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L'instabilità del genoma è una delle caratteristiche delle cellule tumorali e può essere causata da difetti nella riparazione del DNA. In particolare, le rotture del doppio filamento del DNA (DSBs) sono lesioni altamente citotossiche che possono formarsi accidentalmente durante la replicazione del DNA o in seguito all'esposizione ad agenti genotossici, e devono essere correttamente riparate al fine di garantire la stabilità genomica. Per far fronte a queste lesioni del DNA, le cellule eucariotiche attivano la risposta al danno del DNA (DDR) e utilizzano due meccanismi principali per la riparazione dei DSBs: l’unione terminale non omologa (NHEJ) e la ricombinazione omologa (HR). La risposta cellulare ai DSBs ha inizio con il reclutamento dei complessi Ku e MRX/MRN alle due estremità rotte di un DSB. Inoltre, il complesso MRX recluta al DSB anche Tel1/ATM, una chinasi coinvolta nel checkpoint da danno al DNA. Tel1, a sua volta, consente di promuovere e stabilizzare l'associazione del complesso MRX sia ai DSBs che ai telomeri in un ciclo a feedback positivo. Ku, MRX/MRN e Tel1/ATM sono anche necessari per mantenere la lunghezza dei telomeri, strutture nucleoproteiche specializzate situate alle estremità dei cromosomi eucariotici. Il DNA telomerico deve inoltre essere distinto dalle estremità dei DSBs intracromosomici attraverso diversi complessi proteici, i quali vengono reclutati ai telomeri al fine di prevenire l'attivazione della DDR. Nel lievito S. cerevisiae, Rif2 e Rap1 costituiscono due delle principali proteine che compongono tali complessi. Sia Rif2 che Rap1 contrastano l'attivazione di Tel1, la degradazione nucleolitica e l’unione terminale non omologa ai telomeri. Rif2 sembra esercitare tutte queste funzioni inibendo l'associazione del complesso MRX al DNA telomerico; tuttavia, restava ancora da determinare come Rap1 controllasse negativamente l'attività di MRX alle estremità del DNA. Nella prima parte del mio dottorato di ricerca, ho contribuito a, Genome instability is one of the hallmarks of cancer cells and it can be caused by DNA repair defects. Among several types of DNA damage, DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can form accidentally during DNA replication or upon exposure to genotoxic agents. DSBs must be repaired to avoid loss of genetic information and to ensure genomic stability. Eukaryotic cells repair DSBs by activating the DNA damage response (DDR) and by using two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). The cellular response to DSBs is initiated by the recruitment of Ku (Ku70-Ku80) and MRX/N (Mre11-Rad50-Xrs2/Nbs1) complexes at the two DSB broken ends. MRX in turn recruits Tel1/ATM, a kinase involved in the DNA damage checkpoint, a surveillance mechanism that couples DSB repair and cell-cycle progression. Tel1 allows to promote and stabilize MRX association at both DSBs and telomeres in a positive feedback loop. Ku, MRX/MRN, and Tel1/ATM are also required to maintain the length of telomeres, specialized nucleoprotein complexes at the ends of eukaryotic chromosomes. Furthermore, telomeric DNA must be distinguished from intrachromosomal DSBs ends through different protein complexes, which are recruited to telomeres in order to prevent DDR activation. In S. cerevisiae, Rif2 and Rap1 are two of the main proteins that compose these complexes. Both Rif2 and Rap1 counteract Tel1 activation, nucleolytic degradation, and NHEJ at telomeres. Rif2 appears to exert all these functions by inhibiting MRX association with telomeric DNA, however how Rap1 negatively controls MRX activity at DNA ends remained to be determined. In the first part of my PhD, I contributed to show that Rif2 counteracts MRX association at both DSBs and telomeres in a Rap1-dependent manner. Rap1 in turn can inhibit MRX functions in a Rif2-dependent and -independent manner, and Rap1 functions at DNA ends are influenced by its DNA binding mode. An important issue

ATP-dependent chromatin remodelling enzymes play an important role in maintaining genome integrity by modifying chromatin structure to alter nucleosome occupancy or composition and regulate essential genome maintenance pathways such as DNA double-strand break (DSB) repair and DNA replication. The HELLS subfamily of chromatin remodellers has been implicated in DSB repair and the response to replication stress, and human HELLS is mutated or aberrantly expressed in a number of cancers and mutated in a subset of ICF patients, a rare immune disorder characterised by chromosome instability. Previous work from our group has identified a novel role for HELLS in promoting the homologous recombination (HR) pathway of DSB repair within heterochromatin in G2 by facilitating DNA end resection. HR also plays an essential role in the response to replication stress in S-phase while alternative, inaccurate DSB repair pathways share a common end resection mechanism to HR. Consequently, my thesis investigates a role for human HELLS in the response to replication stress and in additional resection-dependent DSB repair pathways. Using CRISPR-Cas9 to generate human immortalised fibroblast cell lines lacking HELLS, HELLS-deficient cells were confirmed to have a defect in HR and display signs of genomic instability. In addition, this thesis provides evidence that HELLS protects cells from replication stress by limiting replication-associated DNA damage and accumulation of stalled replication forks. HELLS is also shown to promote alternative resection-dependent DSB repair pathways in addition to HR, namely the mutagenic microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA) pathways. Furthermore, this work also identifies the potential to use PARP inhibitors as a synthetic lethal therapy for HELLS-defective tumours. The uncovered functions of HELLS in the response to replication stress and DSB repair pathway usage are important for maintenance of genome stabili

It has long been known that the process of transcription is a source of genetic instability. RNA molecules have been implicated in both the generation of DNA damage and, in the recent years, in its repair. These seemingly conflicting roles of RNA motivated us to open this research topic covering transcription-associated genetic instability sources and the role of RNA in the repair of DNA damage. The genome is challenged by both endogenous and exogenous sources of DNA damage. Additionally, failures in DNA metabolic processes, such as replication impairments or chromosomal miss-segregation, can threaten the transmission of the genetic information to the offspring by causing genome and chromosome instability. In particular, DNA transcription increases the frequency of mutation and recombination; this latter being caused, although not exclusively, by increased number of DNA double-strand breaks (DSBs), which are among the most harmful DNA lesions (Gaillard and Aguilera, 2016). RNA transcripts are constantly generated to supply for protein synthesis, RNA interference pathways, and regulation of transcription, translation, splicing as well as DNA repair processes via non-coding RNAs. Even heterochromatic regions can generate RNA molecules at telomeres (Azzalin et al., 2007; Luke et al., 2008; Schoeftner and Blasco, 2008) and centromeres (Saffery et al., 2003), that participate in the regulation of the telomeric (Fukagawa et al., 2004) and centromeric structures, thus impacting chromosome segregation and chromosome instability. The diverse roles of human centromeric RNA in chromosome stability were compiled by Leclerc and Kitagawa in this Frontiers Topic.

The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem. In particular, the use of site-specific nucleases capable of inducing DNA nicks that replication converts into double-strand breaks has enabled the specific study of the repair of such replication-born double strand breaks by sister chromatid recombination. In this chapter, we describe detailed protocols for determining the efficiency and kinetics of this recombination reaction as well as for the genetic quantification of recombination products.

In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2–DDX5 interaction is impaired in cells expressing the BRCA2T207A missense variant found in breast cancer patients.

The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem. In particular, the use of site-specific nucleases capable of inducing DNA nicks that replication converts into double-strand breaks has enabled the specific study of the repair of such replication-born double strand breaks by sister chromatid recombination. In this chapter, we describe detailed protocols for determining the efficiency and kinetics of this recombination reaction as well as for the genetic quantification of recombination products.

In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2–DDX5 interaction is impaired in cells expressing the BRCA2T207A missense variant found in breast cancer patients.

The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem. In particular, the use of site-specific nucleases capable of inducing DNA nicks that replication converts into double-strand breaks has enabled the specific study of the repair of such replication-born double strand breaks by sister chromatid recombination. In this chapter, we describe detailed protocols for determining the efficiency and kinetics of this recombination reaction as well as for the genetic quantification of recombination products.

In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2–DDX5 interaction is impaired in cells expressing the BRCA2T207A missense variant found in breast cancer patients.

The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem. In particular, the use of site-specific nucleases capable of inducing DNA nicks that replication converts into double-strand breaks has enabled the specific study of the repair of such replication-born double strand breaks by sister chromatid recombination. In this chapter, we describe detailed protocols for determining the efficiency and kinetics of this recombination reaction as well as for the genetic quantification of recombination products.

In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2–DDX5 interaction is impaired in cells expressing the BRCA2T207A missense variant found in breast cancer patients.

In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2–DDX5 interaction is impaired in cells expressing the BRCA2T207A missense variant found in breast cancer patients.

The repair of DNA double-strand breaks is crucial for cell viability and the maintenance of genome integrity. When present, the intact sister chromatid is used as the preferred repair template to restore the genetic information by homologous recombination. Although the study of the factors involved in sister chromatid recombination is hampered by the fact that both sister chromatids are indistinguishable, genetic and molecular systems based on DNA repeats have been developed to overcome this problem. In particular, the use of site-specific nucleases capable of inducing DNA nicks that replication converts into double-strand breaks has enabled the specific study of the repair of such replication-born double strand breaks by sister chromatid recombination. In this chapter, we describe detailed protocols for determining the efficiency and kinetics of this recombination reaction as well as for the genetic quantification of recombination products.

In a recent report, we have revealed a new interaction between the BRCA2 DNA repair associated protein (BRCA2) and the DEAD-box helicase 5 (DDX5) at DNA breaks that promotes unwinding DNA-RNA hybrids within transcribed chromatin and favors repair. Interestingly, BRCA2–DDX5 interaction is impaired in cells expressing the BRCA2 missense variant found in breast cancer patients.

Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.

Displacement loops (D-loops) are critical intermediates formed during homologous recombination. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54/Tid1 is also present in somatic cells where its function is less understood. While Rdh54/Tid1 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54. Here, we show that Rdh54/Tid1 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54/Tid1 uniquely restricts the length of Rad51-Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast. We propose that Rdh54/Tid1 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54/Tid1 acts as a physical roadblock to Rad54 translocation, limiting D-loop formation and D-loop length.

The mechanisms that maintain genomic stability safeguard cells from constant DNA damage produced by endogenous and external stressors. Therefore, this thesis aimed to specifically address questions regarding the requirement and involvement of DNA repair proteins in the repair of various types of radiation-induced DNA damage. The first aim was to determine whether the phosphorylation of DNA-PKcs, a major kinase involved in non-homologous end joining pathway, can be utilized to score the DNA double-strand break (DSB) content in cells. DNA-PKcs phosphorylated (pDNA-PKcs) at T2609 was more sensitive to the cellular DSB content than ɣH2AX, as analyzed by flow cytometry. Further, pDNA-PKcs at T2609 could discriminate between DSB repair-compromised and normal cells, confirming that the pDNA-PKcs can be used as a DSB repair marker. In paper II, the DSB repair was assessed in cells with reduced levels of DNA-PKcs. The reduction in DNA-PKcs resulted in decreased cell survival and unaffected DSB repair. These results clearly indicate that DNA-PKcs plays an additional role in promoting cell survival in addition to its function in DSB repair. The second part of the thesis focused on the characterization of complex DNA damage. DNA damage was investigated after exposure to α-particles originating from Ra-223. The Ra-223 treatment induced a nonrandom DSB distribution consistent with damage induced by high-linear energy transfer radiation. The exposure to Ra-223 significantly reduced cell survival in monolayers and 3D cell structures. The last paper unraveled the fate of heat-sensitive clustered DNA damage site (HSCS) repair in cells. HSCS repair was independent of DSB repair, and these lesions did not contribute to the generation of additional DSBs during repair. Prolonged heating of DNA at relatively low temperatures induced structural changes in the DNA that contributed to the production of DNA artifacts. In conclusion, these results demonstrate that DNA-PKcs can be used to monit

The mechanisms that maintain genomic stability safeguard cells from constant DNA damage produced by endogenous and external stressors. Therefore, this thesis aimed to specifically address questions regarding the requirement and involvement of DNA repair proteins in the repair of various types of radiation-induced DNA damage. The first aim was to determine whether the phosphorylation of DNA-PKcs, a major kinase involved in non-homologous end joining pathway, can be utilized to score the DNA double-strand break (DSB) content in cells. DNA-PKcs phosphorylated (pDNA-PKcs) at T2609 was more sensitive to the cellular DSB content than ɣH2AX, as analyzed by flow cytometry. Further, pDNA-PKcs at T2609 could discriminate between DSB repair-compromised and normal cells, confirming that the pDNA-PKcs can be used as a DSB repair marker. In paper II, the DSB repair was assessed in cells with reduced levels of DNA-PKcs. The reduction in DNA-PKcs resulted in decreased cell survival and unaffected DSB repair. These results clearly indicate that DNA-PKcs plays an additional role in promoting cell survival in addition to its function in DSB repair. The second part of the thesis focused on the characterization of complex DNA damage. DNA damage was investigated after exposure to α-particles originating from Ra-223. The Ra-223 treatment induced a nonrandom DSB distribution consistent with damage induced by high-linear energy transfer radiation. The exposure to Ra-223 significantly reduced cell survival in monolayers and 3D cell structures. The last paper unraveled the fate of heat-sensitive clustered DNA damage site (HSCS) repair in cells. HSCS repair was independent of DSB repair, and these lesions did not contribute to the generation of additional DSBs during repair. Prolonged heating of DNA at relatively low temperatures induced structural changes in the DNA that contributed to the production of DNA artifacts. In conclusion, these results demonstrate that DNA-PKcs can be used to monit

DNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.

DNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.

DNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.

DNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.

DNA double strand breaks (DSBs) are the most cytotoxic lesions of those occurring in the DNA and can lead to cell death or result in genome mutagenesis and chromosomal translocations. Although most of these rearrangements have detrimental effects for cellular survival, single events can provide clonal advantage and result in abnormal cellular proliferation and cancer. The origin and the environment of the DNA break or the repair pathway are key factors that influence the frequency at which these events appear. However, the molecular mechanisms that underlie the formation of chromosomal translocations remain unclear. DNA topoisomerases are essential enzymes present in all cellular organisms with critical roles in DNA metabolism and that have been linked to the formation of deleterious DSBs for a long time. DSBs induced by the abortive activity of DNA topoisomerase II (TOP2) are “trending topic” because of their possible role in genome instability and oncogenesis. Furthermore, transcription associated TOP2 activity appears to be one of the most determining causes behind the formation of chromosomal translocations. In this review, the origin of recombinogenic TOP2 breaks and the determinants behind their tendency to translocate will be summarized.

Homologous recombination faithfully restores the sequence information interrupted by a DNA double-strand break by referencing an intact DNA molecule as a template for repair DNA synthesis. DNA synthesis is primed from 3'-OH end of the invading DNA strand in the displacement loop (D-loop). Here, we describe a simple and quantitative proximity ligation-based assay to study the initiation of homologous recombination-associated DNA synthesis initiated at the D-loop and final product formation. The D-loop extension assay overcomes the semiquantitative nature and some limitations of the current PCR-based technique and facilitates the study of the recombination-associated DNA synthesis.

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression

Homologous recombination faithfully restores the sequence information interrupted by a DNA double-strand break by referencing an intact DNA molecule as a template for repair DNA synthesis. DNA synthesis is primed from 3'-OH end of the invading DNA strand in the displacement loop (D-loop). Here, we describe a simple and quantitative proximity ligation-based assay to study the initiation of homologous recombination-associated DNA synthesis initiated at the D-loop and final product formation. The D-loop extension assay overcomes the semiquantitative nature and some limitations of the current PCR-based technique and facilitates the study of the recombination-associated DNA synthesis.

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression

DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression

DNA (DeoxyriboNucleic Acid) Double-Strand Break (DSB) was introduced by the I-Sce I expression vector, pCBASce. Repair of DSB introduced by the I-SceI expression vector at the tk+ allele in human lymphoblstoid TK6 cell line, TSCE5, was measured by induction of TK-deficient mutants. Similarly we also measured the revertants due to such repair in its compound heterozygote (tk-/-) cell line, TSCER2, which carried an additional point-mutation in exon 5. The former and later measurements reflect the DSB repair efficiency due to DNA Non-Homologous End-Joining (NHEJ) and Homologous Recombination (HR), respectively. The pre-treatment, C-ion-irradiation of frozen cells, TSCE5 and TSCER2 with 50 mGy enhanced DSB repair due to both pathways of NHEJ and HR, 2 to 3-fold. This kind of adaptive response might be observed with the cells recovered from the long preservation under the frozen status in ISS (International Space Station).

Proper repair of DNA double strand breaks (DSBs) is vital for the preservation of genomic integrity. There are two main pathways that repair DSBs, Homologous recombination (HR) and Non-homologous end-joining (NHEJ). HR is restricted to the S and G2 phases of the cell cycle due to the requirement for the sister chromatid as a template, while NHEJ is active throughout the cell cycle and does not rely on a template. The balance between both pathways is essential for genome stability and numerous assays have been developed to measure the efficiency of the two pathways. Several proteins are known to affect the balance between HR and NHEJ and the complexity of the break also plays a role. In this review we describe several repair assays to determine the efficiencies of both pathways. We discuss how disturbance of the balance between HR and NHEJ can lead to disease, but also how it can be exploited for cancer treatment.

The interplay between ATM and DNA-PKcs kinases during double strand breaks (DSBs) resolution is still a matter of debate. ATM and DNA-PKcs participate differently in the DNA damage response pathway (DDR), but important common aspects are indeed found: both of them are activated when faced with DSBs, they share common targets in the DDR and the absence of either kinase results in faulty DSB repair. Absence of ATM translates into timely repair that, nevertheless, is incomplete. On the other hand, DNA-PKcs absence translates into slower repair, which in turn gives rise to the accumulation of simple and complex reorganizations. These outcomes confirm that the function of both protein kinases is essential to guarantee genome integrity. Interestingly, V(D)J and CSR recombination events provide a powerful tool to study the interplay between both kinases in DSB repair. Although the physiological DSBs generated during V(D)J and CSR recombination are resolved by the non-homologous end-joining (NHEJ) repair pathway, ATMabsence during these events translates into chromosome translocations. These results suggest that NHEJ accuracy is threatened in the absence of ATM, which may play a role in avoiding illegitimate repair by favouring the joining of the correct DNA ends. Indeed, simultaneous DNA-PKcs and ATM deficiency during V(D)J and CSR recombination translates into a synergistic increase in potentially dangerous chromosomal translocations and deletions. Although the exact nature of their interaction remains elusive, the evidence indicates that ATM and DNA-PKcs play complementary roles that allow complete and legitimate DSB repair to be reached. Faithful repair can only be achieved by the presence and correct functioning of both kinases: while DNA-PKcs ensures fast rejoining, ATM guarantees complete repair.

This thesis focuses on the role of RAD 18 in DNA double-strand break (DSB ) repair. Much is known about the role of RAD 18, and its critical substrate PCNA in replication damage bypass (RDB ) repair. However, the roles of RAD 18 in DSB repair are still elusive, although several interaction partners of RAD 18 have been identified, and the radiation-sensitivity of Rad18 knockout cells has shown that this E3 ligase is active in DSB repair. First, a general introduction on the possible involvement of RAD 18 in DSB repair mechanisms and RDB that operate in mitotic and meiotic cells is presented in Chapter 1. In Chapter 2, we examined the dynamic localization of human RAD 18 during the cell cycle and after DNA damage in living cells. The DNA damage response function