukaryotic cell DNA repair under conditions that retain chromati n structure. The assay can also be adapted to study repair of other ty pes of damage induced in the DNA by ionizing or non-ionizing radiation s, as well as by diverse chemical agents.