Analysis of the role of Rad5 for the regulation of repair of DSB, small deletions and oxidative damage

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.