DSB Repair by Homologous Recombination
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DSB Repair by Homologous Recombination

DSBs (DNA Double-Strand Breaks) are extremely genotoxic DNA lesions that pose problems for DNA Transcription, Replication and Segregation. Improper processing of DSBs gives rise to chromosomal instability that can result in carcinogenesis through activation of proto-oncogenes or inactivation of tumor suppressor genes. DSBs are caused by exogenous sources such as UV Radiations, Mechanical Stress, Ionizing Radiation, and by endogenous sources such as radicals generated during metabolic processes and Genotoxic Chemicals. The adverse effects of DSBs have triggered the evolution of multiple pathways for their repair. Eukaryotes have evolved two distinct pathways of DSB Repair-HR (Homologous Recombination), NHEJ (Non-Homologous End Joining). The fundamental difference between these pathways is their dependence on DNA Homology and accuracy of repair. In general, Homologous Recombination ensures an accurate repair by using the undamaged Sister Chromatid or Homologous Chromosome as a template. DNA End-Joining, on the other hand, uses no or extremely limited sequence homology to rejoin ends in a manner that need not be error free. Homologous recombination includes single-strand annealing, gene conversion and break-induced replication (Ref. 1 & 2).

Homologous recombination is mediated through the Rad52 family of proteins. Rad52 interacts and co-localizes with Rad51, induces Rad51 activity, binds preferentially to DSBs and protects them from exonuclease activity. The initial cellular response to DSBs is mediated through ATM (Ataxia Telangiectasia Mutated) and MRN Complex (Mre11-Rad50-NBS1). The ATM protein is a serine-threonine kinase and a member of the PIKK (Phosphoinositide 3-Kinase-Like Kinase) family, which also includes DNA-PK (DNA Protein Kinase) and ATR (AT and Rad3-related protein). These proteins are associated with DNA damage surveillance, control of cell cycle checkpoints, and cell growth regulation. In response to DSBs, ATM in effect “raises the alarm” to DNA damage, phosphorylating many downstream effector targets such as p53, H2AX, Mdm-2, BRCA11, c-Abl, Chk-2, 53BP1, and SMC-1 (Structural Maintenance Of Chromosome-1). This swift response acts to halt the cell cycle and stop DNA replication ATM; then facilitates DNA repair or triggers apoptosis based on the severity of the damage. In cells of the central nervous system, ATM appears to play an atypical role in that ATM knock-out astrocytes are not radiosensitive and ATM is instead required for apoptosis (Ref. 3). The MRN complex provides paradigm- shifting results of exceptional biomedical interest. MRN is among the earliest respondents to DSBs, and MRN mutations causes’ human cancer predisposition diseases Nijmegen breakage syndrome and ATLD (Ataxia Telangiectasia-Like Disorder). MRNs 3-protein multidomain composition promotes its central architectural, structural, enzymatic, sensing, and signaling functions in DSB responses. To organize the MRN complex, the Mre11 ;exonuclease directly binds NBS1 (Nijmegen Breakage Syndrome 1) DNA, and Rad50. Rad50, which is a SMC related protein, employs it’s ABC (ATP-Binding Cassette) ATPase, Zn hook, and coiled coils to bridge DSBs and facilitate DNA end processing by Mre11. MRN has 3 coupled critical roles in DSB sensing, stabilization, signaling, and effector scaffolding: (1) expeditious establishment of protein--nucleic acid tethering scaffolds for the recognition and stabilization of DSBs; (2) initiation of DSB sensing, cell-cycle checkpoint signaling cascades, and establishment of epigenetic marks via the ATM kinase; and (3) functional regulation of chromatin remodeling in the vicinity of a DSB (Ref. 4).

Subsequent steps of DSB repair through Homologous recombination include DNA-end recognition, possibly by Rad52, and nucleolytic processing of the broken ends of DNA into 3-end single-stranded DNA. This single-stranded DNA is bound by the Rad51 protein which mediates crucial steps in the reaction-the search for a homologous duplex template DNA and the formation of joint molecules between the broken DNA ends and the repair template (Ref. 5). Rad51 is phosphorylated by c-Abl and this response contributes to the down-regulation of Rad51 activity in ATP-dependent DNA strand exchange reactions (Ref. 6). Rad51 protein assembles with single-stranded DNA to form the helical Nucleoprotein filament that promotes DNA strand exchange, a basic step of homologous recombination. Rad54 protein interacts with this Rad51 nucleoprotein filament and stimulates its DNA pairing activity, suggesting that Rad54 protein is a component of the nucleoprotein complex involved in the DNA homology search. The binding of Rad54 protein significantly stabilizes the Rad51 nucleoprotein filament formed on either single-stranded DNA or double-stranded DNA. The Rad54-stabilized nucleoprotein filament is more competent in DNA strand exchange and acts over a broader range of solution conditions. The co-assembly of an interacting partner with the Rad51; nucleoprotein filament represents a novel means of stabilizing the biochemical entity central to homologous recombination, and reveals a new function of Rad54 protein (Ref. 7). The roles played by BRCA1 and BRCA2 in DSB repair by homologous recombination appear to be somewhat different. Despite the apparent dissimilarity in protein sequence and structure, both BRCA1 and BRCA2 have common biological functions. Their levels are highest during S phase, which is suggestive of functions during DNA replication. Both are localised to the nucleus in somatic cells, where they co-exist in characteristic subnuclear foci that redistribute following DNA damage. BRCA2 controls the intracellular transport and function of Rad51. In BRCA2-deficient cells, Rad51 (which does not contain a consensus nuclear localisation signal) is inefficiently transported into the nucleus, which suggests that one function of BRCA2 is to move Rad51 from its site of synthesis to its site of activity. In addition, BRCA2 also appears to control the enzymatic activity of Rad51Addition of peptides containing the Rad51-binding BRC repeat BRC3, BRC4 or BRC7 inhibits nucleoprotein filament formation. BRCA2 might not directly control Rad51 function, since the stoichiometry of their interaction is possibly low and does not appear to be greatly altered following DNA damage (Ref. 8 & 9).

Once the homologous DNA has been identified, the subsequent step leads to Strand Invasion and D-loop formation. Damaged DNA strand invades the undamaged DNA duplex in a process referred to as DNA strand exchange. Upon joint-molecule formation and DNA synthesis, branched DNA structures called Holliday junctions can form as late intermediates in homologous recombination. Holliday junctions can slide, or branch-migrate, along the joined DNAs. Branch migration extends the heteroduplex DNA region between identical recombination partners and might thereby provide a mechanism to prevent recombination between repetitive sequences that are dispersed throughout the genome. A DNA Polymerase then extends the 3 end of the invading strand and subsequent ligation by DNA Ligase-I yields a hetero-duplexed DNA structure. Completion of recombination requires the Resolution of Holliday junctions, in order to separate the recombining partners. One well-characterized way of resolving Holliday junctions requires the enzymatic action of a Resolvase.This recombination intermediate is resolved and the precise, error-free correction of the DSB is complete (Ref.10 & 11).

Ionizing radiation and interstrand DNA crosslinking compounds provide important treatments against cancer due to their extreme genotoxicity for proliferating cells. Both the efficacies of such treatments and the mutagenic potential of these agents modulate the ability of cells to repair the inflicted DNA damage (Ref.1). DNA-PKcs may inhibit alternative DNA repair pathways by regulating access of DNA ends to repair factors. Blocking DNA-PKs kinase activity inhibits both DSB-induced and spontaneous HR inhibits telomere maintenance and inhibits the repair of damage from Topoisomerase-II positions. BRCA2 which acts as a novel client protein for HSP90 and 17-AAG (17-Allylamino-17-Demethoxygeldanamycin) causes the degradation of BRCA2 and in turn altered the behavior of Rad51, a critical protein for HR pathway of DSB repair. Thus, 17-AAG inhibits the HR repair process and could provide a new therapeutic strategy to selectively result in higher tumor cell killing (Ref.12 & 13).