Ilaria Ceppi1,2, Elda Cannavo1, Hélène Bret3, Rosa Camarillo4,5, Francesca Vivalda6, Roshan Singh Thakur1, Amador Romero-Franco4,5, Alessandro A. Sartori6, Pablo Huertas4,5, Raphaël Guérois3 and Petr Cejka1,2 1Institute for Research in Biomedicine, Faculty of Biomedical Sciences, Università della Svizzera italiana (USI), Bellinzona 6500, Switzerland; 2Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), Zürich 8093, Switzerland; 3Institute for Integrative Biology of the Cell (I2BC), Commissariat à l'Energie Atomique, Centre National de la Recherche Scientifique, Université Paris-Sud, Université Paris-Saclay, Gif-sur-Yvette 91190, France; 4Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Sevilla 41080, Spain; 5Centro Andaluz de Biología Molecular y Medicina Regenerativa (CABIMER), Universidad de Sevilla-Consejo Superior de Investigaciones Científicas-Universidad Pablo de Olavide, Sevilla 41092, Spain; 6Institute of Molecular Cancer Research, University of Zürich, Zürich 8057, Switzerland Corresponding author: petr.cejkairb.usi.ch Abstract

DNA double-strand break (DSB) repair is initiated by DNA end resection. CtIP acts in short-range resection to stimulate MRE11–RAD50–NBS1 (MRN) to endonucleolytically cleave 5′-terminated DNA to bypass protein blocks. CtIP also promotes the DNA2 helicase–nuclease to accelerate long-range resection downstream from MRN. Here, using AlphaFold2, we identified CtIP-F728E-Y736E as a separation-of-function mutant that is still proficient in conjunction with MRN but is not able to stimulate ssDNA degradation by DNA2. Accordingly, CtIP-F728E-Y736E impairs physical interaction with DNA2. Cellular assays revealed that CtIP-F728E-Y736E cells exhibit reduced DSB-dependent chromatin-bound RPA, impaired long-range resection, and increased sensitivity to DSB-inducing drugs. Previously, CtIP was shown to be targeted by PLK1 to inhibit long-range resection, yet the underlying mechanism was unclear. We show that the DNA2-interacting region in CtIP includes the PLK1 target site at S723. The integrity of S723 in CtIP is necessary for the stimulation of DNA2, and phosphorylation of CtIP by PLK1 in vitro is consequently inhibitory, explaining why PLK1 restricts long-range resection. Our data support a model in which CDK-dependent phosphorylation of CtIP activates resection by MRN in S phase, and PLK1-mediated phosphorylation of CtIP disrupts CtIP stimulation of DNA2 to attenuate long-range resection later at G2/M.

Received August 4, 2022. Accepted January 12, 2023.