RT Journal Article
SR Electronic
T1 Genome-wide analysis of DNA-PK-bound MRN cleavage products supports a sequential model of DSB repair pathway choice
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.12.07.519501
DO 10.1101/2022.12.07.519501
A1 Rajashree A. Deshpande
A1 Alberto Marin-Gonzalez
A1 Taekjip Ha
A1 Tanya T. Paull
YR 2022
UL http://biorxiv.org/content/early/2022/12/07/2022.12.07.519501.abstract
AB 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 the sites of MRN-dependent processing by isolating and sequencing DNA-PK-bound DNA fragments that are products of MRN cleavage. These intermediates are generated with highest efficiency 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 much greater dispersal. Use of light-activated Cas9 to induce breaks facilitates temporal resolution of DNA-PK and Mre11 binding, showing that Mre11 and DNA-PK both 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.Competing Interest StatementThe authors have declared no competing interest.