Double stranded breaks

DSBs are of greater concern than SSBs, as they can increase the chance of chromosome breakage, neurodegeneration, immunodeficiency and cancer predisposition (Jackson and Bartek, 2009). DSBs can arise during normal physiological processes, for example during replication, when an unrepaired SSB meets the progression replication fork, it is converted into a DSB. Therefore during this time the DNA is stringently monitored for DSBs (and SSBs) by DNA glycosylase, which initiates base excision repair (Almeida and Sobol, 2007, Hitomi et al., 2007, Jacobs and Schar, 2012). Damaged DNA activates processes which halt cell cycle progression, allowing time for DNA repair before the cells continue to go through their cycle. Extrinsically, DSBs can also be induced by: radiation or radiometric compounds such as bleomycin, alkylating agents (e.g. methyl methane sulphate) and topoisomerase II inhibitors (e.g. etoposide), as described in (section 1.5.2) (Vanankeren et al., 1988, Mirabelli et al., 1985).

Introduction The two major pathways involved in sensing and repairing DSBs are homologous recombination (HR) and non-homologous end-joining (NHEJ) (Hartlerode and Scully, 2009, Pardo et al., 2009).

1.6.1.1Non-homologous End-Joining (NHEJ) repair

Non-homologous End-Joining (NHEJ) repair is active throughout the cell cycle but with increased activity as the cells progress through G0/G1 to S and G2/M phase. NHEJ directly ligates broken ends back together without the use of a homologous template to guide repair, therefore this method is error prone and can generate small insertions or deletions in the repaired DNA (Riches et al., 2008, Hartlerode and Scully, 2009, Pardo et al., 2009).

During NHEJ, the first events involve the DNA binding proteins Ku70 and Ku80 associating with the DNA break site (Walker et al., 2001). The Ku70/ Ku80 heterodimer binds each exposed end of the DNA terminus of the break site and its function is to aid the alignment of DNA strands, protect them from degradation plus to prepare the strands for ligation (van Gent and van der Burg, 2007, Weterings and Chen, 2008). The association of Ku70/ Ku80 with the DNA also attracts DNA protein kinase (DNA-PK) to the break site, which later plays a role in both the processing and ligation of DNA. The processing of DNA is performed by the nuclease Artemis, which trims the DNA followed by the DNA ligase IV/XRCC4 complex, which ligates the blunt ends (Yannone et al., 2008). Once the ligation has occurred, the repair process is completed (Figure 1.8A).

1.6.1.2Homologous recombination repair

This mechanism is more complex than NHEJ, but it is more accurate and less prone to errors as it utilises a homologous sister chromatid template. However, this means that the HR repair mechanism is only active during the S and G2/M phase of the cell cycle, when sister chromatids are available (these are identical copies of chromatids generated by DNA replication) (Filippo et al., 2008).

Recognition of DSBs, by the MRN complex, happens within minutes after the damage has occurred. The MRN complex consists of meiotic recombination 11 (MRE11), RAD50 and Nibrin (NBN) (Lavin, 2007). The MRN complex plays a key role in sensing, signalling and promoting repair of DNA breaks. In the case where repair mechanisms are triggered, the DSBs ends are first processed by resection from 5’ to 3’ ends to produce 3’ single strand tails. This is initiated by MRN complex and CtBP-interacting protein (CtIP). With the aid of Rad51, the 3’ ssDNA then undergo strand invasion, branch migration and DNA synthesis.

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Following these processes, the crossover DNAs are resolved and the initial broken DNA are repaired (Figure 1.8B).

Figure 1.8 Mammalian DNA Double Stranded Breaks Repair. (A) NHEJ repair, DSB recognised by KU70/KU80, processed by DNA-PK and ARTEMIS followed by relegation by XRCC4/Lig4 complex. (B) HR repair, DSB recognised by MRN complex followed by resection, strand invasion and extension mediated by RPA and RAD51. After extension, the strands are resolved. Adapted from Lans et al. Epigenetics & Chromatin 2012 5:4

Besides triggering repair, once MRN is associated with the exposed ends of DNA, its NBN component activates the catalytic function of a kinase called ataxia telangiectasia mutated (ATM) (Horejsi et al., 2004, Costanzo et al., 2004). Additionally, it retains the ATM at the site of the DSB, which allows for signal amplification and downstream ATM response

Introduction (Horejsi et al., 2004, Berkovich et al., 2007). When ATM is activated, it phosphorylates targets such as a histone variant called H2AX, as well as carrying out autophosphorylation of itself. Thus, ATM plays a central role in DSBs response where it can phosphorylate other proteins such as Chk2, BRCA1 and p53, involved in repair, cell cycle checkpoint and apoptotic responses (Jowsey et al., 2007, Buscemi et al., 2004, Stiff et al., 2004)(Figure 1.9).

Figure 1.9 Overview of DSB response. A schematic diagram demonstrating the signalling cascade of DSB response. DNA damage is detected by sensor proteins (MRN complex: NBN, Rad50 & Mre11), where signals are transduced by ATM which activate Chk2 (left) or ATR which activates Chk1 (right). Activation of Chk2 or Chk1 will then activate p53 signalling response. (http://www.intechopen.com/books/senescence-and-senescence-related- disorders/molecular-mechanisms-of-cellular-senescence).

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1.6.1.3Pathological relevance of NBN

Nijmegen Breakage Syndrome (NBS) is a rare autosomal recessive disease where the NBN gene is mutated (Weemaes et al., 1981). Patients with NBS display facial dysmorphism, microcephaly and growth retardation. NBS has been documented to be associated with increased cancer incidence, in particular B-cell non-Hodgkin’s lymphoma (Paulli et al., 2000, Hama et al., 2000, Kruger et al., 2007). NBN mutations have been reported in gastrointestinal cancer, breast cancer and lymphoblastic leukemia (Ebi et al., 2007, Bogdanova et al., 2008, Varon et al., 2001). Moreover, NBN is also often found to be mutated in many GBM cases (Watanabe et al., 2009) and in some cases of MB patients (Distel et al., 2003, Bakhshi et al., 2003, Huang et al., 2008). In the study by Huang et al., they screened 42 MB patients and discovered that 7 carried NBN mutations. They then hypothesised that NBN mutations might be involved in MB pathogenesis (Huang et al., 2008).

Introduction