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Phosphorylated CtIP bridges DNA to promote annealing of broken ends

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...

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Autores principales: Öz, Robin, Howard, Sean M., Sharma, Rajhans, Törnkvist, Hanna, Ceppi, Ilaria, KK, Sriram, Kristiansson, Erik, Cejka, Petr, Westerlund, Fredrik
Formato: Online Artículo Texto
Lenguaje:English
Publicado: National Academy of Sciences 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474685/
https://www.ncbi.nlm.nih.gov/pubmed/32817418
http://dx.doi.org/10.1073/pnas.2008645117
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author Öz, Robin
Howard, Sean M.
Sharma, Rajhans
Törnkvist, Hanna
Ceppi, Ilaria
KK, Sriram
Kristiansson, Erik
Cejka, Petr
Westerlund, Fredrik
author_facet Öz, Robin
Howard, Sean M.
Sharma, Rajhans
Törnkvist, Hanna
Ceppi, Ilaria
KK, Sriram
Kristiansson, Erik
Cejka, Petr
Westerlund, Fredrik
author_sort Öz, Robin
collection PubMed
description 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 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.
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spelling pubmed-74746852020-09-18 Phosphorylated CtIP bridges DNA to promote annealing of broken ends Öz, Robin Howard, Sean M. Sharma, Rajhans Törnkvist, Hanna Ceppi, Ilaria KK, Sriram Kristiansson, Erik Cejka, Petr Westerlund, Fredrik Proc Natl Acad Sci U S A Biological Sciences 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 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. National Academy of Sciences 2020-09-01 2020-08-19 /pmc/articles/PMC7474685/ /pubmed/32817418 http://dx.doi.org/10.1073/pnas.2008645117 Text en Copyright © 2020 the Author(s). Published by PNAS. http://creativecommons.org/licenses/by/4.0/ https://creativecommons.org/licenses/by/4.0/This open access article is distributed under Creative Commons Attribution License 4.0 (CC BY) (http://creativecommons.org/licenses/by/4.0/) .
spellingShingle Biological Sciences
Öz, Robin
Howard, Sean M.
Sharma, Rajhans
Törnkvist, Hanna
Ceppi, Ilaria
KK, Sriram
Kristiansson, Erik
Cejka, Petr
Westerlund, Fredrik
Phosphorylated CtIP bridges DNA to promote annealing of broken ends
title Phosphorylated CtIP bridges DNA to promote annealing of broken ends
title_full Phosphorylated CtIP bridges DNA to promote annealing of broken ends
title_fullStr Phosphorylated CtIP bridges DNA to promote annealing of broken ends
title_full_unstemmed Phosphorylated CtIP bridges DNA to promote annealing of broken ends
title_short Phosphorylated CtIP bridges DNA to promote annealing of broken ends
title_sort phosphorylated ctip bridges dna to promote annealing of broken ends
topic Biological Sciences
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7474685/
https://www.ncbi.nlm.nih.gov/pubmed/32817418
http://dx.doi.org/10.1073/pnas.2008645117
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