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• W2892342015 abstract "•PIF1 is involved in DNA resection•G-quadruplexes impair resection•PIF1 is required to resect over sequences prone to forming G-quadruplexes•PIF1 interacts with BRCA1 to resolve G-quadruplexes at DNA breaks DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression. DNA breaks are complex lesions that can be repaired either by non-homologous end joining (NHEJ) or by homologous recombination (HR). The decision between these two routes of DNA repair is a key point of the DNA damage response (DDR) that is controlled by DNA resection. The core machinery catalyzing the resection process is well established. However, little is known about the additional requirements of DNA resection over DNA structures with high complexity. Here, we found evidence that the human helicase PIF1 has a role in DNA resection, specifically for defined DNA regions, such as those prone to form G-quadruplexes. Indeed, PIF1 is recruited to the site of DNA damage and physically interacts with proteins involved in DNA resection, and its depletion causes DNA damage sensitivity and a reduction of HR efficiency. Moreover, G4 stabilization by itself hampers DNA resection, a phenomenon suppressed by PIF1 overexpression. DNA is constantly exposed to different sources of DNA damage that can alter its chemical or physical structure. Within the different types of DNA lesions, DNA double-strand breaks (DSBs) are considered one of the most cytotoxic DNA injuries because they can lead to chromosomal aberrations and cell death. In order to maintain genomic stability, cells have developed a well-coordinated signaling cascade to sense and repair these DNA alterations known as the DNA damage response (DDR), which results in cell cycle arrest, senescence, activation of DNA repair pathways, stress responses, and/or apoptosis. There are two main pathways to repair DSBs: non-homologous end joining (NHEJ) and homologous recombination (HR). On the one hand, NHEJ is based on the direct ligation of the broken DNA ends with little or no DNA end processing and it is the main mechanism to repair DSBs during G0 and G1 phases of the cell cycle (Davis and Chen, 2013Davis A.J.A. Chen D.J. DNA double strand break repair via non-homologous end-joining.Transl. Cancer Res. 2013; 2: 130-143PubMed Google Scholar). On the other hand, HR can accurately restore the DNA molecule using an intact homologous DNA sequence from the sister chromatid as the repair template (Jasin and Rothstein, 2013Jasin M. Rothstein R. Repair of strand breaks by homologous recombination.Cold Spring Harb. Perspect. Biol. 2013; 5: a012740Crossref PubMed Scopus (549) Google Scholar). Because HR prefers the sister chromatid to repair the DSB, this pathway is usually restricted to the S and G2 phases of the cell cycle. If HR uses as donor sequence a DNA molecule different from the sister chromatid, loss of heterozygosity and even chromosome aberrations might be produced. Thus, the choice of the incorrect repair pathway might lead to genomic instability and, in consequence, to different diseases, including cancer. Additionally, DSBs might be sealed by a third type of repair pathways known as alternative-NHEJ (alt-NHEJ) or microhomology-mediated end joining (MMEJ). This alternative repair shares characteristics with both HR and NHEJ, uses short stretches of homology (microhomologies), and is always mutagenic (Sfeir and Symington, 2015Sfeir A. Symington L.S. Microhomology-mediated end joining: a back-up survival mechanism or dedicated pathway?.Trends Biochem. Sci. 2015; 40: 701-714Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). DNA resection is the first step of HR and acts to promote this repair pathway and blocks NHEJ (Huertas, 2010Huertas P. DNA resection in eukaryotes: deciding how to fix the break.Nat. Struct. Mol. Biol. 2010; 17: 11-16Crossref PubMed Scopus (298) Google Scholar, Symington, 2014Symington L.S. End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb. Perspect.Biol. 2014; 6: a016436Google Scholar). MMEJ also requires resection to expose the short homologies implicated in the repair process but to a much shorter extent (Sfeir and Symington, 2015Sfeir A. Symington L.S. Microhomology-mediated end joining: a back-up survival mechanism or dedicated pathway?.Trends Biochem. Sci. 2015; 40: 701-714Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar). During resection, 5′ ends at DSBs are processed to obtain 3′ single-stranded DNA overhangs, which will invade a homologous DNA molecule and will act as primers for DNA synthesis. Resection is initiated by the MRE11-RAD50-NBS1 (MRN) complex that recognizes the DSB. Although MRE11 has endonuclease and exonuclease activities, it needs an additional factor, CtIP, to integrate several cellular signals in order to license resection only when the appropriate criteria are met (Cejka, 2015Cejka P. DNA end resection: nucleases team up with the right partners to initiate homologous recombination.J. Biol. Chem. 2015; 290: 22931-22938Crossref PubMed Scopus (137) Google Scholar, Huertas, 2010Huertas P. DNA resection in eukaryotes: deciding how to fix the break.Nat. Struct. Mol. Biol. 2010; 17: 11-16Crossref PubMed Scopus (298) Google Scholar, Makharashvili and Paull, 2015Makharashvili N. Paull T.T. CtIP: A DNA damage response protein at the intersection of DNA metabolism.DNA Repair (Amst.). 2015; 32: 75-81Crossref PubMed Scopus (66) Google Scholar, Symington, 2014Symington L.S. End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb. Perspect.Biol. 2014; 6: a016436Google Scholar). This initial resection, termed short-range resection, is followed by an extension of the length of single-stranded DNA (ssDNA) in a process denominated long-range resection and catalyzed by either EXO1 or the helicase-nuclease pair BLM-DNA2 (Cejka, 2015Cejka P. DNA end resection: nucleases team up with the right partners to initiate homologous recombination.J. Biol. Chem. 2015; 290: 22931-22938Crossref PubMed Scopus (137) Google Scholar, Huertas, 2010Huertas P. DNA resection in eukaryotes: deciding how to fix the break.Nat. Struct. Mol. Biol. 2010; 17: 11-16Crossref PubMed Scopus (298) Google Scholar, Symington, 2014Symington L.S. End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb. Perspect.Biol. 2014; 6: a016436Google Scholar). This resection machinery is well conserved in all eukaryotes (Cejka, 2015Cejka P. DNA end resection: nucleases team up with the right partners to initiate homologous recombination.J. Biol. Chem. 2015; 290: 22931-22938Crossref PubMed Scopus (137) Google Scholar, Huertas, 2010Huertas P. DNA resection in eukaryotes: deciding how to fix the break.Nat. Struct. Mol. Biol. 2010; 17: 11-16Crossref PubMed Scopus (298) Google Scholar, Symington, 2014Symington L.S. End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb. Perspect.Biol. 2014; 6: a016436Google Scholar). Indeed, the human CtIP-MRN complex, or its counterpart Sae2-MRX in budding yeast, has been proven to constitute the minimal core resection initiation machinery in vitro (Anand et al., 2016Anand R. Ranjha L. Cannavo E. Cejka P. Phosphorylated CtIP functions as a co-factor of the MRE11-RAD50-NBS1 endonuclease in DNA end resection.Mol. Cell. 2016; 64: 940-950Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar, Nicolette et al., 2010Nicolette M.L. Lee K. Guo Z. Rani M. Chow J.M. Lee S.E. Paull T.T. Mre11-Rad50-Xrs2 and Sae2 promote 5′ strand resection of DNA double-strand breaks.Nat. Struct. Mol. Biol. 2010; 17: 1478-1485Crossref PubMed Scopus (170) Google Scholar, Shim et al., 2010Shim E.Y. Chung W.H. Nicolette M.L. Zhang Y. Davis M. Zhu Z. Paull T.T. Ira G. Lee S.E. Saccharomyces cerevisiae Mre11/Rad50/Xrs2 and Ku proteins regulate association of Exo1 and Dna2 with DNA breaks.EMBO J. 2010; 29: 3370-3380Crossref PubMed Scopus (174) Google Scholar). Although the central core of DNA resection is, therefore, well established, only little is known about how the velocity or processivity of DNA resection is modulated. As an illustration, the tumor suppressor BRCA1 affects the processivity of resection (Cruz-García et al., 2014Cruz-García A. López-Saavedra A. Huertas P. BRCA1 accelerates CtIP-mediated DNA-end resection.Cell Rep. 2014; 9: 451-459Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar). Such regulation will impact in the decision between HR and NHEJ but also between different HR subpathways (Ceccaldi et al., 2016Ceccaldi R. Rondinelli B. D’Andrea A.D. Repair pathway choices and consequences at the double-strand break.Trends Cell Biol. 2016; 26: 52-64Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). An important open question is whether the resection machinery needs additional effectors when faced with DNA regions of unusual configurations. One example would be G-quadruplexes (G4s), a DNA-secondary structure formed by four guanines associated through Hoogsteen hydrogen bonding that forms a G-quartet. The planar G-quartets stack on top of each other, giving rise to four-stranded helical structures (Lipps and Rhodes, 2009Lipps H.J. Rhodes D. G-quadruplex structures: in vivo evidence and function.Trends Cell Biol. 2009; 19: 414-422Abstract Full Text Full Text PDF PubMed Scopus (648) Google Scholar). Interestingly, recently it has been described that G-quadruplex-stabilizing compounds, such as pyridostatin (PYR) or CX-5461, are toxic to BRCA1-deficient cells (Xu et al., 2017Xu H. Di Antonio M. McKinney S. Mathew V. Ho B. O’Neil N.J. Santos N.D. Silvester J. Wei V. Garcia J. et al.CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours.Nat. Commun. 2017; 8: 14432Crossref PubMed Scopus (296) Google Scholar, Zimmer et al., 2016Zimmer J. Tacconi E.M.C. Folio C. Badie S. Porru M. Klare K. Tumiati M. Markkanen E. Halder S. Ryan A. et al.Targeting BRCA1 and BRCA2 deficiencies with G-quadruplex-interacting compounds.Mol. Cell. 2016; 61: 449-460Abstract Full Text Full Text PDF PubMed Scopus (146) Google Scholar). Thus, it remains possible that such toxicity might stem of an impairment of DNA resection in the presence of stable G4s and in the absence of processivity factors, such as BRCA1. Several helicases, including FANCJ, BLM, or WRN, have been shown to be able to unwind G4s (Mendoza et al., 2016Mendoza O. Bourdoncle A. Boulé J.B. Brosh Jr., R.M. Mergny J.L. G-quadruplexes and helicases.Nucleic Acids Res. 2016; 44: 1989-2006Crossref PubMed Scopus (270) Google Scholar, Murat and Balasubramanian, 2014Murat P. Balasubramanian S. Existence and consequences of G-quadruplex structures in DNA.Curr. Opin. Genet. Dev. 2014; 25: 22-29Crossref PubMed Scopus (275) Google Scholar, Sanders, 2010Sanders C.M. Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity.Biochem. J. 2010; 430: 119-128Crossref PubMed Scopus (123) Google Scholar), but PIF1 helicase is considered the most specific and active on these structures (Bochman et al., 2012Bochman M.L. Paeschke K. Zakian V.A. DNA secondary structures: stability and function of G-quadruplex structures.Nat. Rev. Genet. 2012; 13: 770-780Crossref PubMed Scopus (959) Google Scholar). The PIF1 family of helicases is highly conserved from yeast to humans and belongs to the superfamily of helicases 1 (Sabouri, 2017Sabouri N. The functions of the multi-tasking Pfh1Pif1 helicase.Curr. Genet. 2017; 63: 621-626Crossref PubMed Scopus (17) Google Scholar). PIF1 plays a role in multiple DNA transactions, including regulation of telomere homeostasis, replication induced by DSBs, transcription, and G4s resolution (Bochman et al., 2010Bochman M.L. Sabouri N. Zakian V.A. Unwinding the functions of the Pif1 family helicases.DNA Repair (Amst.). 2010; 9: 237-249Crossref PubMed Scopus (169) Google Scholar, Gagou et al., 2014Gagou M.E. Ganesh A. Phear G. Robinson D. Petermann E. Cox A. Meuth M. Human PIF1 helicase supports DNA replication and cell growth under oncogenic-stress.Oncotarget. 2014; 5: 11381-11398Crossref PubMed Scopus (24) Google Scholar, Sabouri, 2017Sabouri N. The functions of the multi-tasking Pfh1Pif1 helicase.Curr. Genet. 2017; 63: 621-626Crossref PubMed Scopus (17) Google Scholar). This helicase binds to partially ssDNA and unwinds G4 structures suppressing G4-induced DNA damage (Sanders, 2010Sanders C.M. Human Pif1 helicase is a G-quadruplex DNA-binding protein with G-quadruplex DNA-unwinding activity.Biochem. J. 2010; 430: 119-128Crossref PubMed Scopus (123) Google Scholar). Although the human genome encodes a single PIF1 gene, through alternative splicing, it produces two different transcripts. The long transcript produces PIF1α protein that is located in the nucleus, and the short one produces PIF1β that is found in the mitochondria (Sabouri, 2017Sabouri N. The functions of the multi-tasking Pfh1Pif1 helicase.Curr. Genet. 2017; 63: 621-626Crossref PubMed Scopus (17) Google Scholar). Here, we report several lines of evidence that involve human PIF1α (from here on PIF1) in HR. Indeed, we propose an additional role for this helicase specifically at the resection step of the recombination process. Our data suggest that the helicase activity of PIF1 is particularly relevant for resection when G4 structures are stabilized on the DNA. As previously mentioned, little is known of the additional factors that might help DNA resection machinery when confronted with DNA structures that are problematic. We reasoned that, as for almost every single DNA transaction, helicases would be in charge to reshape such unusual DNA configurations to facilitate the process. To find those helicases, we used an indirect approach and took advantage of the SeeSaw Reporter (SSR) (see Figure 1A, left) and the genome-wide screening we recently published (Gomez-Cabello et al., 2013Gomez-Cabello D. Jimeno S. Fernández-Ávila M.J. Huertas P. New tools to study DNA double-strand break repair pathway choice.PLoS ONE. 2013; 8: e77206Crossref PubMed Scopus (29) Google Scholar, López-Saavedra et al., 2016López-Saavedra A. Gómez-Cabello D. Domínguez-Sánchez M.S. Mejías-Navarro F. Fernández-Ávila M.J. Dinant C. Martínez-Macías M.I. Bartek J. Huertas P. A genome-wide screening uncovers the role of CCAR2 as an antagonist of DNA end resection.Nat. Commun. 2016; 7: 12364Crossref PubMed Scopus (31) Google Scholar) to look for different helicases in the choice between DSB repair pathways. This reporter analyses the choice between HR and NHEJ at very early stages; thus, it is particularly sensitive to changes in DNA resection velocity and/or processivity. Briefly, the SSR measures the balance between NHEJ and HR based on the accumulation of distinct fluorescent proteins (GFP for NHEJ events and red fluorescent protein (RFP) for HR events; in this case, a specific subpathway termed single-strand annealing [SSA]; Ceccaldi et al., 2016Ceccaldi R. Rondinelli B. D’Andrea A.D. Repair pathway choices and consequences at the double-strand break.Trends Cell Biol. 2016; 26: 52-64Abstract Full Text Full Text PDF PubMed Scopus (842) Google Scholar). Alterations of the normal balance toward a relative increase of HR or NHEJ can be detected using this reporter (Gomez-Cabello et al., 2013Gomez-Cabello D. Jimeno S. Fernández-Ávila M.J. Huertas P. New tools to study DNA double-strand break repair pathway choice.PLoS ONE. 2013; 8: e77206Crossref PubMed Scopus (29) Google Scholar, Jimeno et al., 2015Jimeno S. Fernández-Ávila M.J. Cruz-García A. Cepeda-García C. Gómez-Cabello D. Huertas P. Neddylation inhibits CtIP-mediated resection and regulates DNA double strand break repair pathway choice.Nucleic Acids Res. 2015; 43: 987-999Crossref PubMed Scopus (38) Google Scholar, López-Saavedra et al., 2016López-Saavedra A. Gómez-Cabello D. Domínguez-Sánchez M.S. Mejías-Navarro F. Fernández-Ávila M.J. Dinant C. Martínez-Macías M.I. Bartek J. Huertas P. A genome-wide screening uncovers the role of CCAR2 as an antagonist of DNA end resection.Nat. Commun. 2016; 7: 12364Crossref PubMed Scopus (31) Google Scholar). As expected (see Figure 1A, right), depletion of either BLM or RTEL1, proteins with known roles in HR at the level of DNA resection, skewed the balance toward an increase in NHEJ (Gomez-Cabello et al., 2013Gomez-Cabello D. Jimeno S. Fernández-Ávila M.J. Huertas P. New tools to study DNA double-strand break repair pathway choice.PLoS ONE. 2013; 8: e77206Crossref PubMed Scopus (29) Google Scholar, Gravel et al., 2008Gravel S. Chapman J.R. Magill C. Jackson S.P. DNA helicases Sgs1 and BLM promote DNA double-strand break resection.Genes Dev. 2008; 22: 2767-2772Crossref PubMed Scopus (453) Google Scholar, Youds et al., 2010Youds J.L. Mets D.G. McIlwraith M.J. Martin J.S. Ward J.D. ONeil N.J. Rose A.M. West S.C. Meyer B.J. Boulton S.J. RTEL-1 enforces meiotic crossover interference and homeostasis.Science. 2010; 327: 1254-1258Crossref PubMed Scopus (128) Google Scholar). Impairing the activity of the replication helicase minichromosome maintenance (MCM) by downregulation of almost any of its subunits also increased the relative contribution of NHEJ, probably due to an accumulation of S phase cells due to their role in DNA replication (Martinez et al., 2017Martinez M.P. Wacker A.L. Bruck I. Kaplan D.L. Eukaryotic replicative helicase subunit interaction with DNA and its role in DNA replication.Genes (Basel). 2017; 8: E117Crossref PubMed Scopus (7) Google Scholar). In addition, depletion of the chromatin remodeler INO80 showed the opposite effect, with an increased HR, suggesting a role of this helicase favoring NHEJ. This agrees with the fact that mutations in INO80-specific subunits in yeast impair the binding of Mre11, Ku80, and Mec1 kinase at the DSB, resulting in defective error-prone NHEJ (van Attikum et al., 2007van Attikum H. Fritsch O. Gasser S.M. Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks.EMBO J. 2007; 26: 4113-4125Crossref PubMed Scopus (251) Google Scholar, Chambers and Downs, 2012Chambers A.L. Downs J.A. The RSC and INO80 Chromatin-Remodeling Complexes in DNA Double-Strand Break Repair. Elsevier, 2012Crossref Scopus (32) Google Scholar). Interestingly, the depletion of PIF1 had a similar phenotype of RTEL1 or BLM, suggesting a possible additional role of this DNA helicase in the HR branch of DSB repair (Figure 1A). In order to validate this idea, we first performed pathway-specific repair assays (Figures 1B–1D). In all cases, CtIP depletion, which blocks DNA resection, was used as a positive control. Briefly, in all reporters, a DSB is created by expression of the meganuclease I-SceI and its repair through one defined pathway renders the accumulation of GFP-positive cells. We observed that PIF1 depletion (for depletion efficiency, see Figures S1A–S1C) indeed impaired homology-directed repair, both the Rad51-independent single-strand annealing pathway, and also the Rad51-dependent gene conversion pathway (SA-GFP and DR-GFP reporters, respectively; Figures 1B and 1C). On the contrary, the impact on NHEJ was minimal (Figure 1D). Cell cycle is a major regulator of HR, as resection is limited or not existent in G1. However, we discarded that the observed HR defect was caused by an accumulation of G1 cells (Figure S1D). Thus, we conclude that PIF1 affects the balance between HR and NHEJ mainly by facilitating DNA HR. As expected from the repair defect, PIF1-depleted cells were mildly hyper-sensitive to agents that induce DSBs, such as ionizing radiation or camptothecin (Figure 1E). Our results are in agreement with the recently described new role of the PIF1 homolog Rrm3 in Saccharomyces cerevisiae in HR (Muñoz-Galván et al., 2017Muñoz-Galván S. García-Rubio M. Ortega P. Ruiz J.F. Jimeno S. Pardo B. Gómez-González B. Aguilera A. A new role for Rrm3 in repair of replication-born DNA breakage by sister chromatid recombination.PLoS Genet. 2017; 13: e1006781Crossref PubMed Scopus (14) Google Scholar), suggesting that the role of PIF1 in HR might be conserved through evolution. Moreover, also in budding yeast, it has been shown that scPIF1, the other homolog of this helicase, is required for D-loop extension during break-induced replication (Saini et al., 2013Saini N. Ramakrishnan S. Elango R. Ayyar S. Zhang Y. Deem A. Ira G. Haber J.E. Lobachev K.S. Malkova A. Migrating bubble during break-induced replication drives conservative DNA synthesis.Nature. 2013; 502: 389-392Crossref PubMed Scopus (243) Google Scholar, Wilson et al., 2013Wilson M.A. Kwon Y. Xu Y. Chung W.H. Chi P. Niu H. Mayle R. Chen X. Malkova A. Sung P. Ira G. Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration.Nature. 2013; 502: 393-396Crossref PubMed Scopus (225) Google Scholar). Proteins involved in DSB repair are commonly recruited to broken chromatin and can be visualized under the microscope as foci. We tested whether this was also the case for PIF1. Upon the induction of DSBs with the DSB-inducing agent neocarzinostatin (NCS), we readily observed the focal accumulation of GFP-PIF1 using an anti-GFP antibody (Figure 2A; Figure S2A shows a GFP control). The same effect was observed upon treatment with ionizing radiation (IR) (Figure 2B; Figure S2B shows a GFP control). Computer-based automatic scoring of the number of PIF1 foci per cell agreed with an increase of PIF1 accumulation upon DNA damage (Figure 2C). To confirm that such a punctuated pattern reflected the recruitment of PIF1 to the sites of DNA breaks, we used the U2OS19ptight13 cells, in which a single DSB is induced with I-SceI upon the addition of doxycycline at a chromosomal location carrying 256 repeats of the lacO that can be visualized as the accumulation of a cherry-lacI discrete dot (Figures 2D and 2E; Lemaître et al., 2014Lemaître C. Grabarz A. Tsouroula K. Andronov L. Furst A. Pankotai T. Heyer V. Rogier M. Attwood K.M. Kessler P. et al.Nuclear position dictates DNA repair pathway choice.Genes Dev. 2014; 28: 2450-2463Crossref PubMed Scopus (125) Google Scholar). As shown in Figure 2F, there was some background binding of PIF1 prior doxycycline addition, likely due to the DNA structure created by the 256 repeats of the lacO. But importantly, a clear induction of GFP-PIF1 recruitment was observed upon DSB induction with doxycycline. Indeed, this accumulation mirrored DSB appearance, measured as γH2AX accumulation (Figures 2E, 2F, and S2C). One likely explanation of the role of PIF1 in facilitating recombination and its recruitment to broken chromatin is that this helicase might be involved in DNA end resection. To test this idea, we studied replication protein A (RPA) foci formation upon ionizing radiation in PIF1 depleted cells. RPA is an ssDNA binding complex that accumulates at sites of DNA breaks as a direct consequence of DNA resection (Cejka, 2015Cejka P. DNA end resection: nucleases team up with the right partners to initiate homologous recombination.J. Biol. Chem. 2015; 290: 22931-22938Crossref PubMed Scopus (137) Google Scholar, Huertas, 2010Huertas P. DNA resection in eukaryotes: deciding how to fix the break.Nat. Struct. Mol. Biol. 2010; 17: 11-16Crossref PubMed Scopus (298) Google Scholar, Symington, 2014Symington L.S. End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb. Perspect.Biol. 2014; 6: a016436Google Scholar). Thus, the percentage of RPA-foci-positive cells is the gold standard readout of resection in mammalian cells. As shown in Figure 3A, depletion of PIF1 with two different small interfering RNAs (siRNAs) rendered a defect in resection efficiency that resembles, albeit to a lesser extent, downregulation of the key resection factor CtIP. To validate this observation, we used an alternative approach by quantifying the exposure of bromodeoxyuridine (BrdU)-labeled ssDNA in native conditions by fluorescence-activated cell sorting (FACS) as a proxy for ssDNA (Gómez-Cabello et al., 2017Gómez-Cabello D. Checa-Rodríguez C. Abad M. Serrano M. Huertas P. CtIP-specific roles during cell reprogramming have long-term consequences in the survival and fitness of induced pluripotent stem cells.Stem Cell Reports. 2017; 8: 432-445Abstract Full Text Full Text PDF PubMed Scopus (5) Google Scholar). BrdU epitope is hidden in the double-stranded structure of the DNA, so it cannot be detected by an antibody against it unless it is presented in a single-stranded form, either by denaturing the DNA or in native conditions by its exposure during DNA end resection. As seen in Figure 3B, in control cells, an increase of BrdU exposure in non-denaturing conditions after IR was observed. This was dependent on DNA end resection, as was completely abolished upon depletion of CtIP. Strikingly, PIF1 downregulation severely impairs BrdU exposure, as the signal remained close to the untreated control cells. Again, this defect was milder than the observed upon CtIP depletion, in agreement with an accessory role of PIF1 in resection. Both RPA foci formation and BrdU exposure depend at the same time on the number of breaks resected per cell and the extension of DNA resection. In order to analyze in more detail whether only resection initiation was impaired or whether also resection processivity was compromised, we used the single molecule analysis of resection tracks (SMART) technique, a high-resolution approach that measures resected DNA in individual DNA fibers (Cruz-García et al., 2014Cruz-García A. López-Saavedra A. Huertas P. BRCA1 accelerates CtIP-mediated DNA-end resection.Cell Rep. 2014; 9: 451-459Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, Huertas and Cruz-Garcia, 2018Huertas P. Cruz-Garcia A. Single molecule analysis of resection tracks.Methods Mol. Biol. 2018; 1672: 147-154Crossref PubMed Scopus (13) Google Scholar; Figure 3C). Interestingly, not only the number of breaks resected was reduced upon PIF1 depletion, but the average length of ssDNA formed during resection was severely reduced when measured. Indeed, our data suggested that the main role of PIF1 is resection processivity, as in this case the observed defect was similar to that caused by CtIP depletion. This will agree with the idea that PIF1 is not an integral part of the resection machinery but an accessory factor that acts during resection extension, unwinding atypical DNA structures but has a very limited effect in the decision on which breaks will be resected. In order to determine whether PIF1 was acting exclusively in one specific branch of resection, mainly the long-range resection catalyzed by either EXO1 or DNA2/BLM, we dissected its genetic relationship by targeting those factors with siRNA simultaneously to PIF1 depletion. We included also an siRNA against MRE11 as a key factor in the short-range resection machinery. We reasoned that, if PIF1 was exclusively in one of those pathways, its depletion would exacerbate the resection defect caused by downregulation of the other branch. As seen in the Figure 3D, PIF1 was epistatic with both EXO1 and DNA2, indicating it is likely acting on both pathways at the same time and that PIF1 depletion already hampers all long-range resection. Strikingly, PIF1 depletion mildly increased the defect observed upon MRE11 downregulation, likely due to targeting at the same time as both the short and long-range resection. Again, these observations agree with an accessory role of PIF1 during resection extension. Therefore, we conclude that PIF1 is an accessory factor that is helping resection progression mainly at the level of long-range resection, both in the DNA2 and EXO1 branches. To be sure that the observed phenotype was due to PIF1 and not to an off-target effect, we study resection at the level of RPA foci formation in cells bearing siRNA-resistant, GFP-tagged versions of PIF1 gene. Indeed, the resection impairment caused by depletion of PIF1 was rescued by wild-type GFP-PIF1 (Figure 3E). More importantly, this was not observed with expression of a helicase dead version of the protein (Figures 3E and S3A). Thus, we can confirm that PIF1 is acting on long-range DNA end resection through its helicase activity, most likely by unwinding atypical DNA structures. We wondered then why this additional helicase might be needed for DNA end processing, i.e., which kind of atypical DNA species PIF1 is unwinding. Based on the role of PIF1 in facilitating DNA transactions on specific DNA structures, such as those DNA sequences prone to form G-quadruplexes, we reasoned that those structures might act as roadblocks for resection. Indeed, addition of the G4 stabilizer pyridostatin (Rodriguez et al., 2012Rodriguez R. Miller K.M. Forment J.V. Bradshaw C.R. Nikan M. Britton S. Oelschlaegel T. Xhemalce B. Balasubramanian S. Jackson S.P. Small-molecule-induced DNA damage identifies alternative DNA structures in human genes.Nat. Chem. Biol. 2012; 8: 301-310Crossref PubMed Scopus (487) Google Scholar), on its own, reduced the length of resected DNA formed upon induct" @default.
• W2892342015 created "2018-09-27" @default.
• W2892342015 creator A5006814834 @default.
• W2892342015 creator A5018257780 @default.
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• W2892342015 creator A5063041811 @default.
• W2892342015 creator A5084262141 @default.
• W2892342015 date "2018-09-01" @default.
• W2892342015 modified "2023-10-02" @default.
• W2892342015 title "The Helicase PIF1 Facilitates Resection over Sequences Prone to Forming G4 Structures" @default.
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