Double Strand Break Signaling in Health and Diseases

Signaling at DNA double strand breaks2.1 Sensing the DNA double strand breaks When DSBs are generated they are initially recognized by either the Ku70/Ku80 heterodimer, the Mre11-Rad50-NBS1 (MRN) complex or members of the PARP (PARP1/2) family of proteins (Ciccia & Elledge, 2010).The role of these protein sensors is to bind to and tether the DNA ends, thereby preventing further breakage as well as to recruit additional proteins that are required for DSB signaling and repair.The first group of proteins to be recruited to DSBs after initial sensing of the breaks belongs to the phosphatidylinositol-3kinase-like protein kinases family.These include ATM (ataxia-telangiectasia mutated), ATR (ATM and Rad3-related) and the catalytic subunit of DNA-PK known as DNA-PKcs.While ATM and DNA-PK respond only to DSBs, ATR also responds to single strand DNA breaks.www.intechopen.comDNA Repair and Human Health 48 Ku70/80 recruits DNA-PKcs to DSBs where it promotes DNA repair by non-homologous end joining whereas both PARP1/2 and the MRN complex lead to the recruitment of ATM which promotes homologous recombination.The MRN complex is recruited to DSBs in both a PARP1/2 dependent and independent manner (Ciccia & Elledge, 2010).ATM is a central component of the cellular response to DSBs and is predicted to have several hundred downstream targets many of which play a role in the DNA damage response (Matsuoka et al, 2007).Under normal conditions ATM is in a homodimeric form.Following DNA damage, it becomes autophosphorylated at Ser1981 and dissociates into its monomeric form and binds the damaged DNA (Bakkenist & Kastan, 2003).There, it leads to the phosphorylation of many downstream targets involved in the DSB response.The phosphorylation of ATM at Ser1981 and its initial binding to DSBs depend upon the MRN complex.MRN consists of three different proteins Mre11, NBS1 and Rad50.Mre11 is a DNA nuclease that interacts with both Rad50 and NBS1 as well as with other Mre11 molecules to form dimers.When paired with the other components of the MRN complex, Mre11 can have both double strand DNA exonuclease activity and single strand DNA endonuclease activity (D'Amours & Jackson, 2002).In addition, Mre11 has two DNA binding sites and intrinsic DNA binding activity.Rad50 is a protein that bears homology to the structural maintenance of chromosome (SMC) family.It is an ATPase and is needed for tethering of DNA ends together during the process of DNA repair.NBS1 has a fork-head-associated (FHA) domain and two BRCT (BRCA1-tandem repeats) domains at its N-terminus which are used to recognize phospho-threonine and phospho-serine residues respectively in Ser-X-Thr motifs.These domains allow RAD50 to interact with several DNA damage signaling proteins following DSB formation.NBS1 also contains a nuclear localization signal (NLS) that allows the translocation of the MRN complex into the nucleus following DNA damage (Lamarche et al, 2010).NBS1 interacts with ATM thereby leading to its recruitment to DSBs.There, ATM is involved in one of the very early response to the formation of DSBs, mainly the phosphorylation of histone variant H2AX on Ser139 to form γ-H2AX (Figure 1).This phosphorylation can extend over a megabase of DNA from the site of DSBs (Modesti & Kanaar, 2001).The formation of γ-H2AX at the sites of DSBs is key for the recruitment of many effector proteins to the break sites including the regulators of cell cycle checkpoint and DNA repair 53BP1, BRCA1 and Rad51 (Bohgaki et al, 2010).The accumulation of γ-H2AX and other DNA damage signaling and repair proteins at the sites of ionizing-radiation induced breaks leads to the formation of microscopically distinct foci known as IR-induced nuclear foci (IRIFs) which can be used experimentally to study IR-induced DNA damage signaling and repair.The ability of H2AX to recruit DNA damage proteins under normal physiological conditions is hampered by its constitutive phosphorylation at Tyr142 by William's syndrome transcription factor (WSTF).This phosphorylation suppresses the ability of H2AX to recruit downstream signaling and effectors of the DNA damage response to the breaks.However, following DNA damage Tyr142 residue is dephosphorylated by the EYA protein phosphatases (Cook et al, 2009).γ-H2AX recruits MDC1, a mediator of the DNA damage response that functions as an adaptor to recruit downstream effector proteins to the break sites.MDC1 has two BRCT domains at its C-terminus and one FHA domain at its N-terminus that allow it to recognize and interact with other DNA damage response proteins (Stewart et al, 2003).The BRCT domains of MDC1 can recognize phosphorylation sites and were shown to mediate MDC1 binding to γ-H2AX.www.intechopen.com

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