Use of Two-Dimensional Agarose-Gel Analysis to Characterize Processing of UV-Irradiated Plasmids and the Composition of the Replisome Following UV-induced Arrest

摘要

In this thesis, I address two fundamental questions related to our understanding of how DNA damage is processed and repaired during replication. Using Two-dimensional (2-D) agarose gel analysis, I first examine whether DNA damage on plasmids introduced by transformation is processed in a manner similar to that observed on endogenously replicating plasmids and the chromosome. The original intent for using this approach was to develop a technique that could examine how different DNA adducts would be repaired in various sequence contexts. However, I found that distinct differences exist between the processing of DNA damage on transforming plasmids and the chromosome. The 2-D agarose gel analysis shows that RecA-mediated processing does not contribute to the survival of transforming plasmids and that this effect is likely due to inefficient replication of the plasmids after they are initially introduced into cells. These observations, while important, place limitations on the usefulness of transforming plasmids to characterize cellular repair processes. In a second question, I characterize the composition of the replisome following arrest by UV-induced DNA damage. Using 2-D agarose gel analysis the structural changes that occur in DNA during processing and repair have been well characterized, however, little is known about the fate of the replisome itself during these events. I used thermosensitive replication mutants to compare the DNA structural intermediates induced after disruption of specific components of the replisome to those observed after UV damage. The results show that dissociation of subunits required for polymerase stabilization are sufficient to induce the same processing events observed after UV damage. By contrast, disruption of the helicase-primase complex induces abnormal structures and a loss of replication integrity, suggesting that these components remain intact and bound to the template following replication arrest. I propose that polymerase dissociation provides a mechanism that allows repair proteins to gain access to the lesion while retention of the helicase serves to maintain the integrity and licensing of the fork so that replication can resume from the appropriate site once the lesion has been processed.