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Double-stranded DNA break polarity skews repair pathway choice during intrachromosomal and interchromosomal recombination

Published on Mar 13, 2018in Proceedings of the National Academy of Sciences of the United States of America9.58
· DOI :10.1073/pnas.1720962115
Alexanda K. Ling4
Estimated H-index: 4
(U of T: University of Toronto),
Clare C. So3
Estimated H-index: 3
(U of T: University of Toronto)
+ 3 AuthorsAlberto Martin26
Estimated H-index: 26
(U of T: University of Toronto)
Sources
Abstract
Activation-induced cytidine deaminase (AID) inflicts DNA damage at Ig genes to initiate class switch recombination (CSR) and chromosomal translocations. However, the DNA lesions formed during these processes retain an element of randomness, and thus knowledge of the relationship between specific DNA lesions and AID-mediated processes remains incomplete. To identify necessary and sufficient DNA lesions in CSR, the Cas9 endonuclease and nickase variants were used to program DNA lesions at a greater degree of predictability than is achievable with conventional induction of CSR. Here we show that Cas9-mediated nicks separated by up to 250 nucleotides on opposite strands can mediate CSR. Staggered double-stranded breaks (DSBs) result in more end resection and junctional microhomology than blunt DSBs. Moreover, Myc - Igh chromosomal translocations, which are carried out primarily by alternative end joining (A-EJ), were preferentially induced by 5′ DSBs. These data indicate that DSBs with 5′ overhangs skew intrachromosomal and interchromosomal end-joining toward A-EJ. In addition to lending potential insight to AID-mediated phenomena, this work has broader carryover implications in DNA repair and lymphomagenesis.
  • References (50)
  • Citations (4)
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Abstract The nonhomologous end-joining (NHEJ) pathway is the primary repair pathway for DNA double strand breaks (DSBs) in humans. Repair is mediated by a core complex of NHEJ factors that includes a ligase (DNA Ligase IV; L4) that relies on juxtaposition of 3' hydroxyl and 5' phosphate termini of the strand breaks for catalysis. However, chromosome breaks arising from biological sources often have different end chemistries, and how these different end chemistries impact the way in which the cor...
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Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S–S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA po...
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DNA double-strand breaks (DSBs) are known to be powerful inducers of homologous recombination (HR), but single-strand breaks (nicks) have also been shown to trigger HR. Both DSB- and nick-induced HR ((nick)HR) are exploited in advanced genome-engineering approaches based on the bacterial RNA-guided nuclease Cas9. However, the mechanisms of (nick)HR are largely unexplored. Here, we applied Cas9 nickases to study (nick)HR in mammalian cells. We find that (nick)HR is unaffected by inhibition of maj...
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