Rev7 and 53BP1/Crb2 prevent RecQ helicase-dependent hyper-resection of DNA double-strand breaks
DNA repair is an essential process conserved throughout evolution and commonly disrupted in tumor cells ( Jeggo et al . , 2016 ) . Many cancer treatments , including poly ( ADP ribose ) polymerase ( PARP ) inhibitors ( PARPi ) , target DNA repair pathways to kill rapidly dividing , repair-deficient cells ( Farmer et al . , 2005; Fojo and Bates , 2013; Lord et al . , 2015; Mateo et al . , 2015 ) . 5’ end resection , which generates tracts of single-strand DNA ( ssDNA ) at DNA double-strand break ( DSB ) ends dictates repair pathway choice: blocking resection promotes canonical non-homologous end joining ( typically in G1 ) , while initiating resection commits a DSB to repair by homologous recombination ( HR ) , usually in S/G2 ( Chapman et al . , 2012; Hustedt and Durocher , 2016; Symington , 2016 ) . The resection machinery is tightly controlled at both the step of resection initiation ( involving Ctp1/Sae2/CtIP and the MRN/X complex ) and during long-range resection , which is mediated by two parallel pathways catalyzed by either the exonuclease Exo1 or the combination of a RecQ helicase and Dna2 ( Chen et al . , 2012; Croteau et al . , 2014; Nimonkar et al . , 2011; Tkáč et al . , 2016; Zimmermann et al . , 2013 ) . Additional accessory factors play key roles as modulators of resection; for example , loss of Rev7/MAD2L2/FANCV , a small , multifunctional HORMA domain protein ( Bluteau et al . , 2016; Rosenberg and Corbett , 2015 ) derepresses resection inhibition ( Boersma et al . , 2015; Xu et al . , 2015 ) , thereby allowing HR-deficient , Brca1–/–p53–/– cells to become resistant to PARPi . In human cells , Rev7 appears to act in concert with another inhibitor of resection , 53BP1 ( Chapman et al . , 2013; Ochs et al . , 2016; Zimmermann et al . , 2013 ) , loss of which is also sufficient to drive PARPi resistance ( Boersma et al . , 2015; Bouwman et al . , 2010; Jaspers et al . , 2013; Xu et al . , 2015 ) . Importantly , the mechanisms by which Rev7 and 53BP1 inhibit DSB end resection remain poorly understood . To gain insights into how resection is controlled , we have developed a single-cell microscopy-based assay capable of quantitatively measuring DSB end resection rates in the facile genetic model , S . pombe . Leveraging this assay , we find that Rev7 and the 53BP1 orthologue , Crb2 , specifically inhibit the RecQ-helicase-dependent long-range resection pathway . Moreover , through derepression of RecQ helicases , rev7∆ or crb2∆ cells can achieve very fast resection rates ( >20 kb/hr ) – approximately twice as fast as Exo1-dependent long-range resection . As BRCA1 activity has been tied to Exo1-dependent long-range resection ( Tomimatsu et al . , 2012 ) , our findings suggest that PARPi resistance can be driven by compensation through derepression of the RecQ-helicase-dependent resection pathway .
In order to quantitatively measure initial steps in DSB processing in single , living cells , we developed a microscopy-based DSB end resection assay ( Figure 1A ) . In this system , an ectopic 10 . 3 kb , 256-copy LacO array and adjacent HO endonuclease cut site ( HOcs ) are engineered at a euchromatic ( but intergenic ) region near Mmf1 ( Figure 1—figure supplement 1 ) . A single , site-specific DSB is generated by regulating the expression of the HO endonuclease under the control of the Ura-inducible Purg1lox RMCE system ( Watson et al . , 2008; 2011 ) . The timing of on-target DSB events is visualized by the appearance of a Rad52 ( Rad22 ) -mCherry focus that co-localizes with Mmf1:LacO/LacI-GFP ( Figure 1B ) . By tracking cell lineages , we see that HO endonuclease induction produces on-target Rad52-mCherry foci in S/G2 ( G1 is very short in S . pombe ) when the repair machinery is primed for HR ( Symington and Gautier , 2011 ) ( Figure 1B ) . Importantly , we do not observe loading of Rad52-mCherry at the LacO/LacI-GFP array in the absence of HO endonuclease expression ( on-target Rad52 foci in <0 . 2% of uninduced cells , n = 657 ) , suggesting that the LacO array is not sufficient to create a ‘fragile site’ in S . pombe ( Jacome and Fernandez-Capetillo , 2011; Saad et al . , 2014 ) . As resection proceeds , the LacO repeats become single-stranded , disrupting LacI-GFP binding and causing the intensity of the GFP focus to progressively decrease ( Bell and Lewis , 2001 ) ( Figure 1B , Figure 1—figure supplement 2 ) . To verify that loss of GFP focus intensity reflects DSB end resection , we analyzed cells lacking Exo1 , which catalyzes the majority of long-range resection in WT S . pombe , with the Rqh1/Sgs1/BLM and Dna2 resection pathway playing a secondary role ( Langerak et al . , 2011 ) . As expected , cells lacking Exo1 show a persistent LacO/LacI-GFP focus over many hours even after Rad52-mCherry loads ( Figure 1C ) . Importantly , loss of Exo1 does not influence the induction of the site-specific DSB as measured by quantitative loss of a PCR product across the cut site ( Figure 1—figure supplement 3A ) . This assay only visualizes DSB foci after resection initiation by Ctp1/Sae2/CtIP and MRN/MRX ( ~100 nt of resection ) , as Rad52 loads at DSBs by exchanging with RPA on nascent , resected ssDNA ends ( Jensen and Russell , 2016; Lisby et al . , 2004; Ma et al . , 2015; Mimitou and Symington , 2008 ) . Using quantitative imaging with calibration strains , we estimate that visualization of the Rad52-mCherry focus requires loading of ~30 copies ( see Materials and methods ) , representing a Rad52 filament equivalent to at least 90 nt ( but likely several hundred nts , see below ) of resected ssDNA on each sister in G2 ( Gibb et al . , 2014; Grimme et al . , 2010; Kagawa et al . , 2002; Singleton et al . , 2002; Swartz et al . , 2014; Wu and Pollard , 2005 ) . To test the frequency with which resection initiation leads to visible Rad52 foci in this assay , we compared the timing of Rad52-mCherry foci formation within the cell population to an independent measure of resection using a quantitative PCR ( qPCR ) assay in which resection protects from digestion at an ApoI cut site 168 nt downstream of the HO cut site ( Langerak et al . , 2011 ) ( Figure 1—figure supplement 3B ) . Using this qPCR-based assay , we observe similar DSB induction frequency and kinetics for ApoI protection and Rad52 focus formation , with an apparent 30–60 min delay between ApoI protection ( qPCR ) and Rad52-mCherry loading , which likely represents the time required for RPA loading and exchange to Rad52 ( Lisby et al . , 2004 ) ( Figure 1—figure supplement 3C ) . As the visibility of the LacO array is an important endpoint for this assay , we estimate that the LacI-GFP focus is visible down to a LacO array length of <500 bps under these conditions , based on our ability to robustly detect the focus from a 1 kb LacO array integrated into S . pombe ( Figure 1—figure supplement 3D–G ) . By measuring the time interval between Rad52-mCherry focus formation and LacO/LacI-GFP focus disappearance , we can determine the time required to resect through the full LacO array in individual cells ( Figure 1B , Figure 1—figure supplement 2A-D , blue boxes ) . For example , for the two daughter cells in Figure 1B arising from cell division , the time interval from Rad52-mCherry loading until loss of the LacO/LacI-GFP focus is 150 min for the upper cell ( ‘1’ ) , and 140 min for the lower cell ( ‘2’ ) . The progressive loss of LacI-GFP intensity and gain in Rad52-mCherry intensity during the duration of resection is further illustrated by quantitative image analysis ( Figure 1E–F and Figure 1—figure supplement 3A'–B' ) . As we know the genomic separation of the LacO array and the HO cut site ( 13 . 9 kb , Figure 1—figure supplement 1 ) , we can calculate the resection rate from the resection duration . For example , the calculated resection rates for the two cells in Figure 1B are very similar at 5 . 55 and 5 . 94 kb/hr . Across all WT cells , we detect a median , long-range resection rate of 7 . 6 kb/hr ( Figure 1F ) . This rate is somewhat faster than the resection rates measured by previous population-based assays using qPCR in S . pombe ( 4 kb/hr ) or Southern blot in S . cerevisiae ( 4 . 4 kb/hr ) ( Langerak et al . , 2011; Zhu et al . , 2008 ) . As this assay isolates the process of long-range resection after Rad52 loading , one possibility is that resection rates that measure both resection initiation and long-range resection give rise to slower rates . Importantly , using the qPCR approach to compare resection upstream of the DSB ( which contains the LacO array ) and downstream of the DSB ( which does not ) demonstrates that any influence of the LacO array on resection rate is minor ( Figure 1—figure supplement 3H ) . As resection in the absence of Exo1 is very inefficient ( Figure 1C ) , the rate cannot be determined using this assay . However , we can infer an upper bound of the long-range resection rate of ~2 . 8 kb/hr for exo1∆ cells . We also note a strong inhibition of resection ( comparable to ctp1∆ cells ) as close as 300 nts from the DSB in cells lacking Exo1 as detected by qPCR ( Figure 1—figure supplement 3I ) ; this correlates with a defect in Rad52-mCherry loading in the imaging-based resection assay ( Figure 1—figure supplement 3J ) . This observation confirms that the extent of resection required to form visible Rad52 foci in this assay discussed above ( ~30 molecules; <300 nt ) partially requires Exo1-dependent resection in addition to MRN/MRX- and Ctp1/Sae2/CtIP-dependent resection initiation ( Symington , 2016 ) , consistent with a study in budding yeast suggesting a specific requirement for Exo1 in the early phase of resection post-initiation ( Saad et al . , 2014 ) . 53BP1 ( in human cells ) and its orthologue Rad9 ( in budding yeast ) repress resection initiation ( Chapman et al . , 2012; Ferrari et al . , 2015; Symington , 2016 ) ; in budding yeast , loss of Rad9 also increases resection efficiency ( Bonetti et al . , 2015 ) . Applying the live cell resection assay to fission yeast lacking the orthologous Crb2 , we observe a strong increases the median rate of resection to ( 13 . 9 kb/hr ) , with some individual cells demonstrating very fast ( ~40 kb/hr ) resection rates ( Figure 1F ) . Thus , we can readily assess factors that positively and negatively influence long-range resection rate using this new LacO-based assay . Next , we examined how loss of Rev7 influences long-range resection at DSBs during S/G2 . The duration of LacO array resection is shorter in rev7∆ cells than in WT cells ( 80 min or less , Figure 2A–B and Figure 2—figure supplement 1 ) . In the population , the median long-range resection rate for rev7∆ cells is similar to cells lacking Crb2 ( 10 . 4 kb/hr , Figure 2C ) . As Rev7 also functions with Rev3 as part of the polymerase ζ complex in translesion synthesis , we also confirmed that Rev3 does not affect resection ( Figure 2C ) , consistent with previous data showing that repressing resection during HR is a distinct function of Rev7 ( Boersma et al . , 2015; Rosenberg and Corbett , 2015; Xu et al . , 2015 ) . Using the orthogonal qPCR approach ( Figure 1—figure supplement 3B ) , we confirm that in rev7∆ cells , more chromosomes with DSBs have undergone 3 kb and 13 kb of resection than WT between 90 and 180 min after HO induction , respectively ( Figure 2D ) . Again , loss of Rev7 has no influence on the rate of DSB induction ( Figure 2E ) . We next asked if Crb2/Rad9/53BP1 and Rev7 inhibit long-range resection through the Exo1 pathway , the RecQ helicase ( Rqh1 ) -Dna2 pathway , or both ( Symington and Gautier , 2011 ) . Interestingly , we find that the rapid long-range resection rate observed in crb2∆ or rev7∆ cells is entirely Exo1-independent ( Figure 3A–C , Figure 3—figure supplement 1 ) . In stark contrast , the rapid rate of resection in a rev7∆ single mutant is entirely dependent on the presence of Rqh1 ( Figure 3A ) . Consistent with these observations , we find that loss of Rev7 is able to rescue the severe growth defect of exo1∆ cells on rich media plates containing camptothecin , consistent with a derepression of Rqh1-dependent resection in the absence of a functional Exo1 pathway ( Figure 3D ) . When considering only precisely determined resection events , the average long-range resection rate in crb2∆ cells is not statically less than that of crb2∆rqh1∆ cells when correcting for multiple comparisons ( p=0 . 16 ) ( Figure 3A ) . However , in many crb2∆rqh1∆ cells , resection durations extend beyond the timeframe of data acquisition , suggesting that rapid crb2∆ resection also requires Rqh1 ( Figure 3—figure supplement 2 ) . Taken together , these results strongly suggest that loss of either Rev7 or Crb2 drives a shift in resection pathway mechanism from Exo1 to the RecQ helicase-dependent pathway . Unlike Exo1-driven resection that dominates in WT cells , crb2∆ or rev7∆ cells in which the RecQ helicase is derepressed are capable of resection rates in excess of 20 kb/hr , indicating that the RecQ helicases are more capable of driving hyper-resection of DSBs than the Exo1-dependent pathway . The strong inhibition of RecQ helicase-dependent resection by Crb2 and Rev7 also explains why Rqh1 is not a major player in WT S . pombe resection , since Rqh1 can be derepressed to such a large extent ( by loss of Crb2 or Rev7 ) that Exo1 becomes dispensable for long-range resection in crb2∆exo1∆ and rev7∆exo1∆ cells ( Figure 3A-C , Figure 3—figure supplement 1 ) .
Here , we demonstrate a microscopy-based assay capable of quantitatively measuring DSB end resection in living cells , specifically the long-range phase of resection catalyzed by Exo1 or Rqh1/Dna2 . By allowing for the visualization of resection in single cells , this assay reveals individual long-range resection rates , which will provide access to information about the variability in resection efficiency within cell populations . Moreover , as this assay allows the tracking of the location of DSB lesions within the nucleus , it will be able to uniquely interrogate the role of intranuclear architecture on resection rates and pathways in the future . By leveraging the advantages of this LacO-based assay , we show that Crb2/Rad9/53BP1 and Rev7 both act as specific inhibitors of RecQ helicase-mediated long-range resection of DSBs ( Figures 2 and 3 ) , supporting a model in which loss of these resection inhibitors drives a change in resection pathway rather than boosting Exo1-dependent resection . This is consistent with a previous study that identified specific mutations in the budding yeast RecQ helicase , Sgs1 , that can disrupt inhibition by the Crb2 orthologue , Rad9 , leading to a gain in Sgs1-dependent resection ( Bonetti et al . , 2015 ) , as well as a recent study highlighting the ability of Rad9 to antagonize resection at stalled replication forks by repressing a Dna2-dependent pathway ( Villa et al . , 2018 ) . Integrating these findings together with this study , we suggest that Crb2/Rad9 and Rev7 enforce Exo1-dependent long-range resection in yeasts , which prevents the hyper-resection that we find is characteristic of the RecQ helicase/Dna2 pathway ( Figure 3 and Bonetti et al . , 2015 ) . In support of this model , we find that loss of Rev7 is able to restore cell viability of exo1Δ cells on media containing camptothecin , suggesting that derepression of Rqh1 can substitute for loss of Exo1 , consistent with the gain in resection rate in cells lacking both Rev7 and Exo1 ( Figure 3 ) . The ability of Rev7 to antagonize RecQ helicase/Dna2-dependent long-range resection in fission yeast is very likely to be conserved in mammalian cells . Indeed , indirect evidence suggests that Rev7 knock-down promotes a gain in resection , as loss of Rev7 rescues CtIP-dependent RPA and Rad51 loading at irradiation-induced DSBs in cells lacking BRCA1 ( Xu et al . , 2015 ) . Importantly , the resection pathway responsible for the gain in repair factor loading was not investigated in this study , but our work suggests that BLM ( acting with DNA2 ) is a likely candidate . Loss of Rev7 does lead to longer 3’ single-stranded G-rich overhangs at telomeres in cells with inactivated TRF2 ( a component of the shelterin complex ) ; in this case , only a partial rescue was obtained by co-depletion of CtIP or Exo1 ( Boersma et al . , 2015 ) . Again , we hypothesize that this finding likely reflects contributions of BLM and DNA2 , although it remains possible that the impact of Rev7 on long-range resection during G2 figures more prominently in fission yeast . Critically , our findings suggest a new mechanism by which loss of either 53BP1 or Rev7 allows BRCA1-/- p53-/- cells to become resistant to PARP inhibitors ( Jaspers et al . , 2013; Xu et al . , 2015 ) . It has been proposed previously that the compromised CtIP- and Exo1-dependent resection in BRCA1-deficient cells can be restored by loss of 53BP1 or Rev7 ( Boersma et al . , 2015; Xu et al . , 2015 ) , thereby overcoming the increased DSB load caused by PARP inhibitors ( Polato et al . , 2014; Tomimatsu et al . , 2012 ) . Our study reveals that not just the efficiency , but also the molecular mechanism of resection is altered upon loss of Crb2/53BP1 or Rev7 . We expect these insights to have several consequences . First , RecQ helicases , when paired with DNA2 , are capable of exceptionally fast resection in vitro ( Niu et al . , 2010 ) , which when derepressed by loss of 53BP1 or Rev7 could cause extended tracts of ssDNA several kb long , promoting further genome instability ( Hicks et al . , 2010; Ochs et al . , 2016 ) ; indeed , our data point to such a hyper-resection phenotype upon loss of either Crb2 or Rev7 ( Figure 2C ) . Second , our results suggest that inhibitors of RecQ helicases could potentially re-sensitize BRCA1-null cells to PARP inhibitors , as this would make them both Exo1- and RecQ helicase-deficient ( Aggarwal et al . , 2013; Yazinski et al . , 2017 ) .
The strains used in this study are listed in Supplementary file 1 . S . pombe were grown , maintained , and crossed using standard procedures and media ( Moreno et al . , 1991 ) . Gene replacements were made by exchanging open reading frames with various MX6-based drug resistance genes ( Bähler et al . , 1998; Hentges et al . , 2005 ) . The 10 . 3 kb LacO array was inserted between Mmf1 and Apl1 on the right arm of chromosome II ( Chr II: 3 , 442 , 981 ) using a modified two-step integration procedure that first creates a site-specific DSB to increase targeting efficiency of linearized plasmid pSR10_ura4_10 . 3kb ( Leland and King , 2014; Rohner et al . , 2008 ) . A modified MX6-based hygromycin-resistance cassette containing the HO cut site was then inserted between Apl1 and Mug178 on chromosome II ( Chr II: 3 , 446 , 249 ) , 3 . 2 kb distal to the LacO insertion . The total distance between the HO cut site and the beginning of the 10 . 3 kb LacO array is 3 . 57 kb . As the LacO array can contract during the process of transformation , integrants were screened by HincII digest followed by southern blot ( Figure 1—figure supplement 1 ) using standard procedures , a biotin-conjugated LacO probe , and a streptavidin-HRP chemilluminescent detection system ( Thermo #N100 and #34096 ) . We used the uracil-responsive Purg1lox expression system , with slight modifications , to induce HO endonuclease expression and create site-specific DSBs at the HO cut site ( Watson et al . , 2011; Watt et al . , 2008 ) . We performed a fresh integration of the HO gene at the endogenous urg1 locus for each experiment in order to reduce long-term instability at the HO cut site or the development of HO resistance , presumably due to insertion/deletion events caused by basal expression levels of HO . The pAW8ENdeI-HO plasmid ( a gift from Tony Carr ) was transformed into S . pombe , which were then plated onto EMM-leu+thi-ura plates ( -leucine: plasmid selection; +thiamine: Pnmt1-Cre repression; -uracil: Purg1lox-HO repression ) . After 4–5 days of growth at 30°C , 40–100 individual colonies were combined to obtain a reproducible plasmid copy number across the population . Cre-mediate HO gene exchange at the endogenous Urg1 locus ( urg1::RMCEbleMX6 ) was induced by overnight culture in EMM-thi-ura+ade+NPG media ( -thiamine: expression of Cre from pAW8ENdeI-HO; -uracil: Purg1lox-HO repression; +0 . 25 mg/mL adenine: reduce autofluorescence; +0 . 1 mM n-Propyl Gallate ( NPG ) : reduce photobleaching in microscopy experiments , prepared fresh ) . The following day , site-specific DSBs were induced in log-phase cultures by the addition of 0 . 50 mg/mL uracil . This induction strategy resulted in ~15% of cells making a DSB within ~2 hr ( Figure 1—figure supplement 3C ) . Initiation of resection was assessed using a previously described qPCR assay where ssDNA produced by resection causes protection from ApoI digestion ( Langerak et al . , 2011 ) . ApoI cut site positions ( relative to the HO cut cite ( Chr II: 3446192 ) ) and PCR primer sets spanning each ApoI recognition site can be found in Supplementary file 3 . Mock HincII digestions ( do not affect qPCR products ) and additional control primers at Ncb2 were used to normalize for ApoI digestion efficiency ( see Supplementary File 3 ) . All images were acquired on a DeltaVision widefield microscope ( Applied Precision/GE ) using a 1 . 2 NA 100x objective ( Olympus ) , solid-state illumination , and an Evolve 512 EMCCD camera ( Photometrics ) . Slides were prepared ~20 min after adding 0 . 50 mg/ml uracil to log-phase cultures to induce HO endonuclease expression and DSB formation . Cells were mounted on 1 . 2% agar pads ( EMM +0 . 50 mg/mL uracil , +2 . 5 mg/ml adenine , +0 . 1 mM freshly prepared NPG ) and sealed with VALAP ( 1:1:1 vaseline:lanolin:paraffin ) . Image acquisition began between 40 and 80 min after uracil addition . Imaging parameters for all resection assay data acquisition were as follows . Transmitted light: 35% transmittance , 0 . 015 s exposure; mCherry: 32% power , 0 . 08 s exposure; GFP: 10% power , 0 . 05 s exposure . At each time point ( every 10 min for 5–7 hr ) , 25 Z-sections were acquired at 0 . 26 μm spacing . Identical imaging parameters were used to image a strain expressing endogenously tagged Sad1-mCherry ( Sad1 forms a single focus at the spindle pole body that contains between 450 and 1030 molecules ) and relative mCherry foci intensities were used to determine that ~30 molecules of Rad52-mCherry are required to detect a visible focus with these imaging parameters ( Wu and Pollard , 2005 ) . For the LacO resection assay , every cell cycle was tracked and quantified individually , including timing of nuclear division , cellular division , Rad52-mCherry focus formation , and LacO/LacI-GFP focus disappearance . Only on-target Rad52 foci ( that co-localized with LacO/LacI-GFP for at least 2 frames ) were considered , since many DSB events occur throughout the genome spontaneously , especially during S-phase . The number of cells and events used to generate the plots in all Figures is included as Supplementary file 2 . The time between the first frame with an on-target Rad52-mCherry focus and the first frame with complete disappearance of the LacO/LacI-GFP focus is the duration of resection through 3 . 57 kb ( between the HO cut site and the start of the LacO repeats ) plus the full 10 . 3 kb LacO array . All fields from all genotypes were input into custom ImageJ macros that randomized the order of the fields/genotypes , blinded the images by removal of the file names , set the contrast to be identical for every image , and numbered each cell lineage . Each blinded field was then manually assessed for photobleaching of the LacO/LacI-GFP foci in cells without induced DSBs ( >80% of all cells ) to ensure that disappearance of any LacO/LacI-GFP foci in cells with on-target DSBs was due to resection through the LacO array rather than photobleaching of the GFP signal . Next , using the pre-determined contrast settings for mCherry and GFP channels ( to maintain consistency across all images analyzed ) individual cells which had on-target DSB events were manually identified , and scored for the first frame of Rad52-mCherry focus appearance and then the first frame in which the LacO/LacI-GFP focus had completely disappeared . For focus intensity plots ( e . g . Figure 1D , E ) , quantification was performed in ImageJ on the full 5D image stacks ( not maximum intensity projections , which are shown in the image panels throughout for ease of viewing , for example Figure 1B ) . Subpixel measurements were made in a cylinder approximating the point spread function surrounding the manually scored subpixel center of the focus . A cylindrical shell surrounding the focus was used for background subtraction in both the GFP and mCherry channels . Raw data were processed , visualized , and analyzed using R , in particular packages dplyr , ggplot2 , and broom . Raw data , raw analysis for all individual cells included in plots , complete code , and other supporting materials are publically available on GitHub https://github . com/lelandbr/Leland_King_2018_eLife_Rev7_EndResection ( King and Leland , 2018; copy archived at https://github . com/elifesciences-publications/Leland_King_2018_eLife_Rev7_EndResection ) . Cells were grown overnight in YE5S media . Concentrations for each culture were monitored by both OD600 and a Coulter Principle cell counter ( Orflow Moxi Z ) . Cultures were diluted as needed to ensure identical numbers of cells were spotted for each genotype , starting with ~4×106 cell/mL and going down by sixfold dilutions . Plates were prepared using standard procedures ( Moreno et al . , 1991 ) , with the addition of 30 μM camptothecin ( Sigma; ≥95% HPLC purified ) after autoclaving .
Poly ( ADP ribose ) polymerase inhibitors ( PARPi ) target cancer cells deficient in homology-directed repair of DNA double-strand breaks ( DSBs ) . In preclinical models , PARPi resistance is tied to altered nucleolytic processing ( resection ) at the 5’ ends of a DSB . For example , loss of either 53BP1 or Rev7/MAD2L2/FANCV derepresses resection to drive PARPi resistance , although the mechanisms are poorly understood . Long-range resection can be catalyzed by two machineries: the exonuclease Exo1 , or the combination of a RecQ helicase and Dna2 . Here , we develop a single-cell microscopy assay that allows the distinct phases and machineries of resection to be interrogated simultaneously in living S . pombe cells . Using this assay , we find that the 53BP1 orthologue and Rev7 specifically repress long-range resection through the RecQ helicase-dependent pathway , thereby preventing hyper-resection . These results suggest that ‘rewiring’ of BRCA1-deficient cells to employ an Exo1-independent hyper-resection pathway is a driver of PARPi resistance .
Healthy cells can typically repair damage to their DNA with high accuracy , keeping their genetic code intact . In contrast , cancer cells often lose this ability . Inaccurate repair leads to more frequent DNA mutations , which can make a tumor more aggressive . However , DNA repair-deficient tumors can be targeted with cancer therapies , such as PARP inhibitors , which kill cells that do not have working DNA repair mechanisms . PARP inhibitors show great promise clinically , but unfortunately some tumor cells can become resistant to these treatments over time . Recent work has shown that resistance to PARP inhibitors is often caused by further alternations to DNA repair machineries . Being able to visualize DNA repair in living cells is crucial to understanding this process and to find ways to improve cancer treatments . Previous studies have used repetitive DNA sequences called Lac operators ( LacO ) to visualize the dynamic behavior of DNA in live cells . Leland et al . have now adapted this system to watch individual DNA repair events in living yeast cells under the microscope . Their experiments reveal that when cells lose a single protein called Rev7 , an early phase of DNA repair becomes hyperactive . Leland et al . traced the cause of this hyperactivity to an enzyme in the RecQ helicase family . A RecQ helicase becoming hyperactive in cells lacking Rev7 could explain how some cancer cells become resistant to PARP inhibitor treatments . This information could help fine-tune future approaches to treating cancer . For example , using an inhibitor of RecQ helicase alongside PARP inhibitors may help block this type of resistance from developing in the first place . As well as potentially paving the way for better cancer treatments , this method of visualization could improve scientists’ understanding of the basic processes of DNA repair .