A RAPID METHOD TO MONITOR REPAIR AND MIS-REPAIR OF DNA DOUBLE-STRAND BREAKS BY USING CELL-EXTRACTS OF THE YEAST SACCHAROMYCES-CEREVISIAE

We present a rapid in vitro method to scan the repair of DNA double-st rand breaks (DSBs). A DSB was introduced at the EcoRI site within the lacZ gene of the plasmid pUC18 and the plasmid was exposed to cellular extracts from a wild-type repair-competent (RAD) and a mutant (rad52 Delta) strain of the yeast Saccharomyces cerevisiae. The fidelity of r ejoining was determined by the expression of the lacZ gene after bacte rial transformation with the treated plasmid. A cellular extract from the yeast S. cerevisiae was found to be capable of rejoining DNA DSBs. Breaks at the EcoRI site were rejoined by extracts from both wild-typ e and mutant strains to form circular plasmids with almost equal effic iency. However, the fidelity of rejoining was lower for the rad52 Delt a extract than for normal wild-type.