DNA double-strand breaks (DSBs) are particularly deleterious lesions which, if left unrepaired, can lead to cell death, or if repaired aberrantly, can lead to oncogenic chromosomal translocations and deletions (Jackson and Bartek, 2009). Eukaryotic cells utilize two main mechanisms of DSB repair: non-homologous end joining (NHEJ), where the broken DNA ends are ligated together with minimal processing of the DNA termini; and homologous recombination (HR), which uses a homologous sequence, usually on a sister chromatid, as a template for accurate DNA repair. Because HR relies on a homologous template for accurate repair, HR is mostly restricted to S and G₂ phases of the cell cycle when sister chromatids exist. On the other hand, cells can employ NHEJ in any phase of the cell cycle and it is the only option in quiescent (G₀) cells and G₁ phase cells (Scully et al., 2019).

Extensive DNA end resection of the broken DNA ends, which generates long tracts of 3’ ssDNA overhangs at DSBs, is a critical step in committing the cell to use HR to repair DSBs. DNA end resection is initiated by nucleases MRE11 and CtIP, and subsequently extended by nucleases including EXO1 and DNA2/BLM (Paull and Gellert, 1998; Trujillo et al., 1998; Sartori et al., 2007; Gravel et al., 2008; Mimitou and Symington, 2008; Zhu et al., 2008; Bunting et al., 2010). The 3’ ssDNA overhangs are quickly bound by the single-stranded binding protein trimer replication protein A (RPA) to stabilize and protect the ssDNA, and later in repair RPA is replaced by the RAD51 recombinase protein that leads to the homology search to find a homologous template to achieve accurate HR repair (Sugiyama and Kowalczykowski, 2002; San Filippo et al., 2008; Wright et al., 2018). NHEJ is initiated by the KU70/KU80 heterodimer binding to broken DNA ends (Zahid et al., 2021). KU70/KU80 recruits the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) which together form a complex called DNA-PK (Gottlieb and Jackson, 1993; Hammarsten and Chu, 1998). Once the DNA-PK complex is formed, the KU heterodimer translocates inwards along the DNA and DNA-PKcs remains at the DNA ends, undergoing activation via conformational changes mediated by autophosphorylation of the ABCDE cluster (Yaneva et al., 1997; Chen et al., 2021b). Recent cryo-EM structures of DNA-PK also implicate dimerization of DNA-PK as important in recruiting downstream NHEJ factors by bringing broken DNA ends together (Chaplin et al., 2021; Zha et al., 2021). In addition to autophosphorylation, DNA-PKcs phosphorylates members of the NHEJ machinery, including the KU heterodimer, XRCC4, XLF, and Artemis (Bartlett and Lees-Miller, 2018).

The critical bifurcation point in the choice to use HR or NHEJ to repair DSBs is the processing of broken DNA ends to form single-stranded 3’ DNA overhangs, which blocks NHEJ and commits the cell to HR (Symington and Gautier, 2011). Therefore, DNA end resection is tightly regulated to prevent aberrant DNA end resection in G₀ and G₁ phase cells, where NHEJ is the major DSB repair pathway. Several factors have been identified as critical DNA end protection factors that limit resection of DNA DSBs including 53BP1, RIF1, and the Shieldin complex. The proposed mechanism of action of 53BP1 and its downstream effectors include acting as a physical barrier to protect DNA ends from nucleases and promoting DNA polymerase α activity to quickly fill in any resected ends (Bunting et al., 2010; Dev et al., 2018; Mirman et al., 2018; Noordermeer et al., 2018; Setiaputra and Durocher, 2019; Paiano et al., 2021). Additionally, KU70/KU80 has also been shown in budding yeast Saccharomyces cerevisiae to inhibit DNA end resection in G₁ and G₂ phases of the cell cycle, and in S phase in mammalian cells (Lee et al., 1998; Barlow et al., 2008; Clerici et al., 2008; Shao et al., 2012).

While nuclease activity is largely limited in G₀/G₁ phase cells to prevent aberrant DNA end resection, evidence exists suggesting that nuclease-mediated DNA end processing occurs at some DSBs in G₀/G₁. For example, Artemis is required to open hairpin-sealed DNA ends generated during V(D)J recombination in lymphocytes (Menon and Povirk, 2016). Additionally, DNA end resection has been observed in G₁ phase after DNA damage at complex DNA lesions (Averbeck et al., 2014; Biehs et al., 2017), suggesting that DNA end resection is not completely inhibited in the absence of sister chromatids. Moreover, though CtIP phosphorylation by CDKs in G₂ is required for its activity during HR, CtIP also functions in G₁ at DSBs after phosphorylation by PLK3 (Barton et al., 2014). To investigate what additional factors may regulate DNA end resection in cells lacking sister chromatids, we performed a genome-wide CRISPR/Cas9 screen for genes whose inactivation either increases or decreases RPA bound to chromatin after irradiation (IR) in G₀-arrested murine cells. We discovered, unexpectedly, that KU70, KU80, and DNA-PKcs promote extensive DNA end resection in G₀ cells, but not in G₁ or G₂ phases of the cell cycle.

DNA end resection is one of the key events that determines whether cells utilize NHEJ, HR, or other repair pathways utilizing homologous sequences. During G₀ and G₁ phase of the cell cycle, NHEJ is the predominant DSB repair pathway and DNA end resection is largely limited compared to other phases of the cell cycle. However, in this study we revealed that DNA end resection dependent on CtIP and MRE11, which are required for resection in S and G₂ phases of the cell cycle, occurs at DSBs in G₀ mammalian cells (Figure 1B). Because CtIP activity in G₁, G₂ and S phases requires its phosphorylation, this is likely to be the case in G₀ cells and future studies will identify the kinase responsible for any CtIP phosphorylation in G₀ cells. In addition to CtIP and MRE11, we identified additional factors that promote resection in G₀ cells as components of the DNA-PK complex, including KU70, KU80, and DNA-PKcs, in a genome-wide CRISPR/Cas9 screen and showed that the kinase activity of DNA-PK is critical as resection of DSBs diminishes upon DNA-PK inhibitor treatment (Figures 2 and 3). Interestingly, we also found in our genome wide CRISPR/Cas9 screen that inactivating FBXL12, the substrate recognition subunit of the SCF^FBXL12 E3 ubiquitin ligase complex, promotes extensive resection of DNA ends in G₀ cells (Figure 3B). As the SCF^FBXL12 E3 ubiquitin is thought to limit the abundance of the KU70/KU80 heterodimer (Postow and Funabiki, 2013), our data are in line with the notion that loss of FBXL12 results in aberrant accumulation of KU70/KU80 at DSBs, and consequently elevated or prolonged activation of DNA-PK at DSBs which promotes resection in G₀ cells (Figure 5).

Figure 5

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Why would resection occur in G₀ cells? Chemical modifications or secondary structures at DSBs have been identified as requiring DNA end processing to create a more accessible repair environment, which could presumably be the case at DSBs in G₀ cells (Weinfeld and Soderlind, 1991). For example, Artemis is an endo and exonuclease which is activated by DNA-PKcs and uses its nuclease activity to open DNA hairpins at coding ends, which is required for V(D)J recombination, and cleaves 3’ ssDNA overhangs during NHEJ (Ma et al., 2002; Ma et al., 2005). Artemis was also recently shown to contribute to slow, resection dependent NHEJ repair in G₁ phase cells (Biehs et al., 2017). Though Artemis does not have a role in DNA end resection in G₀ (Figure 1—figure supplement 1D). it serves as an example of nuclease activity being critical for DSB repair outside of HR. It is additionally possible that DNA end resection in G₀ results in substrates that are ideal for Pol Theta-mediated end joining (TMEJ). TMEJ occurs after extensive DNA end resection when HR is not possible or when substrates are not suitable for NHEJ, which could be the case at a subset of breaks in G₀ (Yousefzadeh et al., 2014; Wyatt et al., 2016). However, it is notably that TMEJ is KU70/KU80 independent, while the resection that we see in G₀ is KU70/KU80 dependent. Interestingly, DNA end resection has a role in recruiting anti-resection factors to limit extensive DNA end resection. The SHLD2 component of Shieldin binds ssDNA, suppresses RAD51 loading, and ultimately limits DNA end resection by preventing access to resection nucleases (Noordermeer et al., 2018). HELB, a 5’–3’ DNA helicase, binds to RPA and limits EXO1 and BLM-DNA2-mediated DNA end resection (Tkáč et al., 2016). In this way, limited DNA end resection in G₀ cells could be important in preventing more extensive DNA end resection. Altogether, we propose that DNA end resection in G₀ cells is likely not resulting in aberrant HR but may be required to create more accessible DNA ends and/or to recruit anti-resection factors.

Studies investigating the role of KU70/KU80 during DSB repair have found that KU70/KU80 protects DSBs from nuclease activity. For example, at HO endonuclease breaks in budding yeast, deletion of KU70/KU80 leads to ssDNA accumulation in G₁ cells and increased MRE11 recruitment to DSBs compared to wild-type cells (Lee et al., 1998; Clerici et al., 2008). Also in budding yeast, at inducible I-SceI DSBs, deletion of KU70 results in increased RFA1 foci formation in G₁, but deletion of NHEJ factor DNA Ligase IV leads to no defect in RFA1 foci formation compared to wild-type cells, indicating that KU70 itself, not NHEJ, is a barrier to DNA end resection (Barlow et al., 2008). In mammalian cells, complementation of KU70/KU80 knockout cells with a M. tuberculosis KU homolog persistently bound to DSBs in S phase results in reduced RPA and RAD51 foci formation after IR (Shao et al., 2012). Contrary to these roles for KU70/KU80 in protecting DNA ends from nucleolytic attack, we found that in G₀ cells, KU70/KU80 promotes DNA end resection (Figures 3A and 4B). We hypothesize that KU70/KU80 promotes resection through recruitment and activation of DNA-PKcs at DSBs (Gottlieb and Jackson, 1993), as we also found that DNA-PKcs inhibition and genetic deletion of Prkdc leads to less RPA on chromatin after IR and shorter tracts of DNA end resection in G₀ cells (Figure 2C–E, Figure 2—figure supplement 1C and D, 4A, 4C, 4D). It is important to note that most studies establishing the role of KU70/KU80 in protecting DNA ends were performed in S. cerevisiae which do not have a homolog to DNA-PKcs. Therefore, we hypothesize that the function of DNA-PK promoting DNA end resection in G₀ cells may not be evolutionarily conserved. Moreover, previous studies in S. cerevisiae and mammalian cells establishing DNA-PK as a pro-NHEJ complex did not analyze G₀ cells. We found that DNA-PK does not promote DNA end resection in G₁ or G₂ phase cells, only in G₀-arrested cells, indicating that DNA-PK-dependent DNA end resection is unique to G₀, but is not contradictory to its anti-resection function in G₁ or G₂ phase cells (Figure 4). In G₀ cells, KU70/KU80 could protect some DNA ends, but after recruitment and activation of DNA-PKcs, the net effect is DNA end resection. Additional studies may elucidate how the balance between DNA end protection and DNA end resection is regulated in G₀.

ATM and DNA-PK have been shown to have some overlapping functions in DNA damage response and repair, including phosphorylation of H2A.X in response to IR and signal join formation during V(D)J recombination (Stiff et al., 2004; Gapud et al., 2011; Zha et al., 2011). However, we find that this is not the case during DNA end resection in G₀ cells as DNA-PK promotes resection in G₀ cells, but ATM does not have a detectable impact (Figure 2—figure supplement 1B). ATM has been implicated in promoting HR repair by phosphorylating CtIP and promoting KU70/KU80 removal from DSBs, as well as phosphorylating DNA-PKcs at single-ended DSBs to remove it from these breaks that require DNA end resection (Wang et al., 2013; Britton et al., 2020). DNA-PKcs autophosphorylation promotes HR by removing it from DSBs to allow nuclease access but is typically associated with promoting NHEJ by phosphorylating Artemis, XRCC4, and XLF (Zhou and Paull, 2013; Bartlett and Lees-Miller, 2018). So while ATM often promotes DNA end resection and HR, it appears that DNA-PKcs could be acting in place of ATM to promote DNA end resection in G₀ cells. It is additionally possible that DNA-PKcs phosphorylates a unique substrate(s) in G₀ cells that promotes DNA end resection.

In summary, we provide here evidence that DNA-PK promotes DNA end resection uniquely in G₀ cells, and that this DNA end resection is counteracted by FBXL12. We speculate that some aspects of DSB repair in G₀ function differently than DSB repair in cycling cells, and future studies may reveal the mechanism and utility of these key differences.

Sequencing data have been deposited in GEO under accession codes GSE186087.

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Decision letter after peer review:

Thank you for submitting your article "DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G₀ cells" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Wolf-Dietrich Heyer as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Kevin Struhl as the Senior Editor.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.

Recommendations to the authors

All reviewers agree that the authors made a significant discovery that will be of great interest to the field. There was also a consensus that more data are needed in three areas. (1) More evidence is needed to ensure that the observations apply to human cells, in light of the fact that human cells have 50x less DNA-PK than murine cells. (2) More evidence is needed that the phenomenon is truly G0 specific and not occurring in G1 cells. (3) There is some concern that most experiments were conducted in LIG4-deficient cells. Without wanting to be prescriptive, additional data with the LIG4-proficient human MCF10A cell line applying END-Seq to G0 and G1 cells evaluating DSB resection in dependence of DNA PK would nicely address these concerns.

Essential revisions

1) Despite showing a similar increase in the chromatin-bound RPA upon irradiation in WT abl pre-B cells, the authors utilize for the majority of the experiments abl pre-B cells deficient for Lig4. It is important to verify that the inability to complete the NHEJ repair process due to the lack of Lig4 does not cause pathological resection of the DSBs. Additional examination in NHEJ proficient cells would eliminate this concern.

2) The authors use Imatinib treatment for abl pre-B cells or serum deprivation in MCF10A for 48 h to arrest cells in G0 phase. However, further analyses are needed to validate that after only two days of treatment cells have really exited the cell cycle.

3) The authors suggest that the DNA-PK complex promotes DSBs resection uniquely in G0 but not in other cell cycle phases. To draw this conclusion, a few control experiments should be included. END-seq analysis of AsiSI-induced DSBs in Lig4-/- abl pre-B cells should be performed also in G1 phase. Moreover, taking into account that V(D)J recombination is happening in the G0/G1 phase of immune cells, which could explain the high level of resection shown in G1 pre-B cells, the experiments should be replicated in another cell line, like in MCF10A.

4) The paper nicely demonstrates the involvement of the DNA-PK complex in DSBs resection in G0 murine abl pre-B cells. To consolidate the results, the authors should confirm the requirement of the DNA-PK complex (including KU70/80) and its regulation by FBXL12 also in MCF10A. This extends their finding from a murine model to human cells and is important to expand the impact of the paper.

5) The authors should extract more data from the END-seq analyses and report average and median resection tracts for each experiment.

6) In the discussion about the mechanistic significance for G0 DSB resection, the authors may want to consider adding a paragraph on TMEJ and the possibility that end-processing represents a way to shift an NHEJ substrate to a TMEJ substrate. The authors may also want to consider and discuss a relatively recent report on resection dependent classic NHEJ (https://pubmed.ncbi.nlm.nih.gov/28132842/).

7) CtIP is licensed by phosphorylation in S/G2. Can the authors comment on CtIP activity in G0/G1 and the possibility that some phosphorylation exists under these conditions?

8) Chromatin-bound RPA levels have been assessed in various experiments throughout the manuscript. However, the dose of X-rays and the time after irradiation have been frequently changing. Please clarify the reason for those inconsistencies.

9) A genome wide CRISPR/Cas9 screen was performed to identify factors promoting resection in G0 cells. Detailed information regarding the screen is however missing. Please specify how many times it was repeated and clarify how the fold enrichment of the gRNAs is calculated.

10) A clearer description of the Lig4-/- AsiSI abl pre-B cells is needed. Please specify how these cells were generated and how DSB induction was optimized. Please add also information on the concentration of tamoxifen utilized. Furthermore, the location of the DSBs should be analyzed to show if there is a subgroup of them which is prone to resection and is perhaps located in a particular genomic region (genes versus non-genes, transcriptionally active versus inactive, etc.).

11) In the discussion, the authors mention Artemis as an example of a nuclease involved in resection and V(D)J recombination. Since Artemis is known to be involved in NHEJ in G1, and in HR in G2, its function could be tested to assess the difference in NHEJ repair in quiescent and G1 cells and would serve as a nice control to show the processes are entirely different.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "DNA-PK Promotes DNA End Resection at DNA Double Strand Breaks in G₀ cells" for further consideration by eLife. Your revised article has been evaluated by Kevin Struhl (Senior Editor) and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

The revised manuscript strengthened an already strong manuscript, and the revision addresses the concerns of the reviewers. There is one point of clarification needed about Figure 4E. The rebuttal and text describe that G0 wild type MCF10A cells show DNA PK dependent resection. It follows that upon DNAPK inhibition the amount of RPA should decrease, as resection is reduced. As far as I understand Figure 4E, the results show the opposite: the green curve (DNAPKi +IR) shifts to the right (more RPA) compared to IR only. IS there an error in the figure, in the description, or in my understanding?

Essential revisions

1) Despite showing a similar increase in the chromatin-bound RPA upon irradiation in WT abl pre-B cells, the authors utilize for the majority of the experiments abl pre-B cells deficient for Lig4. It is important to verify that the inability to complete the NHEJ repair process due to the lack of Lig4 does not cause pathological resection of the DSBs. Additional examination in NHEJ proficient cells would eliminate this concern.

In addition to showing that chromatin-bound RPA increases upon irradiation in quiescent wild type (WT) abl pre-B cells (Figure 1 —figure supplement 1B) we now show that DNA-PK activity promotes DNA end resection in quiescent WT abl pre-B cells. This is included as new Figure 2 —figure supplement 1C. In addition to showing that chromatin-bound RPA increases upon irradiation in quiescent WT human MCF10A Figure 1 —figure supplement 1E, we show that RPA foci formation increases after IR in quiescent WT MCF10A (Figure 1C), the dependance of DNA-PK activity for RPA foci formation in quiescent WT MCF10A (Figure 2 —figure supplement 1D) and that DNA-PK promotes chromatin bound RPA in quiescent WT MCF10A cells, but not G₁ WT RPA foci formation increases after IR in WT MCF10A (new figure 4E).

2) The authors use Imatinib treatment for abl pre-B cells or serum deprivation in MCF10A for 48 h to arrest cells in G0 phase. However, further analyses are needed to validate that after only two days of treatment cells have really exited the cell cycle.

This is a good point. Accordingly, we recently published an extensive analysis of phospho targets that demonstrates that Imatinib treatment in abl pre-B cells and serum deprivation in MCF10A for 48 h arrests cells in G0 phase not G1 phase (Chen et al. 2021 eLife), using the identical conditions for arrest that we use in the current manuscript.

3) The authors suggest that the DNA-PK complex promotes DSBs resection uniquely in G0 but not in other cell cycle phases. To draw this conclusion, a few control experiments should be included. END-seq analysis of AsiSI-induced DSBs in Lig4-/- abl pre-B cells should be performed also in G1 phase. Moreover, taking into account that V(D)J recombination is happening in the G0/G1 phase of immune cells, which could explain the high level of resection shown in G1 pre-B cells, the experiments should be replicated in another cell line, like in MCF10A.

We had shown that chromatin-bound RPA increases after IR in G₁ phase cells by gating the cycling cells on 2N DNA content at the time of the analysis. RPA only binds to ssDNA, so there is no other conclusion to be made other than this is due to increased DNA end resection. It is not possible for us to perform the reviewers suggested END-seq in G₁ phase cells, because we sort for cycling G₁ cells, and by the time that the END-Seq analysis would be done the cells would no longer be in G₁ phase, given that the induction of AsiSI is by treatment with doxycycline for 24 hours followed by nuclear localization of AsiSI by tamoxifen treatment for 4 and 8 hours to induce AsiSI cutting. Regarding the question of whether the RPA binding is due to V(D)J recombination, we have published extensively that VDJ occurs in the imatinib (G₀) arrested cells while the RPA binding to chromatin and end-resection that we see in G₀ arrested cells is totally dependent on irradiation. Nonetheless, we now show that chromatin-bound RPA increases in both G₀ and G₁ WT MCF10A cells after IR but is only dependent on DNA-PK in G₀ cells (new Figure 4E).

4) The paper nicely demonstrates the involvement of the DNA-PK complex in DSBs resection in G0 murine abl pre-B cells. To consolidate the results, the authors should confirm the requirement of the DNA-PK complex (including KU70/80) and its regulation by FBXL12 also in MCF10A. This extends their finding from a murine model to human cells and is important to expand the impact of the paper.

Unfortunately, human cells are more sensitive to DNA-PK depletion than murine cells, so genetic knockout of DNA-PK was unsuccessful using our lab’s CRISPR/Cas9 technique. Previous literature shows that Ku70 and Ku80 are encoded by essential genes and a homozygous knockout is difficult to achieve (Hendrickson, E.A., et al. 2008 PNAS; Hendrickson, E.A. et al. 2002 PNAS). However, as discussed above, we now include new data showing DNAPK dependent resection in MCF10A cells in G₀ phase (Figure 4E). We attempted to make MCF10A Fbxl12^-/- cells numerous times with different gRNAs, and screened dozens of clones, but were unsuccessful at obtaining homozygous knockout clones. This is what has held up resubmission of our paper for so long. We are still attempting to make homozygous knockout clones but would add at a minimum 2 more months before our resubmission.

5) The authors should extract more data from the END-seq analyses and report average and median resection tracts for each experiment.

This has been done and included as Figure 4D.

6) In the discussion about the mechanistic significance for G0 DSB resection, the authors may want to consider adding a paragraph on TMEJ and the possibility that end-processing represents a way to shift an NHEJ substrate to a TMEJ substrate. The authors may also want to consider and discuss a relatively recent report on resection dependent classic NHEJ (https://pubmed.ncbi.nlm.nih.gov/28132842/).

We now include some discussion about these possibilities. TMEJ requires some DNA end resection and is considered a backup pathway for breaks that cannot complete HR but have undergone DNA end resection or at breaks that do not have suitable ends for NHEJ. It is possible, especially in Lig4^-/- cells, that TMEJ could be responsible for DSB repair after resection in G₀, though we note that TMEJ is Ku-independent and our G₀ DNA end resection is Ku-dependent.

We now mention resection-dependent NHEJ as an example of nuclease activity in DSB repair outside of HR, but the reviewer cited paper shows that Artemis is absolutely required for this process, and our data shows Artemis has no role in G₀ DSB repair (Figure 1—figure supplement 1D), it is unlikely this repair process is what we observe in G₀ cells.

7) CtIP is licensed by phosphorylation in S/G2. Can the authors comment on CtIP activity in G0/G1 and the possibility that some phosphorylation exists under these conditions?

We now mention in the introduction that CtIP has been shown to be phosphorylated by PLK3 in G₁ during DSB repair and have discussed the likelihood that CtIP is also phosphorylated in G₀ cells.

8) Chromatin-bound RPA levels have been assessed in various experiments throughout the manuscript. However, the dose of X-rays and the time after irradiation have been frequently changing. Please clarify the reason for those inconsistencies.

Most flow cytometry experiments were performed with 20 Gray IR and samples were collected 3 hours after irradiation. Figures 1B, 2C, 3D, 4E exhibit either a slightly different dose of IR (15 Gy) or time point (2 hours or 4 hours) due to these experiments being performed at a different facility. The experimental parameters were reassessed and optimized at the new facility. The CRISPR/Cas9 screen was collected at 2 hours instead of 3 due to time constraints of the experiment. We have observed that the extent of resection in G₀ is not significantly different between 2-4 hours after IR. For immunofluorescence experiments doses of IR were experimentally determined to provide resolution of the RPA foci by IF to enable counting, which required a lower IR dose. Immunofluorescence after 15-20 Gray showed RPA foci merging to fill the entire nucleus.

9) A genome wide CRISPR/Cas9 screen was performed to identify factors promoting resection in G0 cells. Detailed information regarding the screen is however missing. Please specify how many times it was repeated and clarify how the fold enrichment of the gRNAs is calculated.

We now include in the figure legend how the fold enrichment was calculated: the ratio of normalized read number of gRNAs in the low RPA population and that in the high RPA population and vice versa. We also indicate that the screen was performed once (n=1). Notably, we validated all the hits that we studied in this manuscript.

10) A clearer description of the Lig4-/- AsiSI abl pre-B cells is needed. Please specify how these cells were generated and how DSB induction was optimized. Please add also information on the concentration of tamoxifen utilized.

We now include this information in the methods section.

Furthermore, the location of the DSBs should be analyzed to show if there is a subgroup of them which is prone to resection and is perhaps located in a particular genomic region (genes versus non-genes, transcriptionally active versus inactive, etc.).

Generally, there are about 200 broken AsisI breaks detectable by ENDseq, so we used the top 200 sites. Most of them are close to TSS: 189 out of 200 are within +/2kb of TSS. And 170 of the 200 AsisI sites that their closest genes are expressed (RPKM>0.1) measured by a nasRNA-seq in abl pre-B cells. These analyses are now included in Figure 1 – supplemental figure 2.

11) In the discussion, the authors mention Artemis as an example of a nuclease involved in resection and V(D)J recombination. Since Artemis is known to be involved in NHEJ in G1, and in HR in G2, its function could be tested to assess the difference in NHEJ repair in quiescent and G1 cells and would serve as a nice control to show the processes are entirely different.

To address this comment, we have now tested Artemis dependency in G₀ arrested abl pre-B cells after IR and show that it is not required for resection in G₀ arrested cells in figure 1 —figure supplement 1D. We have altered the discussion accordingly.

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The revised manuscript strengthened an already strong manuscript, and the revision addresses the concerns of the reviewers. There is one point of clarification needed about Figure 4E. The rebuttal and text describe that G0 wild type MCF10A cells show DNA PK dependent resection. It follows that upon DNAPK inhibition the amount of RPA should decrease, as resection is reduced. As far as I understand Figure 4E, the results show the opposite: the green curve (DNAPKi +IR) shifts to the right (more RPA) compared to IR only. IS there an error in the figure, in the description, or in my understanding?

Thank you for carefully reading our manuscript and looking at the new data. It transpires that somehow upon Figure assembly we did invert the labelling somehow. Thanks so much for seeing this as it avoids an embarrassing correction. We have updated the figure accordingly.

Author details

1. Faith C Fowler

   1. Weill Cornell Medicine Pharmacology Graduate Program, New York, United States
   2. Weill Cornell Medicine, Department of Pathology and Laboratory Medicine, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing - original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7180-8141

2. Bo-Ruei Chen

   Department of Medicine, Division of Hematology and Oncology, O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, United States

   Contribution

   Conceptualization, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6404-2099

3. Nicholas Zolnerowich

   Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States

   Contribution

   Data curation, Investigation

   Competing interests

   No competing interests declared

4. Wei Wu

   Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Raphael Pavani

   Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Jacob Paiano

   Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Chelsea Peart

   Weill Cornell Medicine, Department of Pathology and Laboratory Medicine, New York, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Zulong Chen

   Weill Cornell Medicine, Department of Pathology and Laboratory Medicine, New York, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

9. André Nussenzweig

   Laboratory of Genome Integrity, National Cancer Institute, Bethesda, United States

   Contribution

   Funding acquisition, Project administration

   Competing interests

   No competing interests declared

10. Barry P Sleckman

    Department of Medicine, Division of Hematology and Oncology, O'Neal Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, United States

    Contribution

    Conceptualization, Project administration

    For correspondence

    bps@uab.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-8295-4462

11. Jessica K Tyler

    Weill Cornell Medicine, Department of Pathology and Laboratory Medicine, New York, United States

    Contribution

    Conceptualization, Funding acquisition, Writing - review and editing

    For correspondence

    jet2021@med.cornell.edu

    Competing interests

    Senior editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0001-9765-1659

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