INSPECCIÓN FUNCIONAL DEL PUENTE SOBRE EL RÍO

Survival experiments after gamma irradiation show similar sensitivities of yku70 and WT strains, the sensitivity of the yku70 mutant in the logarithmic phase even being 10 % lower than the sensitivity of the WT. A similar positive effect for cell survival due to YKU70

deletion was observed in haploid strains for the repair of a single HO nuclease-induced DSB (Clikeman, Khalsa et al. 2001). This has been explained either by competition for DSB between HR and NHEJ or by interferences of yKu70 with HR, in which yKu70 would block a certain amount of DNA ends from HR, which are then repaired by NHEJ.

YKU70 deletion suppresses the moderate gamma sensitivity of the rad5 mutant (Figure 18) in the yku70rad5 double mutant in the logarithmic phase. That could be explained taking into consideration the regulatory role of Rad5, which promotes DSB by HR. rad5 mutants repair DSB mostly by inaccurate NHEJ, which had already been observed in a rad5

interruption mutant (75% NHEJ, Ahne, Jha et al. 1997). This reduces its survival capacity after gamma irradiation. In the yku70rad5 mutant, NHEJ is eliminated due to YKU70 deletion and therefore, they must repair DSB by accurate HR, which increases the survival capacity of this mutant to a WT level.

This suppression is not as pronounced in cells in the stationary phase (Figure 19) as in the logarithmic phase. The sensitivities of the yku70 and yku70rad5 mutants strongly increase in the stationary phase in comparison to the logarithmic phase, whereas the sensitivity of the

rad5 mutant remains almost the same. The higher sensitivity of the strains that repair DSB by HR is due to the absence of sister chromatids in the stationary phase (see chapter 5.1.3), having only a small influence in the rad5 mutant, which mainly repairs by NHEJ (Ahne, Jha et al. 1997). Since in PFGE experiments cells are in high stationary growth phase, this would also explain that the DSB repair efficiency of the rad5 mutant is comparable to the efficiency of the WT, yku70 and yku70rad5 strains (Figure 28). Due to this high DSB repair capacity of the rad5 mutant in PFGE experiments, it is not possible to confirm the rad5 phenotype suppression in the yku70rad5 mutant.

To confirm the repair pathways used by the yku70, rad5 and yku70rad5 mutants, plasmids assays would be necessary. However, plasmid assays in high stationary cells have not been successful till now. This can be a consequence of the higher resistance of the cell wall in this growth phase, which impedes the transfection of plasmids. Plasmid assay experiments with cells in the logarithmic phase show that the yku70rad5 mutant repairs mainly by HR, explaining the suppression of the rad5 phenotype in this mutant (Figure 32).

Results on the efficiency of plasmid gap repair show that the rad5 cells present a high effectiveness of rejoining, indicating that NHEJ can be very effective (Ahne, Jha et al. 1997). This high efficiency of NHEJ has been previously shown for repair of intact ends that present 4 nt complementary overhanging ends (Frank-Vaillant and Marcand 2002) and for 3’ cohesive overhanging ends in Ku80 deficient cells (Feldmann, Schmiemann et al. 2000). In this work a high efficiency for end joining was found, even though the DNA ends to be rejoined were non-cohesive. Surprisingly, the HR proficient yku70rad5 double mutant shows a rather low efficiency for plasmid gap repair (Figure 29). This could be explained by the disturbance of the balance between DSB repair pathways in favour of NHEJ in cells lacking Rad5 (Ahne, Jha et al. 1997); the impossibility to repair by this way in the yku70rad5 mutant induces the “redirection” of repair to HR (Figure 32). Thus, although HR is the final repair pathway, it was also the second choice for repair; the loss of decisive time during this redirection process could be the reason of the reduction in repair efficiency in the yku70rad5

mutant.

The rad5 mutant shows a strong decrease in gap repair accuracy in comparaion with WT, yku70 and yku70rad5 strains, which repair almost exclusively by accurate HR. This decrease emight be due to the predominant use of NHEJ by the rad5 mutant for the repair of gaps (Figure 32).This confirms, first, the correctness of HR in general, and second, the minor role of YKU70 for the accuracy of DSB repair in a HR proficient background. In the

yku70rad5 mutant, the possible “redirection” of the repair does not affect its accuracy.

Further studies were carried out by sequencing plasmids that were incorrectly repaired (ura-) by the yku70 and yku70rad5 mutants (Figure 32, Figure 33 and Figure 34). 91% and 95% of the ura- plasmids from the yku70rad5 and yku70 mutants, respectively (100% from WT) were repaired by error-prone HR. Sequence analysis of the 11 recovered ura- plasmids from yku70 and 13 from yku70rad5 strains reveal only minor differences between these

mutants, confirming the rad5 phenotype suppression also for error-prone HR events. The induced point mutations were distributed inside and outside of the repaired gap region; additions were the most widespread mutation (Figure 33-left and Figure 34), deletions were concentrated at the restriction site and transversions outside of the repaired gap region.

Accurate end joining was observed in one clone out of 2 of the yku70rad5 mutant, the recovered plasmid having been repaired by direct end joining without any end processing. The influence of Ku in accurate rejoining has been discussed controversially in xrs6 cells: whereas Kabotyanski found no influence in the accuracy (Kabotyanski, Gomelsky et al. 1998), Feldman indicated a reduction in absence of Ku80 depending on the type of ends being joined (Feldmann, Schmiemann et al. 2000). Feldmann has shown that the accuracy of non- cohesive ends 5’/3’ is completely reduced in xrs6 mutants due to nucleotide deletions at DSB ends. In this work, MMEJ was observed in one clone from the yku70 and one from the

yku70rad5 strains (Table 4).

In summary, a suppression of the rad5 phenotype in the yku70rad5 mutant was found in survival experiments with cells in the logarithmic and stationary growth phase. Plasmid assays revealed that the yku70rad5 mutant repairs plasmidial gaps by HR. This means that the deletion of YKU70 in the rad5 mutant leads to repair by HR in cells that would otherwise repair 75 % by NHEJ, confirming the regulatory role of the Rad5 protein.