Bases de datos SQL vs NoSQL

Mutagenicity experiments allow the study of the genetic stability of a strain. The rad5

mutant shows a similar number of spontaneous locus mutations in comparison with the WT, which confirms previous results (Schüller 1995). In contrast, BER deficient mutants show a strongly increased spontaneous mutagenicity, which is 168 - 240 times higher than that of WT. This confirms previous studies in apn1 mutants which suggest unrepaired AP sites as the cause of the enhanced mutagenicity (Masson and Ramotar 1997). However, the mutagenicity after NTG1 and NTG2 deletion is controversial: Some authors found an increased spontaneous mutation frequency in ntg1 and ntg2 single and double mutants, which they explained by a requirement for repair of endogenous oxidative DNA damage (Alseth, Eide et al. 1999). In contrast, other authors found only a mutation rate enhancement in apn1ntg1ntg2 triple mutants but not in the single and double mutant (Swanson, Morey et al. 1999; Doetsch,

Morey et al. 2001), and this enhancement was even higher after an additional NER or HR elimination. In studies by Swanson, it is also suggested that the majority of spontaneous mutations accumulating in apn1ntg1ntg2 mutants are due the REV3-dependent TLS pathway. Taking into consideration that this pathway is also active in apn1ntg1ntg2rad5 mutants, it would explain the similar number of spontaneous locus mutations in both BER deficient mutants that was found in this work.

In response to UV irradiation, the mutation frequency in the WT and the rad5 mutant increases linearly with the doses, and it is slightly higher in the rad5 mutant, confirming results by Schüller (Schüller 1995). In contrast, the apn1ntg1ntg2 mutant presents a lower mutation frequency than the WT at higher doses. Taking into account that UV damage is repaired mainly by NER and PRR, this suggests that the accumulation of UV lesions in a BER deficient background triggers an alternative repair pathway, which may depend on Rad5. These results contrast with previous studies where an enhancement of the mutation frequency in ntg1 and ntg2 single and double mutants after peroxide treatment was found (Alseth, Eide et al. 1999).

The additional RAD5 deletion leads to a suppression of this phenotype in the

apn1ntg1ntg2rad5 mutant, increasing the mutation frequency up to rad5 level. Moreover, the

apn1ntg1ntg2rad5 mutant presents a 8 times higher mutagenicity in comparison with the WT at 80 J/m2. The lower mutation frequency of the BER deficient apn1ntg1ntg2 mutant could be explained by a repair of the UV-induced lesions by NER. However, the apn1ntg1ntg2rad5

mutant shows a higher mutation frequency, even though this mutant could still repair by NER. This suggests that in the BER deficient mutants, UV-induced lesions are processed by PRR either by the Rad5-dependent subpathways, or in the absence of Rad5, by the error-prone

6.

SUMMARY

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.

7.

APPENDIX