IN-VITRO REJOINING OF DOUBLE-STRAND BREAKS IN CELLULAR DNA BY FACTORSPRESENT IN EXTRACTS OF HELA-CELLS

We described previously a cell-free assay that could be employed to st udy the rejoining of radiation-induced DNA double-strand breaks (dsb) in agarose embedded nuclei by activities present in an extract prepare d from exponentially growing HeLa cells. Here, we extend the study and present an in vitro assay for rejoining of radiation-induced DNA dsb that employs 'naked' DNA prepared from agarose-embedded cells as a sub strate and extract of HeLa cells as an enzyme source. There is no dete ctable residual protein on substrate DNA after extensive lysis with io nic detergents and treatment with proteases, as determined by SDS-PAGE and silver staining. We demonstrate that rejoining of dsb is absolute ly dependent on cell extract and that, under optimal reaction conditio ns, it proceeds to an extent and with kinetics similar to those observ ed in intact cells. Dsb rejoining in this assay requires Mg2+ and is i nhibited by high concentrations of either K+ or Na+. This assay comple ments the nuclei assay for DNA dsb repair previously developed, and ma y be preferable to the latter in the purification of factors involved in DNA dsb repair, as it employs as substrate DNA deprived of proteins .