Cyclin Kinase-independent role of p21CDKN1A in the promotion of nascent DNA elongation in unstressed cells
The p21 protein ( also known as p21CDKN1A and p21Cip1/Waf1 ) , is a member of the family of cyclin-dependent kinase ( CDK ) inhibitors ( CKIs ) which has long been known for its ability to consolidate the G1 and G2 arrest after DNA damage caused by genotoxic agents , such as γ irradiation ( γ IR ) ( Brugarolas et al . , 1995; Bunz et al . , 1998; Deng et al . , 1995; Dulic et al . , 1998 ) . As p21 has no enzymatic domain , it was not surprising to discover that a robust increase in p21 protein levels is required to achieve efficient CDK inhibition in response to DNA damage ( Boulaireet al . , 2000; Cai and Dynlacht , 1998 ) . Such observations have led to the widely-held assumption that the lower amounts of p21 in cycling cells are residual and insufficient to achieve any biological relevant function ( Bertolin et al . , 2015; Soria and Gottifredi , 2010 ) . However , p21 levels in cycling cells are not null . Albeit p21 does not efficiently inhibit CDK activity in cycling cells ( Cai and Dynlacht , 1998 ) it could still regulate CDK-independent processes . CDK-independent functions of p21 could rely on its proliferating cell nuclear antigen ( PCNA ) -interacting region ( PIR ) located on the C-terminus of p21 , which binds the interdomain connecting loop ( IDCL ) of PCNA with high affinity ( [Prives and Gottifredi , 2008] and references there in ) . However , no role for the p21/PCNA complex formation has been yet described . On the contrary , research has focused only on the biological relevance of disrupting the p21/PCNA interaction . As DNA polymerases ( pols ) also bind the IDCL of PCNA through PIR or PIP ( PCNA interacting protein ) boxes , p21 should be capable of negatively regulating all PCNA-dependent DNA synthesis processes in cells ( Moldovan et al . , 2007; Tsanov et al . , 2014 ) . In fact , in vitro experiments demonstrated that the large excess of p21´s PIR inhibits the interaction of PCNA with DNA replication and nucleotide excision repair ( NER ) factors ( for original papers refer to [Prives and Gottifredi , 2008] ) , impairing replication- and repair-associated DNA synthesis respectively ( see examples in [Cooper et al . , 1999; Gottifredi et al . , 2004; Prives and Gottifredi , 2008] ) . However , the amount of p21 used in such experiments were much higher than the maximal p21 levels that can be accumulated in cells , even after genotoxic treatments ( discussed in [Prives and Gottifredi , 2008] ) . The overexpression of p21 to levels in the range of those induced by genotoxins , inhibits neither replication- nor repair- associated DNA synthesis events ( Soria et al . , 2008 ) which are mostly dependent on replicative DNA pols ( Burgers , 1998; Soria and Gottifredi , 2010 ) . These data all together indicate that in cycling cells , physiological levels of p21 are not capable of inhibiting PCNA-dependent DNA synthesis by replicative DNA pols , even when p21 is induced by external stress . However , PCNA-dependent synthesis by other DNA pols could be inhibited by endogenous p21 . In fact , endogenous p21 levels drop dramatically after ultraviolet irradiation ( UV ) and Methyl methane sulfonate ( MMS ) treatments ( Soria et al . , 2006 ) . We have previously shown that p21 downregulation after UV facilitates nascent DNA elongation across UV-damaged DNA templates by enabling the recruitment of alternative ( alt ) DNA pols to replication factories ( Mansilla et al . , 2013 ) . Strikingly , UV irradiation couples translesion DNA synthesis ( TLS ) by alt DNA pols with the activation of the CRL4Cdt2 E3 ligase at replisomes ( Havens and Walter , 2011; Nishitani et al . , 2008; Soria and Gottifredi , 2010 ) . The CRL4Cdt2 E3 ligase binds and degrades p21 only when it is complexed with chromatin-associated PCNA ( Abbas et al . , 2008; Havens and Walter , 2011 ) . The list of genotoxic treatments that triggers p21 proteolysis has expanded lately and includes UV , MMS , cisplatin , hypoxia , hypoxia mimicking factors , hydroxyurea ( HU ) , aphidicolin ( APH ) , hydrogen peroxide , and potassium bromide ( Savio et al . , 2009 ) . In conclusion , the degradation of endogenous p21 at replication sites in S phase allows full TLS activation or fork-restart when required . While the above mentioned reports demonstrate the relevance of disrupting p21-PCNA interaction in cells , no report has ever addressed the relevance of the PCNA-p21 complex in cells . Here we report that endogenous p21 localizes at replication factories through PCNA binding , thereby avoiding DNA polymerase κ ( Pol κ ) to be recruited at replication factories . Surprisingly , in contradiction with its function as a negative regulator of CDKs , p21 facilitates S phase progression; that is p21 promotes nascent DNA elongation . The DNA replication defects caused by p21-depletion caused accumulation of replication stress markers , such as γH2AX and 53BP1 , instability of common fragile sites and micronuclei ( MN ) accumulation . Interestingly , all the replication defects observed in p21-depleted cells were eliminated when Pol κ was depleted , and were also complemented by a p21 mutant with an intact PCNA binding domain and a disrupted CDK binding site . Collectively , our data demonstrate that , although expressed at low levels in S phase , p21 fine-tunes the dynamics of DNA replication by regulating Pol κ loading to replisomes . Therefore , while the CDKs/p21 interaction is crucial to the cellular response to DNA damage , the PCNA/p21 interaction prevents the accumulation of DNA-damage independent genomic instability in unstressed cycling cells .
The limited amounts of p21 in cycling cells allow CDK-dependent cell cycle progression ( Kreis et al . , 2014 ) . Indeed , p21 levels in cycling cells are not null and can be detected on EdU positive cells with p21 specific antibodies ( Figure 1A and B ) as reported recently ( Coleman et al . , 2015 ) . Remarkably , while p21 levels are at the lowest in S phase ( Figure 1—figure supplement 1A , B ) , they are sufficient to impair TLS-dependent DNA synthesis ( Mansilla et al . , 2013; Soria and Gottifredi , 2010 ) if not degraded after UV irradiation ( Figure 1—figure supplement 1A , B ) . Notably , the function of p21 during unperturbed cell cycle progression remained unknown . A hint of such function was revealed by a Proximity ligation assay ( PLA ) which revealed a chromatin bound PCNA/p21 interaction in cycling cells . Such complexes resisted a mild extraction with CSK buffer which removes proteins unbound to chromatin ( Figure 1C–D ) . Consistent with our previous findings , the percentage of cells with PLA spots was reduced by UV irradiation and PLA spots were not detected upon p21 depletion ( Figure 1C–D ) . In agreement , endogenous p21 colocalized with PCNA ( Figure 1E and F ) and EdU-labelled replication factories ( Figure 1—figure supplement 1C ) . The colocalization of p21 and GFP-PCNA became more evident following removal of proteins unbound to chromatin ( Figure 1—figure supplement 2A ) . We next evaluated the requirement of the p21 PIR region for p21-PCNA colocalization . To this end , we transfected cells with either p21 or p21PIPMut* , bearing an intact or a disrupted PIR , respectively ( Mansilla et al . , 2013; Soria et al . , 2008 ) . The disruption of the CDK-binding site by point mutations in both constructs ( Mansilla et al . , 2013; Soria et al . , 2006 , 2008 ) , prevented the arrest outside S phase expected after p21 overexpression ( Figure 1—figure supplement 2B , C ) . Similar to endogenous p21 , overexpressed p21 localized to replication factories ( Figure 1G and Figure 1—figure supplement 3A , B ) . However , p21PIPMut* lost its ability to form foci at replication factories ( Figure 1H and Figure 1—figure supplement 3C ) , did not colocalize with PCNA ( Figure 1—figure supplement 3C ) and showed reduced chromatin retention ( Figure 1—figure supplement 3D and E ) . Hence , the PIR of p21 is required for the localization of p21 to replication factories in cycling cells . 10 . 7554/eLife . 18020 . 003Figure 1 . The PCNA interacting region of p21 facilitates the recruitment of p21 to replication factories in cycling cells . ( A ) Representative images of p21 in EdU positive and negative cells ( left panel ) and from U2OS cells transfected with control siRNA ( siLuc ) or sip21 ( right panel ) . ( B ) p21 intensity in the indicated samples . Nuclei were counterstained with DAPI . 70 nuclei/sample; two independent experiments were performed . ( C ) Representative images from Proximity ligation assay ( PLA ) performed after mild extraction on the indicated samples . ( D ) Quantification of PLA experiments described in C . 100 nuclei/sample; two independent experiments were performed . ( E ) Colocalization of p21 foci with GFP-PCNA which reveal replication factories ( mild extraction was applied ) . ( F ) Profiles of signal intensity along an arbitrary line ( showed in E ) drawn across the nuclei . ( G and H ) Samples were transfected with the indicated p21 mutants . Representative images are shown . Samples treated or not with CSK buffer were classified into the indicated categories . 100 nuclei/ sample; two independent experiments were analysed . For all figures in this manuscript: significance of the differences are: *p<0 . 1; **p<0 . 01; ***p<0 . 001 . When the difference is not statistically significant , the p value is not shown . Error bars represent SEM ( standard error of the mean ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 00310 . 7554/eLife . 18020 . 004Figure 1—figure supplement 1 . Endogenous p21 localizes to replication factories . ( A ) U2OS cells were not treated ( NT ) or UV irradiated with 20 J/m2 and 2 hr later EdU was incorporated for 10 min . EdU and p21 were revealed by Click-IT technology and specific antibodies respectively . Representative panels are showed . Zoomed images correspond to the indicated yellow boxes . ( B ) Intensity of p21 in the experiment shown in zA . 50 nuclei were counted and two independent experiments were performed . ( C ) EdU was incorporated for 10 min to U2OS cells . A CSK extraction buffer was used to retain only chromatin bound proteins . Endogenous p21 was revealed by immunofluorescence and the colocalization between p21 and EdU was determined generating profiles of signal intensity along an arbitrary line drawn across the nuclei . For all supplemental figures in this manuscript: significance of the differences are: *p<0 . 1; **p<0 . 01; ***p<0 . 001 . When the difference is not statistically significant , the p value is not shown . Error bars represent SEM ( standard error of the mean ) . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 00410 . 7554/eLife . 18020 . 005Figure 1—figure supplement 2 . Chromatin bound p21 is localized at replication factories . ( A ) The colocalization of p21 and p21PIPMut* to replication factories was evaluated before and after CSK extraction . While the localization of p21 to replication factories is evident in both conditions , p21PIPMut* does not localize to replication factories before extraction and is removed from the nuclei by CSK extraction . ( B ) The mutation of the CDK-binding domain of p21 allows unperturbed cell cycle progression in the presence of high levels of p21 . U2OS cells were transfected with the indicated expression vectors and the percentage of EdU positive cells was determined by analyzing 200 nuclei in each of the three independent experiments performed . ( C ) Western blot of p21 and p21PIPMut* transfection in U2OS cells . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 00510 . 7554/eLife . 18020 . 006Figure 1—figure supplement 3 . The PIR domain is required for p21 recruitment to replication factoriesU2OS cells were transfected with GFP-PCNA and p21 or p21PIPMut* respectively . ( A and C ) Representative images of panuclear and foci distribution of GFP-PCNA and p21 is shown for each non-extracted condition . ( B and D ) After pre-extraction with CSK buffer , the colocalization of p21 and PCNA was analyzed by confocal microscopy , generating profiles of signal intensity along an arbitrary line drawn across the nuclei . ( E ) p21 and p21PIPMut*expressing cells were subjected to CSK extraction for the indicated times . ( F ) The p21 relative retention into the insoluble fraction was quantified by densitometric analysis . The values on the plot were normalized to the KU70 intensity for each sample . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 00610 . 7554/eLife . 18020 . 007Figure 2 . The depletion of endogenous p21 impairs the choreography of unperturbed DNA replication . ( A ) Western blot ( WB ) analysis showing p21 levels in U2OS cells transfected with the indicated siRNAs . ( B ) EdU positive cells . 200 nuclei/sample were analysed in three independent experiments . ( C ) The percentage of cells with CSK-resistant PCNA nuclear retention . 300 nuclei/sample were analysed in three independent experiments . ( D ) Relative amount of cells with early or mid/late PCNA distribution . 100 nuclei/sample were examined in three independent experiments . ( E ) Representative fibers from control ( siLuc ) or sip21 transfected cells . ( F ) IdU track length . 100 fibers/samples were analysed in three independent experiments . ( G ) Schematic representation of the different structures that can be measured in the fiber analysis . ( H ) Samples in F were used to analyse the frequency of origin firing as the relative number of origins [ ( red-green-red + red only fibers ) /total fibers] . 200 fibers/samples were analysed in three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 00710 . 7554/eLife . 18020 . 008Figure 2—figure supplement 1 . Stable p21 depletion cause alterations in the DNA replication choreography of HCT116 cells . ( A ) Whole cell extracts from isogenic HCT116 p21+/+ and HCT116 p21−/− were subjected to Western Blot analysis to verify p21 elimination . ( * ) represents unspecific band . ( B ) IdU track length was measured after examining 100 fibers in three independent experiments ( C ) Representative field showing DNA fibers in HCT116 p21+/+ and HCT116 p21−/− . Yellow arrows indicate representative of bicolor fibers in each condition . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 00810 . 7554/eLife . 18020 . 009Figure 3 . In the absence of DNA damage , replication stress markers and genomic instability increase when p21 is depleted . ( A ) Quantification of γ H2AX intensity in the nucleus of U2OS transfected with the indicated siRNA . 200 nuclei/sample were examined in three independent experiments . Representative images are shown in the lower part of the panel . ( B ) U2OS cells with more than five 53BP1 foci were quantified . 200 cells/sample were analysed in three independent experiments . Representative images are shown in the lower part of the panel . ( C ) Quantification of cells with RPA foci . 150 nuclei positive for PCNA-resistance to CSK extraction/sample were examined in three independent experiments . ( D ) Quantification of cells with perinuclear DNA ( MN ) accumulation . 300 binucleated U2OS cells/samples were inspected in three independent experiments . Representative images are shown on the right . ( E ) Quantification of CFS expression for the indicated conditions . APH treatment corresponds to 0 . 2 µm for 24 hr . 50 metaphases from HCT116 cells/sample were examined in three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 00910 . 7554/eLife . 18020 . 010Figure 3—figure supplement 1 . Stable p21 depletion cause alterations in the genomic stability of HCT116 cells . ( A ) Representative images of γH2AX intensity in HCT116 p21+/+ and p21−/− cells . ( B ) Quantification of γH2AX intensity in HCT116 p21+/+ and p21−/− cells . 200 nuclei were counted in three independent experiments . ( C ) Quantification of cells with more than five 53BP1 foci in HCT116 cells . 200 nuclei were counted in three independent experiments . ( D ) Quantification of MN accumulation . 300 binucleated cells were analyzed in three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 010 On the basis of the localization of p21 to replication factories , we speculated that p21 could regulate the DNA replication dynamics during S phase . To test this hypothesis , we used p21-depleted U2OS osteosarcoma cells ( Figure 2A ) . The number of cells positive for EdU and with PCNA bound to chromatin increased after p21 depletion ( Figure 2B and C ) . An enrichment in PCNA focal distribution corresponding to mid-to-late S phase ( Essers et al . , 2005; Rey et al . , 2009 ) , was found in p21-depleted samples ( Figure 2D ) which suggested a defect intrinsic to S phase and independent from the G1/S transition . Therefore , we evaluated DNA replication parameters specific to S phase in p21-depleted U2OS cells by means of the DNA spreading technology . Nascent DNA was labelled with a 10-min pulse of CldU ( Chlorodeoxyuridine ) , followed by a 30-min pulse of IdU ( Iododeoxyuridine ) . After denaturalization , stretching and labelling with specific antibodies , DNA track lengths were quantified . Surprisingly , p21 loss resulted in a reduced track length suggesting that p21 is required for a proper elongation of the replication forks ( representative tracks and fields are shown in Figure 2E–F ) . These findings were confirmed in an additional cellular model , the HCT116 p21 null cells which were compared with the HCT116 p21+/+ counterparts ( Figure 2—figure supplement 1A–C ) . Such findings were unexpected from the perspective of the prevalent notion of p21 as a negative regulator of the cell cycle . Since defective fork elongation is generally coupled with increased origin firing ( Blow et al . , 2011; Pillaire et al . , 2007; Techer et al . , 2016 ) , we measured the origin frequency as we did in the past ( Vallerga et al . , 2015 ) [number of red-green-red tracks + red tracks only/total fibers] ( Figure 2G ) . Results indicated that p21 depletion upregulated origin firing in the absence of stress ( Figure 2H ) . Thus endogenous p21 is required for the optimal execution of the DNA replication program during unperturbed S phase . On the basis of the contribution of p21 to unperturbed DNA replication , we tested the effect of p21 depletion on the accumulation of markers of DNA replication stress . The intensity of γH2AX ( Mansilla et al . , 2013; Ward and Chen , 2001 ) increased both in U2OS transfected with p21 siRNA ( Figure 3A ) and in p21 −/− HCT116 cells ( Figure 3—figure supplement 1A–B ) . Also , the number of cells with more than five 53BP1 foci ( Mansilla et al . , 2013; Noon and Goodarzi , 2011 ) ( Figure 3B and Figure 3—figure supplement 1C ) and the number of cells with RPA foci which reveals long stretches of single stranded DNA ( Bergoglio et al . , 2013; Oakley and Patrick , 2010 ) increased when p21 was depleted ( Figure 3C ) . Thus , fork stalling and/or uncoupling events are more frequent in cells attempting to replicate DNA in the absence of p21 than in control samples . Replication-associated defects could trigger the accumulation of perinuclear DNA or micronuclei ( MN ) in binucleated cells that have finished karyocinesis ( Fenech , 2000 ) . Notably , MN acumulated in cells transiently or permanently depleted from p21 ( Figure 3D and Figure 3—figure supplement 1D , respectively ) . Another marker of genomic instability that is highly sensitive to replication defects is the rearrangements of common fragile sites ( CFS ) ( Le Tallec et al . , 2014; Letessier et al . , 2011 ) . Because of low origin density , the replication of CFSs is easily compromised by alterations in the replication program ( Letessier et al . , 2011; Ozeri-Galai et al . , 2011 , 2012 ) . Noteworthy , data in Figure 3E and F revealed that p21 depletion caused the accumulation of the FRA7H CFS instability to an extent similar to that caused by low doses of APH , a known inducer of CFS instability ( Bergoglio et al . , 2013; Sutherland et al . , 1985 ) . It follows that the depletion of p21 jeopardizes the duplication of hard-to-replicate genomic regions triggering replication-associated genomic instability . Besides p21 , novel negative regulators of alt DNA pols have been recently identified ( Bertolin et al . , 2015 ) . One of them , USP1 , has the ability to remove the ubiquitin moiety from PCNA ( Huang et al . , 2006; Niimi et al . , 2008 ) . Mono-ubiquitinated PCNA interacts with the UBM and UBZ domains of alternative DNA pols favouring their loading to the replisomes ( Bienko et al . , 2005; Kannouche and Lehmann , 2004; Plosky et al . , 2006 ) . During unperturbed replication , PCNA ubiquitination is limited by USP1 ( Huang et al . , 2006; Niimi et al . , 2008 ) , as evidenced by increased PCNA ubiquitination upon USP1 depletion ( Figure 4A and B ) . In constrast , the level of PCNA ubiquitination was not modified when p21 was depleted ( Figure 4A ) . Hence , p21 and USP1 regulate the recruitment of alt pols by distinct mechanisms ( Figure 4C ) . Intriguingly , despite such mechanistic differences , both p21 and USP1 depletion caused a similar effect ( both in quality and extent ) on nascent DNA elongation ( Figure 4D ) , origin frequency ( Figure 4E ) , the accumulation of cells with 53BP1 foci ( Figure 4F ) and the number of binucleated cells with MN ( Figure 4G ) . These results reinforce the function of p21 as a negative regulator of alt DNA pols . Moreover , mechanistically distinct regulators of alt DNA pols are equally required to preserve DNA replication and genomic stability during unperturbed replication . 10 . 7554/eLife . 18020 . 011Figure 4 . Mechanistically distinct regulators of alt DNA pols are required to facilitate unperturbed DNA replication . ( A ) W . B . analysis revealing PCNA , ubi-PCNA and p21 levels in U2OS cells transfected with the indicated siRNAs . ( B ) USP1 mRNA levels were examined by quantitative RT-PCR . ( C ) Model depicting the different mechanisms of PCNA regulation by p21 and USP1 . ( D ) IdU track length was measured in 85 fibers/sample in three independent experiments . ( E ) Origin firing frequency . 150 fibers/sample were analysed in three independent experiments . ( F ) Quantification of cells with more than five 53BP1 foci . 200 U2OS cells/sample were analysed in three independent experiments . ( G ) MN accumulation . 300 binucleated cells/sample were analyzed in three independent experiments . Data on the effect of USP1 downregulation on the modulation of nascent DNA elongation ( Figure 4D ) and accumulation of cells with 53BP1 foci ( Figure 4F ) and micronuclei ( Figure 4G ) was reproduced from Jones et al , EMBO . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 011 T . Huang and colleagues have previously reported that MN accumulation induced by USP1 depletion depends on Pol κ ( Jones et al . , 2012 ) . Therefore , we set to explore the effect of p21 on Pol κ recruitment to DNA replication factories . First , we observed that Pol κ foci were formed only in a modest percentage of control cycling cells ( siLuc in Figure 5A–B ) . However , when p21 was depleted , the percentage of cells with Pol κ foci raised significantly ( Figure 5A–B ) . Second , the interaction of PCNA and GFP-Pol κ in the chromatin fraction increased when p21 was depleted ( Figure 5C ) . Third , using PLA an increase in the number of endogenous PCNA/Pol κ interacting foci was revealed in p21-depleted samples ( Figure 5D–E ) . We hypothesized that an increased recruitment of Pol κ to the replication forks in p21-depleted cell may slow down DNA elongation , triggering fork collapse and/or the generation of under-replicated DNA . To test this hypothesis , Pol κ was down-regulated in p21-depleted cells ( Figure 6A ) and different DNA replication parameters were tested . Forty eight hours after siRNA transfection , Pol κ depletion alone had no effect on most parameters , except from a modest increase in RPA foci formation ( Figure 6—figure supplement 1 ) . Such result may be in agreement with the role of Pol κ in the replication of non-B DNA regions such as G4 cuadruplex ( Betous et al . , 2009 ) . Notably however , the simultaneous elimination of Pol κ and p21 prevented all the phenotypes associated with p21 depletion . Specifically , Pol κ depletion rescued the defective nascent DNA elongation ( Figure 6B ) , the origin frequency ( Figure 6C ) , the percentage of EdU positive cells ( Figure 6—figure supplement 1A ) and the increased number of cells with chromatin bound-PCNA ( Figure 6—figure supplement 1B ) . Similarly , markers of replication stress such as RPA foci ( Figure 6—figure supplement 1C ) , γH2AX ( Figure 6—figure supplement 1D ) and 53BP1 foci ( Figure 6D ) were downregulated after simultaneous depletion of p21 and Pol κ . Similar results were obtained when using a second siRNA specific for Pol κ in U2OS cells ( Figure 6—figure supplement 2A–D ) and when employing a different cell line , HCT116 p21 −/− cells ( Figure 6—figure supplement 2E–F ) . 10 . 7554/eLife . 18020 . 012Figure 5 . The recruitment of pol κ to replication-associated structures increases in the absence of p21 . ( A ) U2OS cells were transfected with GFP-polκ . Representative images of cells with and without with GFP-pol κ foci . ( B ) Percentages of cells with GFP-pol κ focal organization . 150 nuclei/sample were analyzed in two independent experiments . ( C ) siLuc and sip21 depleted samples were subjected to chromatin immunoprecipitation using a monoclonal PCNA antibody . GFP-pol κ recruitment to chromatin was revealed by using GFP antibodies . The result was reproduced in three independent experiments . ( D ) Proximity Ligation Assay ( PLA ) between PCNA and endogenous pol κ was performed in U2OS cells . Two representative images of PLA in siLuc and sip21 cells are shown . ( E ) Quantification of PLA foci per nuclei . More than 1000 nuclei were analysed in three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 01210 . 7554/eLife . 18020 . 013Figure 6 . Pol κ depletion prevents the DNA replication defects and the genomic instability caused by p21 downmodulation . ( A ) Western blots showing GFP-pol κ and p21 levels in U2OS cells transfected with the indicated siRNAs . ( B ) Total IdU track length was evaluated in 100 fibers/sample in three independent experiments . ( C ) Frequency of origin firing . 200 fibers were analysed in three experiments . ( D ) Quantification of cells with 53BP1 foci . 200 cells were analysed in three independent experiments . ( E ) Quantification of MN accumulation . 300 binucleated cells/sample were analysed in three independent experiments . ( F ) Quantification of CFS instability in HCT116 cells transfected with the indicated siRNAs . 50 metaphases/sample were analysed in three independent experiments . ( G ) Representative images of the CFS analysed in F . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 01310 . 7554/eLife . 18020 . 014Figure 6—figure supplement 1 . Pol κ depletion prevents the accumulation of DNA replication stress markers caused by p21 downmodulation . ( A ) Quantification of EdU positive cells transfected with the indicated siRNAs . 200 nuclei per sample were analyzed in three independent experiments . ( B ) Quantification of CSK-resistant , PCNA positive U2OS cells transfected with the indicated siRNAs . 250 nuclei per sample were analyzed in three independent experiments . ( C ) Quantification of nuclei with more than 10 RPA foci in PCNA positive cells after transfection with the indicated siRNAs . 150 nuclei per sample were analyzed in three independent experiments . ( D ) Quantification of nuclear γH2AX intensity in cells transfected with the indicated siRNAs . 200 nuclei per sample were analyzed in two independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 01410 . 7554/eLife . 18020 . 015Figure 6—figure supplement 2 . Pol κ prevents the accumulation of DNA replication stress in p21 depleted cells independently of the siRNA used and in cells stably lacking p21 . ( A ) U2OS cells were transfected with sip21 and 2 different siRNA for Pol κ . Western Blot analysis was performed to detect endogenous p21 and GFP- Pol κ . ( B ) RT-PCR was performed to detect mRNA levels of p21 and Pol κ using the indicated siRNAs . ( C ) IdU track length was measured using 2 different siRNAs for Pol κ in siLuc and sip21 depleted cells . 100fibers/sample were counted in 2 independent experiments . ( D ) Cells with more than five 53BP1 foci were analyzed in cells depleted of p21 and using 2 different siRNAs for Pol κ . ( E ) RT-PCR was performed in HCT116 p21+/+ and p21−/− cells to determine mRNA levels of Pol κ . ( F ) IdU track length in HCT116 p21+/+ and p21−/− depleted of Pol κ . 100fibers/sample were counted in 2 independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 01510 . 7554/eLife . 18020 . 016Figure 6—figure supplement 3 . Pol κ but not pol η depletion prevents the DNA replication defects and the genomic instability caused by p21 downmodulation . ( A ) U2OS cells were transfected with the indicated siRNA and GFP-pol η . The percentage of cells with GFP-pol η foci was calculated after analyzing 250 nuclei in three independent experiments . ( B ) Representative images of cells with GFP-pol η foci . ( C ) U2OS cells were transfected with GFP-pol κ and the indicated siRNAs . Western blots were performed to detect GFP-tagged Pol κ , endogenous pol η and p21 . ( D ) Percentage of EdU positive cells were determined with the indicated siRNA . ( E ) DNA fiber analysis was performed in the indicated samples as described in Figure 2 . The total length of the IdU track was evaluated in 100 fibers in three independent experiments . ( F ) Samples in E were used to analyse the frequency of origins as described in Figure 2 . 200 fibers were analysed in three independent experiments . ( G ) Quantification of cells with more than five 53BP1 foci . 200 cells were scored in three independent experiments . ( H ) Quantification of MN accumulation . 300 binucleated cells were scored in three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 016 While MN accumulation was evident when p21 was knocked down , this was not observed after simultaneous depletion of Pol κ and p21 ( Figure 6E ) . CFS instability modestly increased in Pol κ-depleted cells and more pronouncedly in p21-samples . Remarkably , in the context of p21 elimination , instead of increasing CFS instability Pol κ depletion reverted the instability caused by p21 depletion ( Figure 6F–G ) Collectively , these findings indicate that the misuse of Pol κ causes multiple alterations in the DNA replication of p21-deficient cells . The recruitment of the alternative DNA polymerase pol eta ( Pol η ) to replication factories was also stimulated in the absence of p21 ( Figure 6—figure supplement 3A–C ) . However , in contrast to Pol κ , Pol η depletion did not rescue the replication defects triggered by p21 depletion ( see Figure 6—figure supplement 3D–H ) . Hence , the parameters of genomic instability explored in this study are predominantly associated with the misuse of Pol κ in p21-depleted samples . In addition to the well characterized and direct consequences of DNA replication stress , namely chromosomal breakage and aberrations , it has been shown that a fraction of under-replicated/unresolved genomic loci enter into mitosis . When not accurately processed in M phase , such DNA regions are converted into complex broken-DNA structures that are transmitted to daughter cells ( Minocherhomji et al . , 2015 ) . In G1 phase such DNA structures are sequestered and shielded in nuclear compartments described as nuclear 53BP1 bodies ( Bergoglio et al . , 2013; Harrigan et al . , 2011; Lukas et al . , 2011 ) . We therefore explored whether the altered DNA replication dynamics of p21-depleted cells could lead to the mitotic transmission of DNA damage . We first noticed an increase in the percentage of cells positive for the phosphorylated histone H3 , a bona-fide marker of G2/M transition ( Minocherhomji et al . , 2015 ) , upon p21 depletion which was again totally reversed by Pol κ depletion ( Figure 7A ) . To confirm the persistence of under-replicated DNA in mitosis , we used previously reported protocols ( Federico et al . , 2015; Minocherhomji et al . , 2015 ) to quantify 53BP1 body formation in G1 ( EdU-negative ) . We found a significant increase in the number of spontaneous 53BP1 nuclear bodies in G1 in the absence of p21 , as hallmark of incomplete DNA replication during the previous cell cycle , which was again dependent on Pol κ ( Figure 7B–D ) . We conclude from these experiments that the choice of Pol κ in the absence of p21 is sufficient to increase the vulnerability of fragile genomic regions by delaying replication completion at these sequences . Such alteration of the DNA replication dynamics appears to be specific to Pol κ since they were not rescued when Pol η was depleted ( Figure 7E–F ) . 10 . 7554/eLife . 18020 . 017Figure 7 . Pol κ-mediated replication defects of p21-depleted cells are transmitted to the M and G1 phases of the cell cycle . ( A ) Quantification of phospho-H3 positive U2OS . 200 cells/sample were analysed in three independent experiments . ( B ) Representative images of 53BP1 bodies outside S phase . Yellow arrows indicate EdU negative cells with 53BP1 foci ( C ) Percentages of EdU negative cells which are positive for 53BP1 bodies . 200 nuclei/sample were analysed in three independent experiments . ( D ) Distribution of EdU negative cells with increasing number of 53BP1 bodies per cell in the experiments showed in C . 100 nuclei/sample were analysed in three independent experiments . ( E ) Phospho-H3 accumulation was quantified in 200 nuclei/sample of three independent experiments . Samples depleted from pol κ and pol η were compared . ( F ) Percentages of cells with more than five 53BP1 foci were determined after analysing 200 nuclei/sample in three independent experiments . Samples depleted from pol κ and pol η were compared . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 017 Having established that Pol κ triggers replication defects of p21-depleted cells , it was important to determine whether p21 prevents the loading of Pol κ to replication factories and if its ability to interact with PCNA is relevant to such function . We used the p21 mutants described in Figure 1 , which are refractory to an siRNA directed to the 3´UTR of p21 ( Figure 8A ) . Lentiviral transduction allowed the expression of p21 and p21PIPMut* in almost all cells ( Figure 8B ) . A fully functional PCNA binding domain in p21 was required to down-modulate Pol κ foci formation to control levels ( Figure 8C ) . In agreement , the fork elongation defects and the excessive origin firing observed in p21- depleted samples were complemented by p21 , but not by p21PIPMut* ( Figure 8D–E ) . Additionally , the key role of the PIP domain of p21 was supported by experiments performed in HCT116 p21−/− cells . Such experiments revealed that p21 but not the p21PIPMut* mutant complement the replication defects of cells with a null mutation of the endogenous p21 alleles ( Figure 8 F–H ) . Notably , the accumulation of markers of replication stress and genomic instability in p21-depleted cells was abolished when overexpressing p21 , but not p21PIPMut* ( Figure 8I–J ) . Hence , we postulate a key role of the PIP domain of p21 in the preservation of DNA replication homeostasis . 10 . 7554/eLife . 18020 . 018Figure 8 . The PIR domain of p21 and not the CDK-binding domain is required to prevent DNA replication defects in p21 depleted cells during the unperturbed S phase . ( A ) W . B . analysis was performed to evaluate p21 expression in U2OS cells transfected with control or p21 siRNA and infected with p21 or p21PIPMut* lentiviruses 6 hr later . ( B ) Representative panel showing the efficiency of lentiviral infection ( >90% ) . ( C ) GFP-pol κ focal organization in samples depleted from p21 and infected with p21 or p21PIPMut* . ( D ) IdU track length was measured in 100 fibers/sample in three independent experiments . ( E ) Frequency of origin firing . 100 fibers/sample were analysed in three experiments . ( F ) Infection were performed in HCT116 p21+/+ and p21−/− cells . Representative panel showing the efficiency of lentiviral infection in HCT116 p21−/− s . ( G ) IdU track length was measured in 100 fibers/sample in three independent experiments . ( H ) Frequency of origin firing was measured in the experiments shown in G . 200 fibers/sample were analysed . ( I ) Quantification of the percentage of cells with more than 5 53BP1 foci/cell . 200 cells/sample were evaluated in three independent experiments . ( J ) Quantification of MN accumulation . 200 binucleated cells were analysed in three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 018 Given that our results indicate a novel antioncogenic role of p21 in the promotion of DNA replication it was important to determine whether this phenotype was not limited to cancer cells . To address such question we used primary cells from two independent sources: ( a ) human foreskin fibroblast ( HFF ) and ( b ) mesenchyimal stem cells isolated from umbilical cord ( MSC ) . As shown in Figure 9A and B , transfection of p21 siRNA efficiently depleted p21 from both cell types . p21 elimination caused a reduction in the elongation of nascent DNA ( Figure 9A–C ) , which was accompanied with an increase in origin firing ( Figure 9D ) . In turn , such alteration in DNA replication parameters correlated with the accumulation of cells with 53BP1 foci ( Figure 9E–F ) and micronuclei ( Figure 9 G–I ) . Hence , we postulate that p21 regulates protein-complex formation at the replisomes , promoting the choice of the most adequate polymerase and therefore protecting DNA replication homeostasis . Such novel function of p21 depends exclusively on its ability to interact with PCNA and is needed at every S phase to ensure the accurate finalization of DNA replication ( see model in Figure 10 ) . 10 . 7554/eLife . 18020 . 019Figure 9 . p21 depletion promotes replication stress and genomic instability in primary human cells . ( A and B ) IdU track length in Human Foreskin fibroblasts ( HFF ) and Umbilical Cord Mesenchymal Stem cells ( MSC ) . 100 fiber/sample were analysed in three independent experiments . Western blot showing p21 depletion in the indicated cell line . ( C ) Representative fibers in siLuc and sip21 HFF cells . ( D ) Origin frequency in HFF cells . 200 fiber/sample were analyzed in three independent experiments . ( E ) Cells with more than 5 53BP1 foci were analyzed in MSC cells . 200 nuclei/sample were analyzed in three independent experiments . ( F ) Representative images of 53BP1 in siLuc and sip21 MSC cells . ( G and H ) Quantification of MN accumulation . 200 binucleated cells were analyzed in HFF and MSC cells respectively in three independent experiments . ( I ) Representative images of Binulceated cells with and without MN . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 01910 . 7554/eLife . 18020 . 020Figure 10 . Model depicting the implication of our findings . p21 levels in S phase are low but sufficient to prevent pol κ loading to replication forks . When p21 is absent , the abnormal use of pol κ during unperturbed replication impairs nascent DNA elongation . While origin firing increases to compensate the slow fork progression , late replicating and origin-poor DNA regions , that is . CFS , are inefficiently duplicated . The replication defects accumulated in p21-depleted samples lead to genomic instability and transmission of replication defects to G1 . DOI: http://dx . doi . org/10 . 7554/eLife . 18020 . 020
An important implication of our findings is that the idea that p21 acts exclusively as a negative regulator of the cell cycle through the inhibition of cycling kinases ( Warfel and El-Deiry , 2013 ) is simplified , incomplete and limited to specific DNA-damaging scenarios . The results presented herein conclusively show that p21 is more often a positive rather than a negative regulator of the cell cycle , as its contribution is required at every S phase . Remarkably , such contribution relies exclusively on the p21/PCNA interaction . Previous work indicates that p21 can displace alt DNA pols from replicating DNA . First , Z . Livneh and colleagues showed that p21 impairs TLS events on transfected plasmids ( Avkin et al . , 2006 ) . Second , lower levels of p21 in cycling cells must be eliminated to promote nascent DNA elongation across UV damage templates by alt DNA pols ( Mansilla et al . , 2013 ) . Third , endogenous p21 delays the recruitment of alt DNA pols to UV-damaged replication factories in a manner that correlates with the extent of p21 degradation ( Mansilla et al . , 2013; Soria et al . , 2008 ) . Fourth , the PCNA-binding domain of p21 is required to inhibit TLS activation ( Avkin et al . , 2006; Mansilla et al . , 2013; Soria et al . , 2008 ) . It follows that alt DNA pols might be selectively sensitive to p21 levels which are insufficient to inhibit CDKs ( Soria and Gottifredi , 2010 ) . Such a difference in the levels of p21 required to inhibit different cellular processes indicates that p21 is locally concentrated at , and/or has very high affinity for PCNA ( Soria and Gottifredi , 2010 ) . Both replicative and alt DNA pols bind the IDCL of PCNA through PIR or PIP ( PCNA interacting protein ) regions ( Bertolin et al . , 2015; Moldovan et al . , 2007 ) . The PIR of p21 binds PCNA strongly , so that p21 is capable of disrupting the weaker interactions between PCNA and alt DNA pols ( Hishiki et al . , 2009; Mansilla et al . , 2013; Tsanov et al . , 2014 ) without affecting replicative DNA pols which have multiple PIP domains ( Soria and Gottifredi , 2010; Soria et al . , 2008 ) . Such difference may relate to the fact that replicative DNA pols utilize multiple domains and different sites to interact with PCNA ( Johansson et al . , 2004; Moldovan et al . , 2007 ) . There is a tight cross-regulation of PIP box-containing proteins at replication forks which is not yet completely understood . On the one hand , it has been demonstrated that proteins with strong PIP boxes such as p21 can remove alt pols such as Pol κ from replication factories ( this report and [Tsanov et al . , 2014] ) . On the other hand , this and previous reports ( Jones et al . , 2012; Pillaire et al . , 2007 ) suggest that the alt Pol κ can displace replicative pols from replisomes . Conversely however , p21-PCNA interaction in S phase does not impair genomic DNA synthesis ( Soria et al . , 2008 ) suggesting that p21 cannot displace replicative DNA pols from the replisome . Hence , some yet unknown factors/events need to be identified to understand the hierarchy of PCNA partners at forks . Moreover , variables such as the sequence of the DNA which is being replicated and the average retention time of a PCNA partner at different regions of the chromatin might have a role in such a puzzling cross-regulation among PCNA partners . We have shown that Pol κ is required for checkpoint activation at stalled forks ( Betous et al . , 2013 ) and during the replication of repetitive sequences ( Hile et al . , 2012 ) or naturally occurring non-B DNA structures ( Betous et al . , 2009 ) . In agreement , Pol κ depletion affected the CFS expression and RPA accumulation in our experimental settings . It is therefore possible that certain DNA regions may benefit from Pol κ-dependent DNA synthesis ( Betous et al . , 2009 ) while others it might require active displacement of Pol κ by p21 . If that is the case , p21 may prevent Pol κ recruitment to specific regions of the genome while prompt p21 degradation by CLR4Cdt2 might rapidly allow Pol κ binding to others . Such p21 function could be executed either by promoting the dissociation of Pol κ from replisomes or by preventing its recruitment to DNA regions that must be necessarily copied by replicative DNA pols ( such as for example B-regions in the exonic DNA ) . The PIR domain of p21 prevents the recruitment of Pol η , Pol ι , Pol κ and Rev1 to UV-damaged replication factories ( Bertolin et al . , 2015; Mansilla et al . , 2013 ) . Hence , it is unlikely that during unperturbed replication p21 would act as a specific inhibitor of Pol κ . The same is valid for USP1 because the modulation of PCNA ubiquitination should regulate all alt DNA pols ( Huang and D'Andrea , 2006; Jones et al . , 2012 ) . Hence , p21 and USP1 depletion may result in increased loading of all alt DNA pols to undamaged DNA . In fact , others have reported increased spontaneous mutation frequency in the hypoxanthine phosphoribosyl transferase ( hprt ) locus of p21−/− cells , ( McDonald et al . , 1996 ) . Such defects might depend on the unleashed activity of alt DNA pols other than Pol κ ( Yang and Woodgate , 2007 ) . Notwithstanding this , the phenotypes described herein are intimately associated with processive DNA synthesis events , and Pol κ is a very processive alt DNA pol ( the most processive in the Y family ) ( Ohashi et al . , 2000; Zhang et al . , 2000 ) . Because Polκ is less processive than replicative DNA pols ( McCulloch and Kunkel , 2008; Ohashi et al . , 2000 ) , the coupling of a replicative DNA pol and Pol κ at a single replisome may most likely generate asynchronic speed in both DNA strands . In turn , this may cause the transient accumulation of ssDNA in one strand ( see Figure 3C ) . In fact , low doses of APH , which are known to disrupt the DNA pol homeostasis during DNA replication , cause similar levels of CFS expression as p21 depletion ( see Figure 3E ) . Hence , the local degradation of p21 by CLR4CDT2 may maintain the most efficient DNA elongation speed by allowing the rapid and dynamic exchange of replicative DNA pols for Pol κ . In fact , a minor shift in the timing of CLR4CDT2-dependent p21 degradation is sufficient to accumulate cells in S phase ( Coleman et al . , 2015 ) . Notably , the elimination of not only p21 but also USP1 ( Jones et al . , 2012 ) increases the use of Pol κ during undamaged DNA replication , suggesting that the slightest modulation of Pol κ activity perturbs the DNA replication dynamics . In agreement , the expression of a Pol κ mutant with increased affinity for PCNA and the overexpression of Pol κ generate genomic instability and tumor formation ( Bavoux et al . , 2005a; 2005b; Bergoglio et al . , 2002; Hoffmann and Cazaux , 2010; O-Wang et al . , 2001; Jones et al . , 2012 ) . It is relevant to mention that the p21 knockout mice develop spontaneous tumors at 16 months ( Martin-Caballero et al . , 2001 ) , suggesting that modest but accumulative defects eventually trigger oncogenic transformation . Such observation highlights a role of p21 in the face of chronic , rather than acute stress and may correlate with the multiple defects in DNA replication revealed when depleting p21 from primary cells ( Figure 9 ) . While p21 is a negative regulator of TLS , its role in undamaged DNA replication ( revealed in this manuscript ) is most possibly unrelated to TLS inhibition . In fact , if p21 depletion caused the accumulation of DNA lesions , the elimination of an alt polymerase should: ( a ) aggravate the defect ( if the alt pol is essential to promote DNA replication across the replication barrier ) or ( b ) not alter such defect ( if the replication across such barrier is also achieved by another alt polymerase ) ( Bertolin et al . , 2015 ) . As our data show that the elimination of Pol κ promotes DNA replication , it does not support a model in which p21 depletion generates substrates ( DNA lesions ) for alt pols . On the contrary , it suggests that undamaged DNA is being misused by the alternative polymerase pol κ as a replication template . Hence , the contribution of p21 to unperturbed-DNA replication is most probably related to the control of DNA synthesis events on undamaged templates . In such scenario , the colocalization of the p21-CLR4cdt2 complex at replication forks may be crucial to control the composition of the active replisome , allowing the selection of the most adequate polymerase 'on the go' . Such dynamic regulation would favour the most effective DNA replication speed allowing the timely activation of late and hard-to-replicate genomic regions . The coupling of p21 with ongoing replisomes may also favor other p21 functions as homologous recombination ( HR ) events . While such p21 function has been linked to CDK inhibition ( Mauro et al . , 2012 ) , it is still possible that the loading of p21 to PCNA serves to orchestrate multiple transactions at the elongating DNA . While more work is required to reveal yet hidden cross-regulations among members of the replisome , our work unveils a novel tumor suppressor function of p21 dependent on its ability to interact with PCNA . Such a role of p21 is central for genome maintenance in the absence of DNA damage at every S phase and which might be key to prevent spontaneous oncogenic events .
All cell lines were received from reliable sources , either public repositories or directly , from the laboratory that generated them . They were never frozen at passage higher than eight . U2OS ( source: ATCC ) , and HCT116 p21+/+ and HCT116 p21−/− ( gifts from B . Vogelstein-Johns Hopkins University , Baltimore ) were received directly from ATCC and Johns Hopkins University respectively . Samples were minimally amplified and frozen after the reception and were used for limited passages after thawing of those primary stocks . We have verified the identity of the cell lines in terms of the pathways which are key for this study . We have verified the p53 and p21 status by RT-PCR . The three cell lines are positive for p53 and only U2OS and HCT 116 p21+/+ were positive for p21 , while HCT 116 p21−/− was not . None of the used cell lines were in the list of commonly misidentified cell lines maintained by the International Cell Line Authentication Committee ( Capes-Davis et al . , 2010 ) updated in http://iclac . org/databases/cross-contaminations/ ) . Samples were grown in Dulbecco’s modified Eagle’s medium ( Invitrogen , Cambridge , Massachusetts ) with 10% fetal calf serum in a 5% CO2 atmosphere . Samples are routinely tested for mycoplasma by PCR every two weeks . Umbilical cord mesenchymal stem cells ( UC-MSC ) were isolated from Wharton jelly tissues . Small pieces of the umbilical cord , excluding the major vessels , were layered onto plastic culture plates and cultured in D-MEM supplemented with 10% SFB and penicillin-streptomycin . After 14 days , cells started to emerge from the tissue pieces . When plates were almost confluent , umbilical cord pieces were removed and cells trypsinized and frozen . Multilineage differentiation potential of UC-MSC was assessed using StemPro Adipogenesis , Osteogenesis or Chondrogenesis Kit ( Life Technologies , US ) as per instruction of the manufacturer . Human Foreskin Fibroblasts ( HFF ) were obtained under informed consent from his parents from the foreskin of a three-year old boy undergoing a scheduled surgery . HFF were obtained using a similar protocol to the one used with UC-MSC . Both cell lines were grown in DMEM supplemented with 10% FBS and antibiotics up to a 15 passage . All isolations of primary cells were approved by FLENI Ethic Committee after reviewing the research protocol ( Luzzani et al . , 2015 ) . The HFF ( Human Foreskin Fibroblasts ) were analyzed by karyotyping to assess genomic integrity after 10 passages . Experiments were performed using cells up to passage number 15 to prevent the occurrence of abnormalities . In addition , cells surface markers are periodically analized by flow citometry . MSC line were used up to passage number 10 to prevent genomic aberrations and lose of stem cells´ properties . MSC surface marker expression profile and their differentiation potential were periodically analized by flow citometry and by chondregic , adipogenic and osteogenic differentiation , respectively . MSC immunomodulatory properties were also assessed by CFSE lymphocyte proliferation assay . All cell lines were weekly analyzed for mycoplasm contamination by PCR . Transfections of siRNAs were performed using Lipofectamine RNAiMAX ( Thermofisher ) and Jet Prime ( Polyplus , France ) was used when delivering both siRNAs and plasmid expression vectors . GFP-Pol η and GFP-Pol κ were gifts from Dr . A Lehmann ( Kannouche et al . , 2001 ) . GFP-PCNA was kindly provided by Dr . M C Cardoso ( Max Delbrück Center for Molecular Medicine , Berlin , Germany ) ( Leonhardt et al . , 2000 ) siRNAs were purchased from Dharmacon ( Lafayette , Colorado ) : siRNA p21 3'UTR:GGAACAAGGAGUCAGACAU; siRNAUSP1:UCGGCAAUACUUGCUAUCUUA ( Jones et al . , 2012 ) ; siRNA Pol κ1: AAGAUUAUGAAGCCCAUCCAA ( Vallerga et al . , 2015 ) ; siRNA Pol κ2: AACCUCUAGAAAUGUCUCAUA ( Jones et al . , 2012 ) siRNA Pol η: CUGGUUGUGAGCAUUCGUGUA ( Vallerga et al . , 2015 ) . In this work , we used a previously described p21 expression vectors ( CS2-p21 ) and generated the p21PIPMut* ( CS2-p21 with the M147 to A , D149 to A and F150 to A mutations which were shown to disrupt the PCNA/p21 interaction [Gulbis et al . , 1996; Mansilla et al . , 2013] ) Both the p21 and the p21PIPMut* vectors bear three point mutations in its CDK binding domain ( W49 to R , F51 to S and D52 to A ) which disrupt the CDK2/p21 interaction ( Gulbis et al . , 1996; Mansilla et al . , 2013 ) . Primers used to generate p21 mutations were described and validated in our previous work ( Mansilla et al . , 2013 ) and both mutations were characterized previously ( Gulbis et al . , 1996 ) . p21 and p21PIPMut* were cloned into the lentiviral transfer plasmid pLenti CMV/TO Puro ( Plasmid #17482 , Addgene , 3rd generation ) using BamHI and XbaI . Packaging and envelope 2nd generation vectors used were psPAX2 and PMD2 . G respectively . Lentiviral particles were obtained by transfecting with Jet prime Hek293T cells in 60 mm dishes in a 5:5:1 ratio ( pLenti: psPAX2:PMD2 ) . 48 hr after transfection lentiviral particles were collected from the supernatant of Hek293T cells , centrifuged , filtered with a 0 . 45 μm filter and stored at −80°C . Lentiviral preparations were slowly thawed and used to infect U2OS cells in 24/12 well dishes in the presence of 8 µg/ml of polybrene . Infection efficiency was approximately 90% ( Figure 7B ) . For the quantification and inmunodetection of specialized GFP tagged Pol κ and Pol η , 53BP1 and γH2AX respectively , cells were fixed in 2% paraformaldehyde ( PFA ) /2% sucrose and permeablized with 0 . 1% Triton X-100 in phosphate buffered saline ( PBS ) as described previously ( Mansilla et al . , 2013 ) . For the detection of chromatin-bound protein ice cold , 0 . 5% Triton CSK buffer was used ( 10 mM Pipes , pH 7 . 5 , 100 mM NaCl , 300 mM sucrose , 3 mM MgCl2 ) . Proteins were preextracted 1 min or 5 min when detecting p21 or RPA/PCNA respectively . EdU was detected following manufacturer’s instructions ( Click-iT EdU kit– C10338 Invitrogen ) . Blocking was performed overnight in PBS 2% donkey serum ( Sigma , St . Louis , Missouri ) . Coverslips were incubated for 1 hr in primary antibodies: 53BP1 ( Santa Cruz , Dallas , Texas ) , γH2AX ( EMD Millipore , Massachusetts ) , p21 ( c-19 Santa Cruz ) , RPA ( NA18 EMD Millipore ) , PCNA ( Abcam ) , pH3 ( ser10 Millipore ) . Secondary anti-mouse/rabbit-conjugated Cy2/Cy3 antibodies were from Jackson Immuno Research and anti-rabbit alexa 488 from Invitrogen . GFP-tagged specialized Y polymerases and GFP-PCNA were detected by GFP autofluorescence . Nuclei were stained with DAPI ( SIGMA ) . Images were obtained with a Zeiss Axioplan confocal microscope or a Zeiss Axio Imager A2 . For Western blot analysis , samples were lysed in Laemmli buffer . Antibodies used were anti-p21 ( c-19; Santa Cruz Biotechnology ) anti-polη ( H-300; Santa Cruz Biotechnology ) , anti-KU70 ( A9; Santa Cruz Biotechnology ) , and anti-GFP ( Santa Cruz Biotechnology ) . Secondary antibodies ( Sigma ) and ECL detection ( Amersham GE Healthcare , Milwaukee , Wisconsin ) were used according to the manufacturers’ instructions . Western blot images were taken with Image QuantLAS4000 ( GE Healthcare ) , which allows capture and quantification of images within a linear range . These images were then quantified with the ImageJ software when indicated . DNA fibers were analysed using a protocol previously used by us ( Mansilla et al . , 2013 ) with a minor change in the time of labelling . Exponentially growing cells were pulse labeled with CldU ( 20 µM ) for 10 min , washed twice , and incubated with IdU ( 200 µM ) for additional 30 min . Cells were trypsinized and lysed with 6 µl of 0 . 5% SDS , 200 mM Tris–HCL ( pH 7 . 4 ) and 50 mM EDTA buffer onto clean glass slides , which were tilted , allowing DNA to unwind . Samples were fixed in 3:1 methanol/acetic acid and denatured with HCL ( 2 . 5 N ) for 1 h , blocked in PBS 5%Bovine serum albumin ( BSA ) for 15 min and incubated with the mouse anti-BrdU ( Becton Dickinson , USA ) to detect IdU , donkey anti-mouse Cy3-conjugated secondary antibody ( Jackson Immuno Research , West Grove , Pennsylvania ) , rat anti-BrdU ( Accurate Chemicals , Westbury , New York ) to detect CldU and donkey antirat Alexa 488 secondary antibody ( Invitrogen ) . Slides were mounted with Mowiol 488 ( Calbiochem ) , and DNA fibers were visualized using a Zeiss Axioplanconfocal microscope . Images were analysed using Zeiss LSM Image Browser software and Image J software . Each data set is derived from measurement of 85–100 fibers . Chromatin immunoprecipitations were performed as described in ( Soria et al . , 2008 ) . U2OS cells were plated in 100 mm culture dishes , transfected with either Luc or p21 siRNA and 24 hr later transfected with GFP-Pol Kappa . Cells were rinsed twice with cold PBS and then extracted with 5 mL of CSK buffer ( 250 mM sucrose , 25 mM KCl , 10 mM HEPES at pH 8 . 0 , 1 mM EGTA , and 1 mM MgCl2 ) for 10 min in ice . The CSK-extracted cells were fixed with 1% formaldehyde in PBS ( 4 . 5 mL ) for 12 min . Then , 0 . 5 mL of 1 M glycine was added for 5 min to quench the cross-linking reaction . The cross-linked nuclei were rinsed with PBS and then lysed in 750 μL of IP lysis buffer ( 10 mM Tris·HCl at pH 7 . 5 , 25 mM FNa , 20 mM NaCl , 1% Nonidet P-40 , 1% Na-Deoxicholate , and 0 . 1% SDS ) freshly supplemented with protease and phosphatase inhibitors . Lysates were scraped from the plates and transferred into 1 . 5-mL Microfuge tubes . Samples were sonicated ( Bioruptor Sonication System , Diagenode ) and clarified by centrifugation at 12 , 000 × g for 30 min at 4°C . PCNA was immunoprecipitated overnight at 4°C with 5 μL of monoclonal PCNA antibody ( PC-10 AC; Santa Cruz Biotechnology ) . Samples were washed , lysed in Laemmli buffer and heated at 99°C for 30 min to revert the crosslink , and resolved in SDS/PAGE for Western blot analysis . U2OS cells were seeded onto 22 mm x 22 mm coverslips in 6 well plates . 24 hr later cells were pre-extracted with ice cold CSK buffer ( PIPES 10 mM , NaCl 100 mM , sucrose 300 mM , MgCl2 3 mM , triton 0 . 5% ) for 2 min and fixed with PFA 4% for 10 min . After pre-extraction and fixation , interaction between endogenous p21 and PCNA was detected following manufacturer’s instructions Duolink In Situ – Fluorescence ( SIGMA ) . Briefly PLA’s principle is based on detecting proteins in close proximity ( 30–40 nm ) by using different species of primary antibodies . A pair of oligonucleotide ( PLA probes ) detect the primary antibodies bound the proteins of interest and generates a signal only when the two PLA probes have bound in close proximity , meaning that the samples are localized in close proximity . The signal from each detected pair of PLA probes is visualized as an individual fluorescent spot . These PLA signals can be quantified ( counted ) and assigned to a specific subcellular location based on microscopic images . Images were obtained with a Zeiss Axioplan confocal microscope and PLA spots were counted using Image J software , cells were counted as positive when more than 3 PLA spots were detected . When analyzing pol κ/PCNA interaction a pol κ 'home made' monoclonal antibody from mice purified by Biotem ( clone n°16A7-3A11 ) and PCNA rabbit polyclonal ( Abcam ref ab18197 ) were used . For the quantification of number of foci per nuclei the Cell Profiler program was used to analyze more than 1000 nuclei . After transfection with the indicated siRNAs , cells were lysed and total RNA was extracted using TRIzol Reagent ( Invitrogen ) . Total RNA ( 1 μg ) was used as a template for cDNA synthesis utilizing an ImProm-II Reverse Transcription System ( Promega ) and oligo-dT as a primer . Quantitative real-time PCR was performed using the MX3005P quantitative PCR instrument ( Stratagene ) with Taq DNA poly-merase ( Invitrogen ) and SyberGreen and ROX as reference dyes ( Invitrogen ) . Samples were normalized using GAPDH primers . Both target gene and GAPDH amplification reactions approached 100% efficiency as determined by standard curves . Three independent biological samples were analyzed , and one representative set of results is shown . Primer sequences were as follows: USP1 ( Forward ) : 5’- GGACGCGTTGCTTGGAATGT-3’ ( Reverse ) 5’-TGCCCATCTCAGGGTCTTCA-3’ . GADPH: ( Forward ) 5’-AGCCTCCCGCTTCGCTCTCT-3’ ( Reverse ) 5’-GAGCGATGTGGCTCGGCTGG-3’ ( Vallerga et al . , 2015 ) . Transfected cells were replated at low density . 24 hr after replating cytochalasine B ( 4 . 5 μg/ml-Sigma ) was added to the media and 40 hr later cells were washed 1 min with hypotonic buffer ( KCl 0 . 0075 M ) diluted 1/10 from stock solution in PBS 1X , twice with PBS 1X and fixed with PFA/sucrose 2% for 20 min . DAPI staining served to visualize cell nuclei . 300 binucleated cells were analyzed . Cells were treated with 0 . 1 μg/ml colcemid ( Gibco ) for 3 hr . Cells were trypsinized for 1 min trying to take off only the mitotic cells . After centrifugation cell pellets were resuspended in hypotonic solution ( 0 . 075 M KCl ) and incubated for 15 min at 37°C . Samples were then fixed in a methanol/acetic acid solution ( 3:1 ) , washed three times in the fixative solution and dropped on slides to obtain spread chromosomes . Metaphase slides were incubated with Rnase ( 10 μg/ml in 2× SSC ) for 1 hr at 37°C followed by dehydration in successive ethanol baths ( 70 , 85 , and 100% ) . The RP 11 36B6 BAC ( corresponding to FRA7H locus ) was labeled by nick translation ( Abbott ) with green dUTP ( Vysis Spectrum ) and ethanol precipitated with human Cot-1 DNA ( Roche , California ) overnight at −20°C . Precipitated DNA was incubated for 1 hr at 37°C in hybridization mix . The probe was denatured for 8 min at 70°C and applied to denatured metaphases . Samples were incubated over night at 37°C and , after washing ( 1X SSC , 5 min at 72°C and SCC 1X 5 min at RT ) and mounted in VECTASHIELD Antifade Mounting Medium with DAPI . The images were acquired by using an inverted wide-field Zeiss Axio Observer Z1 microscope fitted with a x63 oil NA 1 . 4 objective and an Axiocam MRm CDD camera . Frequency distributions of DNA track length were determined with GraphPad Prism 5 software . In non-Gaussian distributions , Mann–Whitney and Kruskal–Wallis tests were used for statistical analyses when comparing two and more than three variables , respectively . Other statistical analyses were performed with GraphPad in Stat software using the Student’s t test and the one-way ANOVA test when applicable .
The levels of the cyclin-dependent kinase ( CDK ) inhibitor p21 are low in S phase and insufficient to inhibit CDKs . We show here that endogenous p21 , instead of being residual , it is functional and necessary to preserve the genomic stability of unstressed cells . p21depletion slows down nascent DNA elongation , triggers permanent replication defects and promotes the instability of hard-to-replicate genomic regions , namely common fragile sites ( CFS ) . The p21’s PCNA interacting region ( PIR ) , and not its CDK binding domain , is needed to prevent the replication defects and the genomic instability caused by p21 depletion . The alternative polymerase kappa is accountable for such defects as they were not observed after simultaneous depletion of both p21 and polymerase kappa . Hence , in CDK-independent manner , endogenous p21 prevents a type of genomic instability which is not triggered by endogenous DNA lesions but by a dysregulation in the DNA polymerase choice during genomic DNA synthesis .
Cancer develops when cells in the body mutate in ways that allow them to rapidly grow and divide . To protect cells from becoming cancerous , various molecules act like guardians to prevent cells from dividing when their DNA is damaged , or if they are short of energy . Other guardian molecules monitor the DNA copying process to ensure that the newly-made DNA is as identical as possible to the original DNA template . A protein called p21 belongs to the first group of guardian molecules: DNA damage triggers the production of p21 , which prevents the cell from copying its DNA . This role relies on a section of the protein called the CDK binding domain . Cells that have already started to copy their genetic material also have low levels of p21 . Mansilla et al . used human cells to investigate whether p21 is also involved in the process of copying DNA . The experiments show that the low levels of p21 act to increase the speed at which the DNA is copied . This activity helps to ensure that all of the cell’s DNA is copied within the time available , including sections of DNA that are harder to copy because they are more fragile and prone to damage . This newly identified role does not involve the CDK binding domain , but instead requires a different section of the p21 protein known as the PCNA interacting region . Mansilla et al . propose that p21 plays a dual role in protecting us from developing cancer . The PCNA interacting region is also found in other proteins that are involved in copying DNA . Therefore , a future challenge is to find out how these proteins interact with each other to ensure that cells accurately copy their DNA in a timely fashion .