6 STATE OF THE ART 8 UV-induced DNA damage 8 Inside the chemistry of UV-induced DNA lesions 10 Sensing and repair of UV-induced DNA lesions 12 Bypass of UV-induced DNA lesions 16 The Proliferating Cell Nuclear Antigen (PCNA) 16 Enzymology of the RAD6 epistasis group 19 Rad6 and Rad18 20 53 CONCLUSIONS AND FUTURE PERSPECTIVES 55 REFERENCES 59 Index 3 PART II 73 CONTENT 74 Published paper I Use of the optimized protocol for in vivo detection of ubiquitylated PCNA in the presence of hybrid RNA-DNA

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... structures in the genome 75 Published paper II Crosstalk among UV-induced DNA damage checkpoint, NER and PRR 76 Published paper III A Systems Biology approach to describe the DDR 78 Abstract 6 ABSTRACT My research activity has been supported by the bioinformatic platform of the Fondazione Cariplo NOBEL Project named "Understanding DNA damage checkpoint and repair". The aim of this platform was to investigate the molecular mechanisms involved in the DNA Damage Response (DDR), through a Systems Biology approach. Among the DDR pathways, the focus of my research was the DNA Damage Tolerance (DDT) pathway called Post-Replication Repair (PRR) using Saccharomyces cerevisiae cells as a model system. This pathway leads to the bypass of UV-induced DNA lesions through a mechanism, which is poorly characterized at molecular level in comparison with the Nucleotide Excision Repair (NER) process, the mechanism leading to the effective repair of UV-induces DNA lesions. The data presented in this thesis led to the production of the first mathematical model of PRR in eukaryotic cells. In the work carried out in this thesis I had the difficult task to manage both in silico and in vivo aspects of a Systems Biology project. Given the complexity of the PRR pathway and the lack of critical experimental data, the attempt to build a mathematical model of PRR was an ambitious aim not free from difficulties. Before this study, the crosstalk between PRR and other DDR pathways were unknown and, indeed, the discrepancy between the in vivo data and the in silico simulations, observed under certain experimental conditions, led to additional experiments that uncovered new unpublished aspects of PRR and others that need to be done. Abstract 7 In this way the so called "Circle of Systems Biology" applied to PRR can be considered closed and promising for the future: the limit of the model to particular experimental conditions is leading to a new batch of experiments to do and new hypothesis to test. State Of The Art 8 STATE OF THE ART UV-induced DNA damage Sun radiation is one of the elements, which allowed and continues to support life on t he earth. However, its energy is so strong that we can easily identify a second essential element for life in our planet: the ozonosphere, a layer of ozone enveloping earth in the stratosphere, which is absorbing part of this energy. Life on e arth depends upon a sort of equilibrium between sun radiation and its protection by the ozone layer. This layer is subjected to periodic reduction, but since the half of the seventies we know that this reduction is increasing because of the increase of Chloro Fluoro Carbon (CFC) gases in the atmosphere. The reduction of the ozone layer is decreasing the shielding against sun radiation, which ranges from infrared to ultraviolet (UV) wavelength (3000 to 100 nm , respectively). Inside the UV region three sub-regions can be distinguished: the UV-A (400-315 nm), the UV-B (315-280 nm) and the UV-C (280-100 nm). The physiological relevance of this project arises from the damaging effects of UV radiations present particularly in the lower region of the spectrum. UV radiations act on biologic materials, in particular DNA, so enhancing mutagenesis, affecting genome stability in living organisms and, ultimately, they increase cancerogenesis in human skin. UV radiations cause covalent DNA modifications, such as Cyclobutane Pyrimidine Dimers (CPDs) and 6-4 photoproducts (6-4 PPs). CPDs are usually the most abundant lesions: the CPDs: 6-4 PPs ratio is on average about 3:1 (Friedberg et al., 2005), however, it may change in different State Of The Art 9 organisms. Moreover, 6-4 PPs can be converted into their Dewar isomers by sunlight induced photo-isomerization (Taylor and Cohrs, 1987; Taylor et al., 1990) and the carcinogenicity of the last UV-induced DNA lesion on human cells is still under investigation. The level of CPDs/Kbp of DNA has been calculated for UV-A, UV-B and UV-C: the first relevant aspect of these studies was the linearity between the UV dose and the number of UV-induced lesions on D NA. It was discovered that UV-C radiations were about 100 times more effective than UV-B and 100000 more effective than UV-A in inducing CPDs on DNA (Kuluncsics et al., 1999). The physiological DNA damage caused by shielded sunlight (which is a mixture of about 6% UV-A, 0.8% UV-B, 44.5% visible light, 48.7% infrared and no U V-C) could be due only to UV-B (Kuluncsics et al., 1999). Most of the experimental data on t he State Of The Art 20 Rad6 and Rad18 The RAD6 and RAD18 genes were identified in a screening for UV sensitive mutants in S.cerevisiae (Cox and Parry, 1968). Further analysis highlighted that yeast strains carrying deletions of these genes show an increase in spontaneous mutagenesis and a decrease in UV induced mutagenesis (Lawrence and Christensen 1976; Armstrong et al., 1994). The Rad6 protein is one of the 13 E2 enzymes in budding yeast and it is involved in many cellular processes, such as PRR, induced mutagenesis, sporulation, silencing and N-end rule protein degradation (Raboy et al., 1999). Rad6 receives the activated ubiquitin moiety from the unique ubiquitin-activating enzyme (E1) Uba1 and transfers it to the target protein through an E3 ubiquitin ligase, which provides the target specificity. During post-replicative repair, the E3 ubiquitin ligase for PCNA is Rad18 (Hibbert et al., 2011). Rad18 is a RING finger E3 enzyme with ATPase activity and ssDNA binding properties (Bailly et al., 1997). Deletions of RAD6 or RAD18 causes sensitivity not only to UV irradiation, but also to X-rays, γ-rays, alkylating agents like Methyl Metan Sulphonate (MMS), UV mimetic drugs like 4-Nitro Quinoline Oxide (4-NQO), anti-tumoral drugs (DNA damaging agents) like Bleomicin and Cysplatin. Co-immunoprecipitation experiments showed that Rad6 and Rad18 are able to form stable dimer of hetero-dimers (Bailly et al., 1994; Bailly et al., 1997; Notenboom et al., 2007). Even if Rad18 has intrinsic ssDNA binding properties, its localization at the level of stalled replication forks requires interaction with RPA (Davies et al., 2008); therefore, both DNA damage checkpoint and PRR share the same activating substrate, namely RPA-coated ssDNA. The presence of Rad18 at the level of a s talled State Of The Art 21 replication fork targets Rad6, carrying an active ubiquitin moiety, on PCNA and through its RING domain promotes PCNA monoubiquitylation on K 164 (Hoege et al., 2002). Even if this complex was isolated as a s table hetero-dimer, a r ecent study explained that the most probable stoichiometry of the full-length Rad18-Rad6 complex is 2:1, respectively (Fig. 6). The asymmetry of the Rad18-Rad6 complex seems to force Rad6 to exclusively mono-ubiquitylate PCNA, by inhibiting the Rad6 degradative poly-ubiquitylation activity (Huang et al., 2011). Fig. 6. The last published structure of the Rad18-Rad6 complex. The Rad18 RING alone is able to form a 2:2 dimer, but in full-length Rad18, only one Rad6 is bound. 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