KINETICS AND MECHANISM OF DNA-REPAIR - AN AUTOMATED PROGRAMMABLE APPARATUS FOR FAST TIME-RESOLVED STUDIES OF THE REPAIR OF MAMMALIAN DNA AFTER UV IRRADIATION

An automated apparatus, designed and constructed for use in fast time- resolved studies of mammalian DNA repair after u.v. irradiation, is de scribed. The methodology makes use of caged DNA-break-trapping reagent s, e.g. [alpha-P-32]dideoxyadenosine triphosphate [see Meldrum, Shall, Trentham and Wharton (1990) Biochem J. 266, 885-890] and the apparatu s incorporates excimer lasers both for the delivery of u.v. damage and for the photoactivation of the caged reagents. The design is based on a sample-changing turntable which permits the sequential irradiation and quenching of samples. A maximum of eight samples can be processed in a single experiment, the sequence of events being programmed on a m icrocomputer, which permits a very generalized experimental sequence. Pipettes for addition of cells, nucleotides and quenching agent are dr iven pneumatically. A pair of pneumatically operated platinum electrod es allows electroporation of cells for loading of negatively charged r eagents prior to irradiation. The time resolution of the apparatus is dependent upon the experimental scheme used and can be very short (e.g . 1 ms) when caged reagents are used; a more usual period is 1 s for t he shortest incubation.