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Phosphorylated CtIP bridges DNA to promote annealing of broken ends

Robin Öz, Sean M. Howard, Rajhans Sharma, Hanna Törnkvist, Ilaria Ceppi, Sriram KK, Erik Kristiansson, Petr Cejka, Fredrik Westerlund
2020 Proceedings of the National Academy of Sciences of the United States of America  
The early steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The roles of these proteins in nucleolytic DSB resection are well characterized, but their role in bridging the DNA ends for efficient and correct repair is much less explored. Here we study the binding of phosphorylated CtIP, which promotes the endonuclease activity of MRN, to single long (∼50 kb) DNA molecules using nanofluidic channels and
more » ... idic channels and compare it to the yeast homolog Sae2. CtIP bridges DNA in a manner that depends on the oligomeric state of the protein, and truncated mutants demonstrate that the bridging depends on CtIP regions distinct from those that stimulate the nuclease activity of MRN. Sae2 is a much smaller protein than CtIP, and its bridging is significantly less efficient. Our results demonstrate that the nuclease cofactor and structural functions of CtIP may depend on the same protein population, which may be crucial for CtIP functions in both homologous recombination and microhomology-mediated end-joining.
doi:10.1073/pnas.2008645117 pmid:32817418 fatcat:wulk64dvjfb4zotdcclpa3pane