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Gomez-Paramio, Idoia (2007): Analysis of the role of Rad5 for the regulation of repair of DSB, small deletions and oxidative damage. Dissertation, LMU München: Fakultät für Biologie



Rad5 is a decisive protein in S. cerevisiae due to its role in the Post-replication repair (PRR) pathway, in which Rad5 is necessary for at least one error-free and one error-prone repair subpathway. In addition, Rad5 plays a role in other repair pathways; for instance, Rad5 regulates the balance between the double strand break (DSB) repair pathways, favoring the Rad52-dependent Homologous Recombination (HR) over the yKu70-dependent Non-Homologous-End Joining (NHEJ). Furthermore, since UV-induced damages are substrates for Rad5 but also for Base Excision Repair (BER) proteins, Rad5 is possibly involved directly or indirectly in the BER pathway. To get a deeper insight into the interaction of Rad5 with HR, NHEJ and BER proteins, survival curves, plasmid assays, and mutagenicity experiments were carried out in this work. In addition, a new software tool has been developed for the quantification of DSB. This software, called Geltool, allows the quantification of DSB in haploid cells from PFGE gels, even if the number of DSB is small. This represents a decisive advantage in comparison with previous programs. The sensitivity of Geltool has permitted the quantification of DSB repair during the stationary growth phase in haploid cells, detecting a repair of 46 %- 57 % of the gamma-induced DSB in HR proficient strains against 6 % - 16 % in HR deficient strains. Studies of the functional interactions of Rad5 with HR and NHEJ proteins revealed a synergistic effect between Rad5 and Rad52 proteins for the repair of DSB at chromosomal and plasmidial level. Differences in the repair of plasmids from the rad52 and the rad5 mutants revealed different end joining mechanisms for gap repair. Severe degradations found in plasmids from rad52 and rad52rad5 mutants could indicate an end protection function for Rad52 and also for Rad5, when Rad52 is absent. Moreover, the regulatory role of the Rad5 protein is confirmed, since the additional deletion of YKU70 suppresses the rad5 phenotype and forces the yku70rad5 mutant to repair by HR. The further study of the interplay of Rad5 with BER proteins shows that while BER only plays a minor role for the repair of gamma-induced damage, the rad5 phenotype is suppressed in the BER deficient apn1ntg1ntg2rad5 mutant. The same phenotype of suppression is also found for survival after UV irradiation. An enhanced mutagenicity of the apn1ntg1ntg2rad5 mutant indicates a possible repair through the REV3-dependent Translesion Synthesis Repair (TLS) pathway, suggesting that an error-prone tolerance of UV-induced damage can be very effective for survival.