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

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Abstract
Using 'caged' DNA break trapping agents as well as the equivalent uncaged 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 after 120 secs 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 ddATP, which suggests that an excision activity which results in removal of recently incorporated bases is not solely provoked by the incorporation of an unnatural base, but also by the incorporation of an incorrectly paired base in a phase of what may be low fidelity repair. The second phase consists of a more steady state of incorporation. Both phases are dose dependent and show higher incorporation at higher doses. The transient is most apparent at does which cause some lethality. It may 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 phase of repair. Since excision of newly incorporated radiolabel takes place very rapidly during the first phase this would imply that a polymerase with an associated proof-reading nuclease is active at this stage. Polymerases alpha, delta, and epsilon all have this property but delta and epsilon have a higher sensitivity to dideoxythymidine than does alpha. Since the transient burst phase shows significant inhibition by dideoxythymidine, it is more likely that delta or epsilon are active at this stage. The putative panic response discussed in relation to proof reading mechanisms in aminoacyl-tRNA and DNA synthesis.

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