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DNA double-strand break repair

Radiotherapy using ionizing radiation is the most employed form of therapy against cancer. This treatment shows severe cytotoxic effects on actively growing cancer cells by inducing DNA damage such as DSBs. DSBs in human cells are repaired by non-homologous end joining (NHEJ) or homologous recombination. NHEJ repair DSBs during G1 to early S phase, while HR is used in late S to G2 when newly replicated chromosomes are available as homologous templates.  NHEJ leads to the joining of DNA broken ends in a mechanism dependent on the activity of DNA-dependent protein kinase and the Ku70/Ku80 proteins. This pathway promotes rapid DSB repair but can lead to mutations in the DNA. The HR pathway is a faithful way of repairing DSBs and is the major mechanism for the error-free homology-directed repair of DSBs. HR utilizes the presence of the sister chromatid as a template to repair DNA double-strand breaks. HR can be divided in three major steps: the presynaptic stage involves RAD51 nucleoprotein filament assembly and strand stabilization, the synaptic stage is characterized by strand invasion and branch migration, and the postsynaptic stage includes Holliday junction formation and resolution. In eukaryotes, HR is initiated when the MRE11-RAD50-NBS1 (and the associated proteins such as CtIP) complex enters DNA at a DSB. MRE11 is a conserved protein with a N-terminal nuclease domain that has both 3'-to-5' exonuclease and endonuclease activity. The nuclease travels along the DNA leading to the resection of the DSB and the formation of single-stranded DNA tails. These are then bound by the ssDNA binding protein RPA, which must be displaced by a recombination mediator (such as BRCA2) and then acted upon by the RAD51 recombinase (an homolog of E. coli RecA) to form a nucleoprotein filament catalyzing the invasion of the single-strand DNA into an intact sister chromatid to create a D-loop structure . Following DNA synthesis, the Holliday junction can be moved along the DNA (a process called branch migration) and cleaved by a resolvase complex to complete the exchange of genetic information and DNA repair. Alternatively, synthesis-dependent strand annealing can occur where the extended DNA tails can reanneal with a complementary sequence leading to DSB repair. Our lab focuses mostly on homologous recombination and we are dissecting each step from DNA resection to strand invasion. 

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