Human U2OS DNApk GLASS-ChIP LC-MS/MS

ABSTRACT: The Mre11-Rad50-Nbs1 (MRN) complex recognizes and processes DNA double-strand breaks for homologous recombination by performing short-range removal of 5ʹ strands. Endonucleolytic processing by MRN requires a stably bound protein at the break site—a role we postulate is played by DNA-dependent protein kinase (DNA-PK) in mammals. Here we interrogate sites of MRN-dependent processing by identifying sites of CtIP association and by sequencing DNA- PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated most efficiently when DNA-PK is catalytically blocked, yielding products within 200 bp of the break site, whereas DNA-PK products in the absence of kinase inhibition show greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA- PK and Mre11 binding, showing that both complexes bind to DNA ends before release of DNA- PK-bound products. These results support a sequential model of double-strand break repair involving collaborative interactions between homologous and non-homologous repair complexes.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Cell Culture

SUBMITTER: Nicolette Ender  

LAB HEAD: Tanya T. Paull

PROVIDER: PXD045033 | Pride | 2023-10-24

REPOSITORIES: Pride