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Mortusewicz, Oliver (2007): Analysis of the DNA damage response in living cells. Dissertation, LMU München: Fakultät für Biologie



DNA lesions arising from environmental and endogenous sources induce various cellular responses including cell cycle arrest, DNA repair and apoptosis. Although detailed insights into the biochemical mechanisms and composition of DNA repair pathways have been obtained from in vitro experiments, a better understanding of the interplay and regulation of these pathways requires DNA repair studies in living cells. In this study we employed laser microirradiation and photobleaching techniques in combination with specific mutants and inhibitors to analyze the real-time accumulation of proteins at laser-induced DNA damage sites in vivo, thus unravelling the mechanisms underlying the coordination of DNA repair in living cells. The immediate and faithful recognition of DNA lesions is central to cellular survival, but how these lesions are detected within the context of chromatin is still unclear. In vitro data indicated that the DNA-damage dependent poly(ADP-ribose) polymerases, PARP-1 and PARP-2, are involved in this crucial step of DNA repair. With specific inhibitors, mutations and photobleaching analysis we could reveal a complex feedback regulated mechanism for the recruitment of the DNA damage sensor PARP-1 to microirradiated sites. Activation of PARP-1 results in localized poly(ADP-ribosyl)ation and amplifies a signal for the subsequent rapid recruitment of the loading platform XRCC1 which coordinates the assembly of the repair machinery. Using similar techniques we could demonstrate the immediate and transient binding of the RNA Polymerase II cofactor PC4 to DNA damage sites, which depended on its single strand binding capacity. This establishes an interesting link between DNA repair and transcription. We propose a role for PC4 in the early steps of the DNA damage response, recognizing and stabilizing single stranded DNA (ssDNA) and thereby facilitating DNA repair by enabling repair factors to access their substrates. After DNA lesions have been successfully detected they have to be handed over to the repair machinery which restores genome integrity. Efficient repair requires the coordinated recruitment of multiple enzyme activities which is believed to be controlled by central loading platforms. As laser microirradiation induces a variety of different DNA lesions we could directly compare the recruitment kinetics of the two loading platforms PCNA and XRCC1 which are involved in different repair pathways side by side. We could demonstrate that PCNA and XRCC1 show distinct recruitment and binding kinetics with the immediate and fast recruitment of XRCC1 preceding the slow and continuous recruitment of PCNA. Introducing consecutively multiple DNA lesions within a single cell, we further demonstrated that these different recruitment and binding characteristics have functional consequences for the capacity of PCNA and XRCC1 to respond to successive DNA damage events. To further study the role of PCNA and XRCC1 as loading platforms in DNA repair, we extended our analysis to their respective interaction partners DNA Ligase I and III. Although these DNA Ligases are highly homologous and catalyze the same enzymatic reaction, they are not interchangeable and fulfil unique functions in DNA replication and repair. With deletion and mutational analysis we could identify domains mediating the specific recruitment of DNA Ligase I and III to distinct repair pathways through their interaction with PCNA and XRCC1. We conclude that this specific targeting may have evolved to accommodate the particular requirements of different repair pathways (single nucleotide replacement vs. synthesis of short stretches of DNA) and thus enhances the efficiency of DNA repair. Interestingly, we found that other PCNA-interacting proteins exhibit recruitment kinetics similar to DNA Ligase I, indicating that PCNA not only serves as a central loading platform during DNA replication, but also coordinates the recruitment of multiple enzyme activities to DNA repair sites. Accordingly, we found that the maintenance methyltransferase DNMT1, which is known to associate with replication sites through binding to PCNA, is likewise recruited to DNA repair sites by PCNA. We propose that DNMT1, like in DNA replication, preserves methylation patterns in the newly synthesized DNA, thus contributing to the restoration of epigenetic information in DNA repair. In summary, we found immediate and transient binding of repair factors involved in DNA damage detection and signalling, while repair factors involved in the later steps of DNA repair, like damage processing, DNA ligation and restoration of epigenetic information, showed a slow and persistent accumulation at DNA damage sites. We conclude that DNA repair is not mediated by binding of a preassembled repair machinery, but rather coordinated by the sequential recruitment of specific repair factors to DNA damage sites.