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  1. Evaluation Summary:

    This manuscript will be of interest for scientists interested in cell cycle, DNA repair, and genome stability reporting the unexpected discovery that the DNA-dependent protein kinase (DNA-PK) is required for DSB resection in G0 cells, whereas it is known and confirmed here that it inhibits resection in G1 and G2 cells. This finding has important implications for the clinical application of DNA-PK-targeted inhibitors. The data are of high quality and derive from two independent cell lines, genetic requirements were mostly established by gene knockouts, and the latest genome-wide sequencing techniques were applied to measure resection tracts. The key claims of the manuscript are supported by the data presented by the authors; however, further validations are needed to strengthen the quality and impact of the paper.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  2. Joint Public Review:

    Fowler et al. report on hits of a CRISPR-Cas9 FACS-based screen for chromatin associated RPA in quiescent murine pre-B cells that lack DNA ligase 4 (to prevent NHEJ) identifying component required for DSB resection or inhibiting DSB resection in G0 cells. The screen is well validated by previously published results (Chen et al. 2021 eLife) and controls reported in this manuscript. Unexpectedly, the authors identify all components of the DNA-dependent protein kinase complex, Ku70, Ku80 and DNA-PK catalytic subunit, as being required for DSB resection in G0 cells. This was surprising as DNA PK inhibits DSB resection in G1 and G2 cells, which was confirmed in this work. The results are verified by END-seq, showing strand specificity, and processing is dependent on MRE1 and CtIP, assuring that the RPA signal reports on DSB resection. Independent confirmation is derived from results with FBXL12, an DNA-PK-specific ubiquitin E3 ligase, which leads to DNA-PK turnover and counteracts DSB resection is G0 cells. The genetic dependencies were established by gene knockouts, and key results confirmed in a human cell line (MCF-10A). The specificity of the effect for DNA-PK was confirmed using inhibitors against ATM, which showed no effect on DSB resection, whereas DNA-PK inhibitors mimicked the genetic dependency.

    The manuscript is well structured and describes an interesting finding for the DNA repair community, speculating that DSBs repair in quiescent cells functions differently than in cycling cells. This has implications on how non-cycling cells in the body, such as neurons, could handle DNA damage, but remains to be validated in the corresponding model. In addition, more experiments need to be performed to adequately support the key conclusions of the manuscript with respect to the applicability in human cells and with regards to the distinction between resection in G0 and G1 cells. Some data are lacking a precise description of the methods which need to be extended.

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