IN-VITRO REJOINING OF DOUBLE-STRAND BREAKS INDUCED IN CELLULAR DNA BYBLEOMYCIN AND RESTRICTION ENDONUCLEASES

We have previously described a cell-free assay that can be employed to study rejoining of radiation-induced DNA double-stranded breaks (dsb) in 'naked' DNA prepared from agarose-embedded cells using an extract of HeLa cells as a source of enzymes. Rejoining of dsb in this assay i s absolutely dependent on cell extract and proceeds, under optimal rea ction conditions, to an extent and with kinetics similar to those obse rved in intact cells. Here, we extend these experiments and demonstrat e that the assay also supports rejoining of bleomycin and restriction endonuclease-induced dsb, agents that generate dsb with known ends. Re joining of bleomycin-induced dsb proceeds to an extent and with kineti cs similar to those observed with radiation-induced dsb. The kinetics of rejoining of restriction endonuclease-induced dsb are also similar to those of radiation-induced dsb. However, more and more dsb remain u nrejoined as the extent of DNA fragmentation increases when enzymes cu tting the DNA al frequent intervals are used. Dsb with blunt ends are rejoined with a similar efficiency to dsb with cohesive ends. Rejoinin g of restriction endonuclease-induced dsb is, in the presence of cell extract, more efficient than in the presence of T4 DNA ligase, suggest ing the action in the overall reaction of activities in addition to DN A ligases. The experiments presented generalize the utility of the ass ay in studying the enzymology of dsb rejoining after treatment with ra diomimetic drugs and restriction endonucleases and should be useful in the elucidation of the enzymatic requirements of dsb repair.