THE KINETICS AND MECHANISM OF REPAIR OF UV INDUCED DNA-DAMAGE IN MAMMALIAN-CELLS - THE USE OF CAGED NUCLEOTIDES AND ELECTROPORATION TO STUDY SHORT-TIME COURSE EVENTS IN DNA-REPAIR

Using 'caged' DNA break trapping agents as well as the equivalent unca ged reagents and an automated apparatus, we have measured time courses of incorporation of radiolabelled nucleotides into HL60 cellular DNA in the early stages after 248 UV laser damage. These time courses show two distinctive phases, one between 0 and 120 seconds and another aft er 120 sees following damage. The first phase consists of a transient which shows a rapid initial incorporation of radiolabel followed by a sharp fall in incorporated label. This occurs with TTP as well as ddAT P, which suggests that an excision activity which results in removal o f recently incorporated bases is not solely provoked by the incorporat ion of an unnatural base, but also by the incorporation of an incorrec tly paired base in a phase of what may be low fidelity repair. The sec ond phase consists of a more steady state of incorporation. Both phase s are dose dependent and show higher incorporation at higher doses. Th e transient is most apparent at does which cause some lethality. It ma y represent a form of emergency or 'panic' repair where it seems that there may be an immediate effort to maintain strand continuity in the damaged DNA. Results of experiments with polymerase inhibitors suggest that a polymerase which is sensitive to aphidicholin and which shows some sensitivity to dideoxythymidine is active during the transient ph ase of repair. Since excision of newly incorporated radiolabel takes p lace very rapidly during the first phase this would imply that a polym erase with an associated proof-reading nuclease is active at this stag e. Polymerases alpha, delta, and epsilon all have this property but de lta and epsilon have a higher sensitivity to dideoxythymidine than doe s alpha. Since the transient burst phase shows significant inhibition by dideoxythymidine, it is more likely that delta or epsilon are activ e at this stage. The putative panic response discussed in relation to proof reading mechanisms in aminocyl-tRNA and DNA synthesis.