**2. Consequences of genomic instability**

Loss or insufficiency of DDR and genome repair can lead to an increased susceptibility to cancer due to the consequential genomic instability. Ineffective repair may result in subsequent mutations of genes required for cellular replication and division. The genome repair pathways also communicate with processes involved in induction of senescence and apoptosis when the damage cannot be repaired. Carefully balanced signalling cascades and regulatory systems are implicated in the maintenance of healthy cell survival in order to unfavour tumorigenesis and maintain stem and progenitor cells for renewal (anti-ageing) (Seviour & Lin 2010). Therefore, an effectively repaired genome is crucial not only for cancer prevention but also for lifespan extension. This notion is even more enhanced by the emerging benefit of the response of HR defective tumors to double strand break (DSB) producing therapies a promising and continuously evolving field. A clearer understanding of the biochemical, structural and genetic processes in conjuction with clinical data will lead to the development of more effective treatment strategies for both cancer and ageing processes.

#### **2.1 Genomic instability and cancer**

It is generally accepted that tumors are derived from a single genetically unstable cell, and that the unstable cell population as a whole continues to acquire further chromosomal abnormalities over time, although the precise mechanisms of acquisition of these abnormalities still remain unclear. Hereditary cancers are often characterized by the presence of a specific type of genomic instability, termed chromosomal instability. In these cancers, chromosomal instability can often be attributed to mutations in DNA repair genes, suggesting that the driving force behind tumor development is an increase in spontaneous genetic mutations resulting from lack of appropriate management of DNA damage. A second form of genomic instability, termed microsatellite instability, is also associated with defects in DNA repair, namely the mismatch repair system. However, in non-hereditary sporadic tumors, the picture is less clear. It should be emphasized that cancer is an extremely complex set of diseases, and that cancer cells develop many different mechanisms to achieve a similar phenotype of independent and uncontrolled growth (Hanahan & Weinberg, 2000; Luo et al., 2009 as reviewed by Schild & Wiese 2010).

Many of the DDR components including BRCA1 are known to be lost or mutated in human tumors. While loss of BRCA1 has been shown to lead to the development of mammary tumors in mouse models, the genetic diversity within those tumors suggests that the loss of BRCA1 may not directly be responsible for tumorigenesis. It is more likely, therefore, that the role of BRCA1 in the initiation of cancer is a result of its effects on DNA repair and the maintenance of genomic integrity. BRCA1 -/- tumors are shown to display numerous

Structure-Function Relationship of DNA Repair

DNA damage over time to encourage cancer growth.

replicative senescence.

al., 2010).

**2.2.2 Apoptosis and DNA damage** 

Proteins: Lessons from BRCA1 and RAD51 Studies 107

Replicative senescence results from progressive shortening of telomeres with repeated rounds of cell replication. p53–dependent senescence serves as a tumor suppressor mechanism and is activated by the uncapping of critically shortened telomeres which are recognised as damaged DNA (Feldser and Greider, 2007). Recent studies argue that p53 can either activate or suppress senescence in cells, depending on their specific transcriptional activities and its interaction with partner molecules. As described by Vigneron & Vousden, 2010, the role of p53 in cell fate determination is even more complex as it involves epigenetic modifications of chromosomal DNA and relates chronic DDR signalling with increased levels of p53 acetylation. In addition to p53 other DNA damage response proteins like ATM have been associated with replicative senescence. ATM depletion in mice results in an increase in both chromosomal end-to-end fusion events and cell cycle-dependent telomere loss. These mice exhibit a premature ageing phenotype as defined by increased hair graying, alopecia and marked weight loss. Expression of mutant BRCA1 in mice also results in premature ageing, accompanied by an increase in cellular senescence and an increased susceptibility to certain cancers. The enhanced senescence observed in these mice may interfere with the fact that senescent cells have been noted to modify their tissue microenvironment. This phenomenon is thought to synergize with the accumulation of

Oncogene-induced senescence can be induced by the overexpression of oncogenes by among others the induction of DNA damage resulting from both the generation of reactive oxygen species (ROS) and the hyper-replication of DNA. Both of these mechanisms activate the DNA damage response, which result in senescence by similar processes that induce

The accumulation of DNA damage can also lead to apoptosis. Activation of p53 by DNA damage and its role in the regulation of expression of pro-apoptotic proteins has been well documented. This is further supported by the fact that functional p53 is not detected to the majority of tumors. In cases of decline of the immune system an increase in p53 mediated apoptosis has been observed, linking ageing with apoptotic function. Moreover, constitutively activated p53 in mice also showed that, while high levels of p53 protect against cancer, it also accelerates the ageing process by reducing the mass of various tissues. The human condition Ataxia telangiectasia, which results from mutations in ATM, is associated with substantial neuro-degeneration. This has been shown in a mouse model to result from an accumulation of neurons harboring genomic damage, due to the inability of the mutant ATM protein to stimulate the p53 apoptotic cascade. Chk2 has also been shown to regulate apoptosis in a p53-dependent manner *in vitro* and *in vivo* in response to DNA damage (Seviour & Lin, 2010). Notably, the major recombinase of HR, Rad51, seems also to interact with p53, possibly serving as a tool for monitoring the extension as well the effectiveness of DNA repair processes (Henning & Sturzbecher Toxicology, 2003; Morita et

Consequently, impaired DDR appears to have dramatic effects on both tumorigenesis and premature ageing. At the molecular level, DDR impairment could be attributed to irregular interactions between the complexes involved in each process due to structural changes of DDR components resulting from either mutagenesis, modified post-translational modifications of altered protein levels, guiding equilibrium in favour of abnormal decisions.

chromosomal aberrations. Analysis of BRCA1 -/- mouse models, coupled with the study of human BRCA1 -/- tumors, has revealed prevalence for p53 mutations in these tumors, which is likely to be caused by the decrease in genomic stability associated with the defects in DNA repair. Overall, these and many other data suggest that the loss of cell cycle checkpoints confers a selection advantage to cells with DNA repair defects, thereby triggering tumorigenesis in genetically unstable cells. Moreover, an increase in genomic instability is significantly correlated with the metastatic potential of the tumor. Further studies are required to determine whether this involvement in metastasis is a result of acquired genetic mutations resulting from DNA repair defects, or whether other mechanisms are required for this process (Murphy & Moynahan, 2010).

DNA repair by the high fidelity mechanism of homologous recombination, termed as Homologous Recombinational Repair (HRR) is practically the only 'error free' repair mechanism of the cell and as it requires a sister chromatid, normally is active in late S and G2 phases of the cell cycle. HRR involves a compex network of recombination mediators and comediators. Defects in recombination mediators and co-mediators, leading to impaired HRR, are indicated as major contributors in carcinogenesis and particularly in breast cancer (reviewed by Pierce et al., 2001; Henning & Stuerzbecher, 2003; Murphy & Moynahan, 2010). Nevertheless, up to now not a single mutation in the coding region of RAD51, the central recombinase in the HRR pathway, has been found in many tumor types examined. However, many primary tumor cells and cancer cell lines express significantly modified levels of RAD51 (Maacke et al., 2000; Henning & Stuerzbecher 2003; Klein 2008) and at least partly, this misregulation in protein expression levels is correlated with the polymorphism G->C in the 5'untranslated region of rad51 mRNA, as shown in some cases of hereditary breast tumors with BRCA2 mutation. As extensively discussed in the excellent and comprehensive review of Schild & Wiese 2010, RAD51 overexpression presumably complements initial HRR defects, thereby limiting genomic instability during carcinogenic progression and may explain the high frequency of *TP53* mutations in human cancers, as wild-type p53 represses RAD51 expression. Notably, both positive and negative regulations of HRR are required to maintain genomic stability by precise repair and suppression of deleterious rearrangements.

#### **2.2 Genomic instability and ageing**

DNA damage is a prominent cause of cancer in frequently dividing cells since cell proliferation is a prerequisite for the manifestation of genetic changes as permanent mutations. In contrast, DNA damage in infrequently dividing cells is likely a prominent cause of ageing (Best, 2009). Therefore, in addition to its role in the maintenance of genomic integrity, the DDR has been hypothesized to play a critical role in organismal ageing. Supporting to this hypothesis is the observation that DNA repair disorders such as Werner's syndrome, Bloom's syndrome and Ataxia telangiectasia, syndromes also characterised by premature ageing and / or retarded growth, are often called "segmental progerias" ("accelerated ageing diseases"). Individuals suffering from such diseases appear elderly and suffer from ageing-related diseases at an abnormally young age, while not manifesting all the symptoms of old age.

Ageing, resulting from the accumulation of damage to molecules, cells, organs and tissues over time, is believed to be caused by two cellular processes: senescence and apoptosis.

#### **2.2.1 Senescence and DNA damage**

Senescence, a phenomenon describing the irreversible cease of cell division, was initially described by Hayflick and Moorhead in 1961 and includes replicative senescence and oncogene-induced senescence, both of which involve aspects of the DDR.

chromosomal aberrations. Analysis of BRCA1 -/- mouse models, coupled with the study of human BRCA1 -/- tumors, has revealed prevalence for p53 mutations in these tumors, which is likely to be caused by the decrease in genomic stability associated with the defects in DNA repair. Overall, these and many other data suggest that the loss of cell cycle checkpoints confers a selection advantage to cells with DNA repair defects, thereby triggering tumorigenesis in genetically unstable cells. Moreover, an increase in genomic instability is significantly correlated with the metastatic potential of the tumor. Further studies are required to determine whether this involvement in metastasis is a result of acquired genetic mutations resulting from DNA repair defects, or whether other

DNA repair by the high fidelity mechanism of homologous recombination, termed as Homologous Recombinational Repair (HRR) is practically the only 'error free' repair mechanism of the cell and as it requires a sister chromatid, normally is active in late S and G2 phases of the cell cycle. HRR involves a compex network of recombination mediators and comediators. Defects in recombination mediators and co-mediators, leading to impaired HRR, are indicated as major contributors in carcinogenesis and particularly in breast cancer (reviewed by Pierce et al., 2001; Henning & Stuerzbecher, 2003; Murphy & Moynahan, 2010). Nevertheless, up to now not a single mutation in the coding region of RAD51, the central recombinase in the HRR pathway, has been found in many tumor types examined. However, many primary tumor cells and cancer cell lines express significantly modified levels of RAD51 (Maacke et al., 2000; Henning & Stuerzbecher 2003; Klein 2008) and at least partly, this misregulation in protein expression levels is correlated with the polymorphism G->C in the 5'untranslated region of rad51 mRNA, as shown in some cases of hereditary breast tumors with BRCA2 mutation. As extensively discussed in the excellent and comprehensive review of Schild & Wiese 2010, RAD51 overexpression presumably complements initial HRR defects, thereby limiting genomic instability during carcinogenic progression and may explain the high frequency of *TP53* mutations in human cancers, as wild-type p53 represses RAD51 expression. Notably, both positive and negative regulations of HRR are required to maintain genomic

DNA damage is a prominent cause of cancer in frequently dividing cells since cell proliferation is a prerequisite for the manifestation of genetic changes as permanent mutations. In contrast, DNA damage in infrequently dividing cells is likely a prominent cause of ageing (Best, 2009). Therefore, in addition to its role in the maintenance of genomic integrity, the DDR has been hypothesized to play a critical role in organismal ageing. Supporting to this hypothesis is the observation that DNA repair disorders such as Werner's syndrome, Bloom's syndrome and Ataxia telangiectasia, syndromes also characterised by premature ageing and / or retarded growth, are often called "segmental progerias" ("accelerated ageing diseases"). Individuals suffering from such diseases appear elderly and suffer from ageing-related diseases at an

Ageing, resulting from the accumulation of damage to molecules, cells, organs and tissues over time, is believed to be caused by two cellular processes: senescence and apoptosis.

Senescence, a phenomenon describing the irreversible cease of cell division, was initially described by Hayflick and Moorhead in 1961 and includes replicative senescence and

mechanisms are required for this process (Murphy & Moynahan, 2010).

stability by precise repair and suppression of deleterious rearrangements.

abnormally young age, while not manifesting all the symptoms of old age.

oncogene-induced senescence, both of which involve aspects of the DDR.

**2.2 Genomic instability and ageing** 

**2.2.1 Senescence and DNA damage** 

Replicative senescence results from progressive shortening of telomeres with repeated rounds of cell replication. p53–dependent senescence serves as a tumor suppressor mechanism and is activated by the uncapping of critically shortened telomeres which are recognised as damaged DNA (Feldser and Greider, 2007). Recent studies argue that p53 can either activate or suppress senescence in cells, depending on their specific transcriptional activities and its interaction with partner molecules. As described by Vigneron & Vousden, 2010, the role of p53 in cell fate determination is even more complex as it involves epigenetic modifications of chromosomal DNA and relates chronic DDR signalling with increased levels of p53 acetylation. In addition to p53 other DNA damage response proteins like ATM have been associated with replicative senescence. ATM depletion in mice results in an increase in both chromosomal end-to-end fusion events and cell cycle-dependent telomere loss. These mice exhibit a premature ageing phenotype as defined by increased hair graying, alopecia and marked weight loss. Expression of mutant BRCA1 in mice also results in premature ageing, accompanied by an increase in cellular senescence and an increased susceptibility to certain cancers. The enhanced senescence observed in these mice may interfere with the fact that senescent cells have been noted to modify their tissue microenvironment. This phenomenon is thought to synergize with the accumulation of DNA damage over time to encourage cancer growth.

Oncogene-induced senescence can be induced by the overexpression of oncogenes by among others the induction of DNA damage resulting from both the generation of reactive oxygen species (ROS) and the hyper-replication of DNA. Both of these mechanisms activate the DNA damage response, which result in senescence by similar processes that induce replicative senescence.

#### **2.2.2 Apoptosis and DNA damage**

The accumulation of DNA damage can also lead to apoptosis. Activation of p53 by DNA damage and its role in the regulation of expression of pro-apoptotic proteins has been well documented. This is further supported by the fact that functional p53 is not detected to the majority of tumors. In cases of decline of the immune system an increase in p53 mediated apoptosis has been observed, linking ageing with apoptotic function. Moreover, constitutively activated p53 in mice also showed that, while high levels of p53 protect against cancer, it also accelerates the ageing process by reducing the mass of various tissues. The human condition Ataxia telangiectasia, which results from mutations in ATM, is associated with substantial neuro-degeneration. This has been shown in a mouse model to result from an accumulation of neurons harboring genomic damage, due to the inability of the mutant ATM protein to stimulate the p53 apoptotic cascade. Chk2 has also been shown to regulate apoptosis in a p53-dependent manner *in vitro* and *in vivo* in response to DNA damage (Seviour & Lin, 2010). Notably, the major recombinase of HR, Rad51, seems also to interact with p53, possibly serving as a tool for monitoring the extension as well the effectiveness of DNA repair processes (Henning & Sturzbecher Toxicology, 2003; Morita et al., 2010).

Consequently, impaired DDR appears to have dramatic effects on both tumorigenesis and premature ageing. At the molecular level, DDR impairment could be attributed to irregular interactions between the complexes involved in each process due to structural changes of DDR components resulting from either mutagenesis, modified post-translational modifications of altered protein levels, guiding equilibrium in favour of abnormal decisions.

Structure-Function Relationship of DNA Repair

or senescence decisions.

aspects of DDR pathways (Murphy & Moynahan, 2010).

**3.1 The BRCA1 structure – function relation paradigm** 

maintenance of genomic stability (Rowling et al., 2010).

Proteins: Lessons from BRCA1 and RAD51 Studies 109

The fundamental proteins involved in the HRR pathways are highly conserved in almost all organisms ranging from bacteria to human. The significance of this repair system is also indicated by the fact that defects in HRR cause human hereditary cancers as well as sporadic tumors. In many cases the dysfunction of proteins observed in many tumors helps to elucidate all three categories of the mechanisms mentioned above and to clarify different

Herein we will focus on current information regarding the structure – function relationship of two key players in regulation and performance of DSB repair – the most deleterious reported lesion of the genome – BRCA1 and RAD51. BRCA1 is a core component of many multi-molecular complexes involved in DNA damage detection, HR regulation, cell cycle regulation and genome transcription. RAD51, a key factor of HRR, replaces RPA on the produced single strands of damaged DNA and performs the search for homologous DNA strand and exchange in order to restore the damaged DNA sequence according to its sister chromatid. Moreover, RAD51 is implicated in telomere maintenance via ALT pathway and is also involved in mitochondrial DNA repair. Via at least its indirect interaction with BRCA1 as well as with direct p53 complex formation, RAD51 seems to be an interplayer responsible for communication between DNA repair effectiveness, cell replication, apoptosis

The Breast Cancer Susceptibility Gene 1 protein (BRCA1) is a multifunctional nuclear phosphoprotein of 1863 residues (220-240 kDa). BRCA1 was attributed the role of a tumor suppressor involved in multiple cellular functions (Starita & Parvin, 2003). Most of BRCA1 is located in the cell nucleus and is phosphorylated in a cell cycle-dependent manner by a number of kinases (reviewed by Ouchi, 2006). Depending on the position and the number of phosphorylated residues, BRCA1 participates in different multiprotein complexes performing diverse tasks. Therefore, BRCA1 has been implicated in a variety of functions required for the

Regarding DDR, BRCA1 has been attributed many roles in regulation of genome integrity including DNA replication, cell cycle checkpoint control, apoptosis, regulation of transcription, chromatin unfolding and protein ubiquitination. The ascribed functions are exerted through an extensive number of protein interactions reported (Jasin M. 2002 as cited in Murphy & Moynahan 2010). In brief, upon detection of chromatid relaxation due to breakage of both strands of the double helix of DNA, BRCA1 – being activated by ATR kinase – is recruited to the damage breakpoint assisting assembly of the BRCA2 – RAD51 complex in order to replace RPA and restore damage by the high fidelity process of HRR. In parallel, BRCA1 interaction with Fanconi Anemia (FA) and other complexes regulates G1/S and G2/M checkpoints. BRCA1 implication in cell cycle regulation is assisted by complex formation with BRCA1 interacting protein C-terminal helicase (BRIP1) and CtIP which are activated in S-phase by post-translational modifications. A graphical representation of the BRCA1 protein, including sites of both post-translational modifications and regions involved in protein-protein interactions, is depicted in Fig 1. The amino-terminal region of BRCA1 contains a distinct ~100aa RING finger motif involved in ubiquitin ligase activity and enables BRCA1 to mono- or poly-ubiquitinate cellular proteins. BARD1 (another RING and BRCT domain-containing protein) is the 'permanent' partner of BRCA1 in the formation of the ubiquitine ligase complex. Phosphorylation of specific residues of BRCA1 appears to regulate its participation in transcription regulation and ubiquitination of substrate proteins. As many different BRCA1 species are produced by alternative splicing of its mRNA, the phosphorylated residues each form contains may
