DNA Ends Alter the Molecular Composition and Localization of Ku Multicomponent Complexes

Guillaume Adelmant,Anne S Calkins,Brijesh K Garg,Joseph D Card,Manor Askenazi,Alex Miron,Bijan Sobhian,Yi Zhang,Yoshihiro Nakatani,Pamela A Silver,J Dirk Iglehart,Jarrod A Marto,Jean-Bernard Lazaro

doi:10.1074/mcp.m111.013581

Guillaume Adelmant, Anne S Calkins + Show 11 more

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https://doi.org/10.1074/mcp.m111.013581

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The Ku heterodimer plays an essential role in non-homologous end-joining and other cellular processes including transcription, telomere maintenance and apoptosis. While the function of Ku is regulated through its association with other proteins and nucleic acids, the specific composition of these macromolecular complexes and their dynamic response to endogenous and exogenous cellular stimuli are not well understood. Here we use quantitative proteomics to define the composition of Ku multicomponent complexes and demonstrate that they are dramatically altered in response to UV radiation. Subsequent biochemical assays revealed that the presence of DNA ends leads to the substitution of RNA-binding proteins with DNA and chromatin associated factors to create a macromolecular complex poised for DNA repair. We observed that dynamic remodeling of the Ku complex coincided with exit of Ku and other DNA repair proteins from the nucleolus. Microinjection of sheared DNA into live cells as a mimetic for double strand breaks confirmed these findings in vivo.

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The Ku heterodimer is composed of 70 kDa (Ku70) and 86 kDa (Ku86) subunits that associate in stoichiometric quantities and bind DNA ends with high affinity as a first step in the nonhomologous end-joining DNA repair pathway
Ku Switches From a Ribonucleo- to a Deoxyribonucleo-Protein Complex in Response to UV-Induced DNA Damage— DNA-PK is best known for its role in response to double strand breaks (DSB) resulting from ionizing radiation, recent evidence has shown that its catalytic subunit
We recently reported that the Ku heterodimer, which is essential for the recruitment of DNA-PKcs to sites of DNA damage, re-localizes from the nucleolus to the nucleoplasm after UV irradiation [34]

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Introduction

The Ku heterodimer (hereafter referred to as “Ku”) is composed of 70 kDa (Ku70) and 86 kDa (Ku86) subunits that associate in stoichiometric quantities and bind DNA ends with high affinity as a first step in the nonhomologous end-joining DNA repair pathway. These observations, taken together with recent findings [35], suggest that Ku protein complexes exit the nucleolus in response to UV-induced DNA damage, and that this change in localization is accompanied by remodeling of Ku protein associations from largely RNA-binding to primarily DNA-binding proteins.

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