Mutational phospho-mimicry reveals a regulatory role for the XRCC4 and XLF C-terminal tails in modulating DNA bridging during classical non-homologous end joining
In mammalian cells , classical non-homologous end joining ( c-NHEJ ) is the primary pathway that repairs DNA double-strand breaks ( DSBs ) . This pathway is specialized in the repair of two-ended DSBs like those that are induced by ionizing radiation or by many radiomimetic drugs . The c-NHEJ pathway is also exploited by developing lymphocytes to repair DSBs that are introduced by the RAG1/2 endonuclease during antibody and T-cell receptor V ( D ) J gene rearrangement . The c-NHEJ machinery is versatile and adapts to efficiently repair DSBs with diverse DNA end structures ( Lieber , 2010; Reid et al . , 2017 ) . Core c-NHEJ effectors include the heterodimeric Ku DNA end sensor , the DNA-dependent protein kinase catalytic subunit ( DNA-PKcs ) , DNA Ligase 4 ( LIG4 ) , and the structurally related XRCC4 and XLF proteins . Current dogma advocates a sequential model for c-NHEJ as follows: ( 1 ) Ku performs the recognition step , rapidly and avidly binding and protecting broken DNA ends; ( 2 ) Ku recruits DNA-PKcs and promotes DNA end synapsis , regulating DNA end access; ( 3 ) if required , DNA end processing is carried on by non-core factors ( Artemis , X-family polymerases , or other DNA modifying enzymes ) ; and 4] the LIG4/XRCC4 complex , assisted by XLF , catalyzes the ligation step directly . Emerging data are challenging this sequential model , and suggest a more dynamic and adaptive model for c-NHEJ . These paradigm shifting data include structural , biochemical , and functional studies revealing that LIG4 , XRCC4 and XLF form filamentous structures that bridge DNA and promote ligation in vitro ( Andres et al . , 2012; Hammel et al . , 2011; Ropars et al . , 2011; Roy et al . , 2015 , 2012; Wu et al . , 2011 ) . This DNA tethering , which has been observed in vitro , implies an early function for XRCC4/XLF filaments during DNA repair; we have proposed that XRCC4/XLF complexes rapidly and robustly ‘splint’ broken DNA as ‘sleeve-like’ protein bandages ( Brouwer et al . , 2016 ) . In fact , a recent study showed that in living cells , XRCC4 and XLF , independently of LIG4 , form filamentous super-structures at bleomycin-induced DSBs that adopt various configurations ( Reid et al . , 2015 ) . Currently it is unclear exactly how these filaments function in the context of c-NHEJ; however , specifically disrupting XRCC4-XLF filament formation ( by mutagenesis ) affects c-NHEJ function in living cells ( Roy et al . , 2015 ) . Thus , addressing the dynamics and regulation of XRCC4-XLF super-structures is an important area of investigation with regard to a better understanding of the c-NHEJ mechanism . Here we focus on understanding the functional contribution of the XRCC4 and XLF C-terminal regions , which have so far been considered to be dispensable for c-NHEJ . XRCC4 and XLF both have disordered C-terminal tails that are phosphorylated by DNA-PK and/or ATM in response to DSBs [of note , numerous studies have reported substantial hyper-phosphorylation of both XRCC4 and XLF in response to genotoxic stress in living cells] . Early functional studies ( using phospho-blocking and phospho-mimetic mutants ) suggested that these phosphorylations do not alter the efficacy or fidelity of c-NHEJ in living cells ( Yu et al . , 2003 , 2008 ) . However , potential functional consequences of these phosphorylations might be masked in studies focusing on either XRCC4 or XLF individually , if phosphorylation of these sites is functionally redundant , as it has been shown for many DNA-PK dependent phosphorylations ( Cui et al . , 2005; Ding et al . , 2003; Neal et al . , 2014 ) . Thus , here we utilize biochemical and cell-based approaches to address the impact of XRCC4 and XLF phospho-blocking or phospho-mimicking mutations in concert . Whereas , blocking or mimicking eight phosphorylation sites in XRCC4 and 6 sites in XLF does not abate stimulation of LIG4 in vitro , nor diminish end-joining of episomal substrates in living cells , phospho-mimicking dramatically affects XRCC4/XLF dissociation from DNA and stability of DNA tethering in vitro , and also impedes cellular survival after exposure to radio-mimetic drugs , especially drugs that induce complex DNA lesions . Moreover , phospho-mimicking mutations alter repair of chromosomal DNA DSBs in living cells , resulting in increased nucleotide loss and greater dependence on end-terminal short sequence homologies .
The phosphorylation of XRCC4 and XLF has been addressed by several investigators . Briefly , we identified nine phosphorylation sites in XRCC4 ( Yu et al . , 2003 ) ; Lees-Miller and colleagues identified six sites in XLF ( Yu et al . , 2008 ) . Studies using phospho-specific antibodies and numerous phospho-proteome reports have confirmed that eight of the nine sites ( residues S193 , S260 , S304 , S315 , S320 , T323 , S327 , and S328 – using UniProt Q13426 isoform 1 as reference ) in XRCC4 and minimally four of the six sites ( residues S132 , S203 , S245 and S251 – using UniProt Q9H9Q4 isoform 1 as reference ) in XLF are phosphorylated in vivo ( http://www . phosphosite . org ) . Remarkably , all the above-mentioned phosphorylation sites are located in the C-terminal tails of XRCC4 and XLF ( Figure 1 ) . 10 . 7554/eLife . 22900 . 003Figure 1 . Structural organization of XRCC4 and XLF homodimers . ( A ) Structure of XRCC4 homodimer based on PDB 3II6 , including residues 1–203 of XRCC4 and a schematic representation of the C-terminal disordered tails . In the bottom panel , also residues 654–911 of LIG4 are shown ( green ) . ( B ) Structure of XLF homodimer based on PDB 2R9A , including residues 1–224 and a schematic representation of the C-terminal tails . Purple stars indicate the residues modified for phospho-mimicking or -blocking , and all were mutated , respectively , to Asp or Ala in this study . Residues are numbered using as reference Uniprot Q13426 isoform 1 and Q9H9Q4 isoform 1 for XRCC4 and XFF , respectively . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 00310 . 7554/eLife . 22900 . 004Figure 1—figure supplement 1 . Coomassie stained reducing SDS-PAGE analysis of recombinant XRCC4 and XLF variants produced in bacteria ( 1 μg/lane ) . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 004 To test whether the disordered C-terminal tails of XRCC4 and XLF are implicated in regulation of the DNA interaction dynamics of XRCC4-XLF complexes , phospho-mimetic ( Asp ) and phospho-ablating ( Ala ) variants of XRCC4 and XLF targeting the multiple DNA-PK or ATM dependent phosphorylation sites were designed and generated ( Figure 1—figure supplement 1 ) . To study the impact of these modifications , several different activity assays were optimized and validated using wild-type ( WT ) XRCC4 and XLF proteins ( Figure 2 ) . These assays include electrophoretic-mobility shift assays ( EMSAs , Figure 2A and B ) , phage T4 DNA ligase assays ( as a way to indirectly measure DNA bridging activity , Figure 2C , D and E ) , and protein-mediated DNA pull-down assays to directly measure DNA bridging activity ( Figure 2F and G ) . Overall , analyses of the activity of the WT proteins confirmed that XRCC4 and XLF cooperatively form super-complexes detectable by EMSA ( Figure 2A and B ) that strongly promote intermolecular DNA cohesive and blunt end ligation ( Figure 2C , D and E ) , by favoring molecular collision rates during ligation through a robust DNA bridging activity as can be seen in Figure 2F and G . In the latter experiment , it can be also observed that XLF has an intrinsic DNA bridging activity that depends on the presence of its C-terminal tail ( Figure 2G , lanes 3 and 5 , XLF ( 1-224 ) lacks the 75 C-terminal residues ) . This intrinsic XLF DNA bridging activity is strongly stimulated by XRCC4 ( Figure 2G , lanes 3 and 7 ) in a manner that depends on physical interaction between XLF and XRCC4 , as tested using an XLF mutant ( L115D ) described previously that does not interact with XRCC4 but has an intact C-terminal tail ( Figure 2G , lanes 4 and 8 , Malivert et al . , 2010; Roy et al . , 2015 ) . Furthermore , as seen in Figure 2—figure supplement 1 , the importance of the XRCC4 and XLF C-terminal tails , and especially XLF’s tails in promoting DNA bridging and increasing the rate of molecular collision during ligation in vitro , is demonstrated using the truncated tail-less XRCC4 ( 1–157 ) and XLF ( 1-224 ) variants . 10 . 7554/eLife . 22900 . 005Figure 2 . Wild-type XRCC4 and XLF form super-complexes in vitro that bridge DNA favoring molecular collision rates that enhance DNA ligation . ( A ) EMSA using a 2 . 7 kb DNA fragment with protruding ends and the indicated proteins resolved by agarose gel electrophoresis and DNA detected by ethidium bromide staining . ( B ) EMSA using a blunt-ended 2 . 7 kb DNA fragment and the indicated proteins resolved by agarose gel electrophoresis and DNA detected by ethidium bromide staining . ( C ) ( D ) ( E ) T4 DNA ligase assay using a 2 . 7 kb DNA fragment with cohesive ( left ) or blunt ends ( right ) and the indicated proteins . Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by detection by ethidium bromide staining . NC = nicked circle , CCC = covalently closed circle . ( F ) Schematic of the DNA pull-down/bridging assay . A one-end biotinylated 1 kb DNA fragment is first attached to streptavidin-coated magnetic beads and then incubated with a blunt-ended 2 . 7 kb DNA fragment in the presence of the proteins of interest . ( G ) Bridging assays for the indicated proteins resolved by agarose gel electrophoresis after deproteinization followed by DNA detection by ethidium bromide staining . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 00510 . 7554/eLife . 22900 . 006Figure 2—figure supplement 1 . T4 DNA ligase assay using a 2 . 7 kb DNA fragment with cohesive ( left ) or blunt ends ( right ) at three different protein equimolar concentrations . ( A ) 2 μM each , ( B ) 1 μM each , and ( C ) 0 . 5 μM each . Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by DNA detection by ethidium bromide staining . NC = nicked circle , CCC = covalently closed circle . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 006 We first analyzed XRCC4 and XLF phosphorylation site mutants for DNA binding by EMSA ( Figure 3 ) . Neither the intrinsic DNA binding of XRCC4 nor its ability to form super-complexes with XLF-WT is affected by phospho-blocking eight sites in XRCC4 ( Figure 3A , XRCC4-Ala ) . In contrast , phospho-mimicking these eight sites in XRCC4 strongly reduces XRCC4’s intrinsic DNA binding and modestly reduces cooperative DNA binding with XLF-WT ( Figure 3A , XRCC4-Asp ) . Neither phospho-blocking nor phospho-mimicking six sites in XLF alters either the intrinsic DNA binding activity of XLF or its ability to cooperatively form super-complexes with XRCC4-WT ( Figure 3B ) . 10 . 7554/eLife . 22900 . 007Figure 3 . Formation of DNA/XRCC4/XLF super-complexes is not disrupted by phosphorylation site mutation . ( A ) EMSAs showing DNA binding of XRCC4 variants in isolation ( top panel ) and cooperatively with XLF-WT ( bottom panel ) resolved by agarose gel electrophoresis and detected by ethidium bromide staining . ( B ) EMSAs showing DNA binding of XLF variants in isolation ( top panel ) and cooperatively with XRCC4-WT ( bottom panel ) resolved by agarose gel electrophoresis and detected by ethidium bromide staining . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 007 Protein-protein cross-linking experiments in the absence of DNA suggest that the XRCC4 and XLF variants interact at the protein-protein level similarly to WT XRCC4 and XLF ( Figure 4 ) . To further investigate these interactions , we used Isothermal Titration micro-Calorimetry ( ITC ) to quantify the thermodynamic parameters of the XRCC4-XLF protein-protein interaction in the absence of DNA . Experiments were performed at 37°C ( after optimization tests at 10 , 25 , and 37°C ) because this condition provided the highest heat signature and optimal signal to noise ratio ( Figure 5 and Figure 5—figure supplement 1 ) . Two experimental replicas were performed in duplicate using two protein concentrations ( Table 1 ) . The dissociation constant values ( Kd ) obtained for the three different combinations ( WT-WT , Ala-Ala , and Asp-Asp ) are equal within the experimental errors , and are approximately 2 μM ( Figure 5D , top panel , and Table 1 ) . Nevertheless , the XRCC4 and XLF variants have different enthalpic ( ΔH0 , dark grey bars ) and entropic ( -TΔS0 , light grey bars ) contributions ( Figure 5D , bottom panel , and Table 1 ) that compensate one another resulting in similar free energies ( ΔG0 , black bars ) . We conclude that although the phospho-blocking and -mimicking mutations in the disordered tails do not affect the overall equilibrium of the XRCC4-XLF interaction , these mutations affect the thermodynamics of the interaction . Moreover , the disordered tails may contribute to stabilizing the XRCC4-XLF interaction . In fact , the Kd measured with full-length ( WT ) proteins is about half of that measured for XRCC4 and XLF truncations missing the disordered tails ( Malivert et al . , 2010 ) . Also , ITC experiments showed that for all variants ( and conditions ) the stoichiometry of the XRCC4 and XLF interaction is 1:1 ( Table 1 ) . In sum , ITC experiments confirm that , in the absence of DNA , the Ala and Asp variants interact in solution with similar affinity than WT XRCC4 and XLF , despite the fact that the underlying molecular interactions have different thermodynamic contributions . We suggest that this may indicate phosphorylation induced conformational changes ( see Discussion ) . 10 . 7554/eLife . 22900 . 008Figure 4 . Phosphorylation site mutations do not alter XRCC4-XLF direct interaction . XRCC4 and XLF were cross-linked in isolation or in equimolar combination with BS3 . Cross-linked species were resolved by reducing and denaturing SDS-PAGE and detected by Coomassie staining . ( A ) XRCC4 and XLF WT-WT combination ( left panel ) and XRCC4-WT combined with XLF-L115A , that has a point mutation that diminished affinity for XRCC4 ( right panel ) . ( B ) XRCC4-WT ( left panel ) , XRCC4-Ala ( middle panel ) and XRCC4-Asp ( right panel ) respectively cross-linked to all the XLF variants . M = protein molecular weight size ladder . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 00810 . 7554/eLife . 22900 . 009Figure 5 . Thermodynamic analysis of wild-type and phospho-mimetic XRCC4-XLF interaction . Isothermal titration micro-calorimetry data obtained at 37°C injecting ( A ) XLF-WT ( 154 μM ) into XRCC4-WT ( 12 μM ) , ( B ) XLF-Ala ( 250 μM ) into XRCC4-Ala ( 19 μM ) , ( C ) XLF-Asp ( 250 μM ) into XRCC4-Asp ( 19 μM ) . Top panels display the thermogram of the heat exchanged as a function of time after baseline ( red lines ) subtraction . Bottom panels show the result of the ( numerical ) integration of the peaks as a function of the molar ratio between the titrant ( XLF ) and the analyte ( XRCC4 ) ; black lines are non-linear least squares fits of the data . Data are representative results out of two experimental replicas each composed of a technical duplicate ( see Table 1 for details ) . Source data are included in Table 1—source data 1 . ( D ) Equilibrium constant ( Kd ) ( top ) and thermodynamic parameters ( free energy ΔG0 , black bars; enthalpy ΔH0 , dark grey bars; and entropy –TΔS0 , light grey bars ) ( bottom ) obtained from sigmoidal fit ( black lines in bottom panels of ( A , B , C ) of ITC curves . Bars show mean values and error bars represent the standard error of the mean ( SEM ) of the four experimental runs performed per each condition . Thermodynamic parameter values are reported in Table 1 . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 00910 . 7554/eLife . 22900 . 010Figure 5—figure supplement 1 . Isothermal titration micro-calorimetry thermograms . Top panels display the heat exchanged as a function of time , after baseline ( red lines ) subtraction . Bottom panels show the result of the ( numerical ) integration of the peaks as a function of the molar ratio between the titrant ( XLF ) and the analyte ( XRCC4 ) . Thermograms have been obtained ( A ) at 10°C injecting XLF-WT ( 168 μM ) into XRCC4-WT ( 17 μM ) and at 25°C injecting ( B ) XLF-WT ( 330 μM ) into XRCC4-WT ( 25 μM ) , ( C ) XLF-Ala ( 330 μM ) into XRCC4-Ala ( 25 μM ) , and ( D ) XLF-Asp ( 330 μM ) into XRCC4-Asp ( 25 μM ) . Control isothermal titration micro-calorimetry thermograms obtained at 37°C injecting ( E ) buffer into XRCC4-WT ( 12 μM ) and ( F ) XLF-WT ( 154 μM ) into buffer ( the first large peak might represent XLF homodimer dissociation heat due to XLF dilution ) . Source data are included in Table 1—source data 1 ( panels A–D ) and reported in Figure 5—figure supplement 1—source data 1 ( panels E and F ) . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 01010 . 7554/eLife . 22900 . 011Figure 5—figure supplement 1—source data 1 . Thermograms of control experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 01110 . 7554/eLife . 22900 . 012Table 1 . Impact of phospho-mimicking or phospho-blocking mutations on the thermodynamics parameters of XRCC4-XLF protein-protein interaction by micro-calorimetry . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 01210 . 7554/eLife . 22900 . 013Table 1—source data 1 . Thermograms of XRCC4-XLF WT-WT , Ala-Ala , and Asp-Asp experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 013T ( °C ) 2-Me ( mM ) [XRCC4] ( μM ) [XLF] ( μM ) RatioNKa ( M−1 ) ΔH0 ( kcal/mol ) Kd ( μM ) −ΔG0 ( kcal/mol ) −TΔS0 ( kcal/mol ) Thermogram shown in Source data<Kd> ( μM ) <ΔH0> ( kcal/mol ) <-ΔG0> ( kcal/mol ) <-TΔS0> ( kcal/mol ) ITC cellITC syringeXRCC4-WT XLF-WT10101716810n . d . n . d . n . d . n . d . n . d . n . d . Figure 5—figure supplement 1AA251017168100 . 97536000−1 . 71 . 8−7 . 8−6 . 1--B3 . 2 ± 0 . 8−2 . 4 ± 0 . 4−7 . 5 ± 0 . 2−5 . 2 ± 0 . 5251033330100 . 91306000−2 . 93 . 2−7 . 5−4 . 6Figure 5—figure supplement 1BC251025330130 . 84222000−2 . 64 . 5−7 . 3−4 . 7--D37023300130 . 85273000−6 . 03 . 7−7 . 7−1 . 7--E2 . 4 ± 0 . 7−7 . 1 ± 1−8 . 0 ± 0 . 2−1 . 0 ± 0 . 837023300130 . 89278000−4 . 93 . 6−7 . 7−2 . 9--F37012154130 . 92848000−8 . 61 . 2−8 . 40 . 2Figure 5AG37012154130 . 84760000−8 . 81 . 3−8 . 30 . 4--HXRCC4-Ala XLF-Ala251025330131 . 21300000−1 . 73 . 3−7 . 5−5 . 8Figure 5—figure supplement 1CI37025325131 . 07637000−3 . 11 . 6−8 . 2−5 . 1--J2 . 3 ± 0 . 4−5 . 8 ± 1 . 4−8 . 0 ± 0 . 1−2 . 2 ± 1 . 437025325131343000−3 . 72 . 9−7 . 8−4 . 1--K37019250130 . 92345000−8 . 42 . 9−7 . 80 . 6--L37019250130 . 92582000−8 . 11 . 7−8 . 2−0 . 1Figure 5BMXRCC4-Asp XLF-Asp251035350101 . 27672000−0 . 91 . 5−7 . 9−7 . 0--N251025330130 . 82263000−1 . 23 . 8−7 . 4−6 . 2Figure 5—figure supplement 1DO37032416131 . 06289000−2 . 43 . 4−7 . 8−5 . 4--P2 . 4 ± 0 . 4−3 . 7 ± 0 . 8−8 . 0 ± 0 . 1−4 . 3 ± 0 . 737032416130 . 95425000−2 . 32 . 4−8 . 0−5 . 7--Q37019250130 . 88566000−4 . 71 . 8−8 . 2−3 . 5Figure 5CR37017220130 . 85538000−5 . 51 . 8−8 . 1−2 . 6--S2-Me stands for 2-MercaptoethanolRatio indicates the titrant over analyte concentration ( [XLF]/[XRCC4] ) Source data specifies the column corresponding to each thermogram in Table 1—source data 1 . Last four columns report mean values ± Standard Error of the Mean ( SEM ) from four experimental runs at 37°C ( grey cells ) and 3 runs at 25°C ( white cells ) n . d . denotes no detectable heat transfer ( at 10°C ) To assess the role of the XRCC4 and XLF C-terminal tails , we first used T4 ligase assays to indirectly infer DNA bridging by XRCC4-XLF complexes . The XRCC4-Ala variants combined with either XLF-WT or XLF-Ala variants modestly enhanced ligation as compared to the WT counterpart ( Figure 6 , lanes 5 , 6 , 8 , and 9 ) suggesting more stable DNA-protein complexes . In contrast , stimulation of T4 ligase activity is minimal with the Asp mutants; this is most dramatic when aspartate mutants of both XRCC4 and XLF are utilized concomitantly ( Figure 6 , lanes 13 ) . The DNA bridging activity of XRCC4 and XLF variants was also investigated via a protein-mediated DNA pull-down assay ( Figure 7 ) . When both XRCC4-Asp and XLF-Asp variants are combined , DNA bridging activity is severely impaired ( Figure 7 , lanes 18 ) . Of note , the ability of the XLF-Asp variant to bridge long DNA on its own is also strongly reduced ( Figure 7B , lane 8 ) although its ability to bind to DNA is not affected when tested by EMSA ( Figure 3B ) . From these data , we conclude that XRCC4-Asp and XLF-Asp variants are defective in cooperative DNA bridging , even though the proteins interact with one another analogously ( i . e . with similar Kd ) to WT XRCC4 and XLF . We therefore hypothesized that XRCC4 and XLF with aspartate substitutions in the C terminal tails might generate complexes that dissociate faster from DNA as compared to WT XRCC4-XLF complexes . 10 . 7554/eLife . 22900 . 014Figure 6 . Blocking XRCC4 and XLF phosphorylation sites enhances DNA bridging; phospho-mimicking mutations abate DNA bridging . All combinations of XRCC4 variants with XLF variants tested in ability to stimulate T4 DNA ligase cohesive end ligation ( A ) or blunt end ligation ( B ) . Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by detection by ethidium bromide staining . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 01410 . 7554/eLife . 22900 . 015Figure 7 . Blocking XRCC4 and XLF phosphorylation sites enhances DNA bridging; phospho-mimicking mutations abate DNA bridging . DNA bridging assays using a short blunt ended 0 . 5 kb DNA ( A ) or a long blunt ended 2 . 7 kb DNA ( B ) and the indicated proteins . Samples were deproteinized and resolved by agarose gel electrophoresis followed by detection by ethidium bromide staining . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 015 To assess dissociation rates of XRCC4-XLF complexes from DNA , a Surface Plasmon Resonance ( SPR ) assay was established . One-ended biotinylated 400 bp-long dsDNA molecules were captured on streptavidin-coated chips; protein/DNA association and dissociation was monitored using a Biacore instrument ( Figure 8A ) . WT proteins were first characterized in isolation and at equi-molar concentrations ( Figure 8B ) . We observed that XRCC4-WT efficiently binds DNA only when flushed at high concentrations , but once bound , the protein remains stably attached to DNA ( Figure 8B , left panel ) . As compared to XRCC4 , XLF’s association with DNA is much faster , but XLF also dissociates faster from DNA than XRCC4 ( Figure 8B , middle panel ) . When WT XRCC4 and XLF are mixed together in solution , at an equimolar ratio , and flushed , a strong enhancement of DNA association was observed followed by dissociation with comparable kinetics to XLF alone ( Figure 8B , right panel ) . Probably as a consequence of polymerization of the filamentous structures formed by XRCC4 and XLF on the DNA , the SPR association and dissociation curves could not be perfectly fitted with a simple 1:1 Langmuir model to compute first order association and dissociation rate constants ( kon and koff , respectively ) . Nevertheless , we estimate that XRCC4-WT’s association rate is at least 10-times slower than that of XLF-WT alone or that of the XRCC4-WT + XLF-WT combination . Moreover , also XRCC4’s dissociation rate is at least 10-times slower than that of XLF-WT alone or in combination with XRCC4-WT . When the XRCC4 and XLF Asp and Ala variants were analyzed in isolation at a fixed concentration ( 1 μM ) , association and dissociation kinetics were strongly affected for XRCC4-Ala , XRCC4-Asp and XLF-Asp but just minority for XLF-Ala ( Figure 8—figure supplement 1A ) . Next , given the above-mentioned limits in correctly quantifying the association and dissociation rate constants , we decided to systematically analyze all the possible combinations of the different variants at a fixed concentration ( [C] = 1 μM each , Figure 8C ) . To inspect and compare the behavior of the different combinations during the association phase , we considered the equilibrium affinity ( defined as the response unit values averaged over 5 s taken 3 s before the start of the dissociation phase , gray band in Figure 8C ) , which reports on the ratio: [C]·kon / koff . The XRCC4-Ala and XLF-Ala variants together , or in combination with the WT variant , exhibited equilibrium affinities similar to the WT-WT combination ( Figure 8D ) . The XRCC4-Asp and XLF-Asp variants combined with the WT and Ala variants also showed modest deviations from the WT-WT combination . In contrast , the combination of the two Asp variants resulted in a 4-fold drop in the equilibrium affinity as compared to the WT-WT combination . To understand the observed drop for the XRCC4-Asp + XLF-Asp complexes , we analyzed the dissociation phase . The dissociation curves of all combinations have a biphasic character , with an initial rapid dissociation followed by a slower dissociation phase ( Figure 8—figure supplement 1B ) . The slower dissociation kinetic component ( koff_slow ) was similar for all combinations of XRCC4 and XLF variants , but , interestingly , the initial rapid dissociation phase ( koff_fast ) was accelerated 4-fold in the case of the XRCC4-Asp + XLF-Asp complexes as compared to the WT-WT complexes ( Figure 8E ) . Thus , the decrease in equilibrium affinity detected for the XRCC4-Asp + XLF-Asp complexes can be explained by the increase in the rate of dissociation without a major change in the rate of association of XRCC4-Asp + XLF-Asp complexes . Taken together , these analyses indicate that simultaneous aspartate substitutions within the C-terminal tails of both XRCC4 and XLF results in a shifted DNA binding equilibrium , such that complexes bind to DNA similarly to WT XRCC4/XLF complexes , but detach much faster . This likely explains the impairment in DNA bridging , as assessed by pull-down and T4 DNA end ligation assays . Lastly , SPR experiments using C-terminal tail-less variants XRCC4 ( 1–157 ) and XLF ( 1-224 ) confirmed that C-terminal tails are required ( and especially those of XLF ) for stable DNA binding ( Figure 8—figure supplement 1C and D ) , consistently with DNA bridging results obtained with the T4 DNA ligation assays ( Figure 2—figure supplement 1 ) . 10 . 7554/eLife . 22900 . 016Figure 8 . Phospho-mimicking XRCC4 and XLF mutants associate with DNA , but dissociate rapidly . ( A ) Scheme of the SPR assay with 400 bp blunt end DNA anchored to the streptavidin coated chip via biotin-streptavidin linkage . ( B ) From left to right , sensorgrams of XRCC4-WT , XLF-WT , and equi-molar mix of XRCC4-WT and XLF-WT at the indicated concentrations . Source data are provided in Figure 8—source data 1 , 2 and 3 , respectively . ( C ) Sensorgrams of all XRCC4 and XLF variant combinations each at 1 μM concentration . Source data are provided in Figure 8—source data 4 . ( D ) Equilibrium affinity of the different combinations of XRCC4 and XLF variants . Source data are provided in Figure 8—source data 5 . ( E ) Dissociation rates for the different combinations of XRCC4 and XLF variants . koff_fast ( dark grey symbols ) and koff_slow ( light grey symbols ) correspond , respectively , to the initial ( fast ) and late ( slow ) phase in the biphasic dissociation curves . Error bars fall within symbol dimension . Source data are provided in Figure 8—source data 5 . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 01610 . 7554/eLife . 22900 . 017Figure 8—source data 1 . Sensorgrams of XRCC4 WT tritation . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 01710 . 7554/eLife . 22900 . 018Figure 8—source data 2 . Sensorgrams of XLF WT tritation . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 01810 . 7554/eLife . 22900 . 019Figure 8—source data 3 . Sensorgrams of XRCC4-XLF WT-WT tritation . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 01910 . 7554/eLife . 22900 . 020Figure 8—source data 4 . Sensorgrams of all XRCC4-XLF variant combinations . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 02010 . 7554/eLife . 22900 . 021Figure 8—source data 5 . Equilibrium affinity and dissociation rates of all XRCC4-XLF variant combinations . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 02110 . 7554/eLife . 22900 . 022Figure 8—figure supplement 1 . Sensorgrams of XRCC4 and XLF variants and truncations . ( A ) Sensorgrams of XRCC4 variants ( left ) or XLF variants ( right ) at 1 μM concentration in isolation . Source data are provided in Figure 8—figure supplement 1—source data 1 . ( B ) Sensorgrams comparison between XRCC4 and XLF WT-WT ( black , left scale ) and Asp-Asp ( red , right scale ) at equimolar concentration ( 1 μM each ) showing the faster dissociation of the Asp-Asp combination and the deviation in both cases from a single exponential decay ( dashed lines ) and the biphasic behavior ( solid lines ) . Source data are provided in Figure 8—figure supplement 1—source data 2 . ( C ) Sensorgrams of XRCC4-WT and truncation XRCC4 ( 1–157 ) lacking the C-terminal tail at 1 μM concentration in isolation ( left ) ; and sensorgrams of XLF-WT and truncation XLF ( 1-224 ) lacking the C-terminal tails at 1 μM concentrations in isolation ( right ) . Source data are provided in Figure 8—figure supplement 1—source data 3 . ( D ) Sensorgrams of equimolar combinations ( 1 μM each ) of the WT and truncated XRCC4 and XLF proteins as indicated . Source data are provided in Figure 8—figure supplement 1—source data 3 . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 02210 . 7554/eLife . 22900 . 023Figure 8—figure supplement 1—source data 1 . Sensorgrams of XRCC4 and XLF variants in isolation . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 02310 . 7554/eLife . 22900 . 024Figure 8—figure supplement 1—source data 2 . Sensorgrams and mono- and double-exponential fits of XRCC4-XLF WT-WT and Asp-Asp combinations . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 02410 . 7554/eLife . 22900 . 025Figure 8—figure supplement 1—source data 3 . Sensorgrams of XRCC4-XLF full-length and truncations in isolation and in combination . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 025 XRCC4 is in large excess over LIG4 ( Mani et al . , 2010 ) . On the one hand XRCC4 associates with XLF to form complexes that bridge broken DNA during c-NHEJ ( Brouwer et al . , 2016 ) , and on the other hand , as a separate function , it associates with LIG4 to form a 2:1 XRCC4/LIG4 complex that performs the ligation step of c-NHEJ , which is further stimulated by XLF ( Lu et al . , 2007; Tsai et al . , 2007 ) . Therefore , to assess the role of the XRCC4 and XLF C-terminal tails in this distinct function of XRCC4 , we next considered whether the XRCC4 and XLF Ala and Asp phosphorylation site mutants could support LIG4 function . Recombinant LIG4-XRCC4 complexes were produced incorporating XRCC4-WT , XRCC4-Ala or XRCC4-Asp ( Figure 9—figure supplement 1 ) ; and these complexes were systematically analyzed for cohesive end ligation activity in combination with XLF-WT , XLF-Ala , or XLF-Asp ( Figure 9A , B and C ) . As can be seen , alanine or aspartate modifications have no major impact on LIG4 activity , even when LIG4-XRCC4-Asp was combined with XLF-Asp . These data suggest that the disordered C-terminal tails of XRCC4 and XLF regulate the DNA bridging activity of XRCC4-XLF complexes , but do not directly affect the ability of XLF to stimulate the XRCC4/LIG4 complex . 10 . 7554/eLife . 22900 . 026Figure 9 . XRCC4 and XLF phospho-mimetics do not bridge DNA ends but are completely sufficient in stimulating LIG4-XRCC4 activity . Stimulation of LIG4/XRCC4-WT , LIG4/XRCC4-WT-eGFP , LIG4/XRCC4-Ala-eGFP or LIG4/XRCC4-Asp-eGFP cohesive end ligation by XLF-WT ( A ) , XLF-Ala ( B ) or XLF-Asp ( C ) . Ligation products were deproteinized and resolved by agarose gel electrophoresis followed by detection by ethidium bromide staining . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 02610 . 7554/eLife . 22900 . 027Figure 9—figure supplement 1 . Coomassie stained reducing SDS-PAGE analysis of recombinant LIG4/XRCC4 variants purified after overexpression in bacteria ( 1 μg/lane ) . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 027 Our biochemical analyses suggest that XRCC4 and XLF C-terminal tails may be redundant with one another . For example , the Asp variants of either XRCC4 or XLF paired with WT ( and Ala ) partners bind and dissociate from DNA analogously to the WT-WT combinations ( Figure 8 ) , but when the Asp variants are combined together , DNA dissociation is markedly more rapid . Thus , any functional impact of the C-terminal tails in living cells might be masked by this redundancy when XRCC4 or XLF are studied individually . To facilitate functional analyses of XRCC4 and XLF C-terminal disordered tails in concert in living cells , we used 293 T cells where genes encoding both proteins were disrupted using CRISPR/Cas9 ( Neal et al . , 2016 ) . To co-express WT or mutant forms of XRCC4 and XLF , expression constructs were prepared so that C-terminal myc-tagged XRCC4 and XLF ( interrupted by an IRES sequence ) are expressed from the same promoter . Constructs were prepared encoding XRCC4 alone , or XRCC4 and XLF together , with all 14 DNA-PK/ATM phosphorylation sites intact , or with all sites substituted to Ala or Asp . We have recently described a panel of episomal substrates ( Neal et al . , 2016 ) in which two pairs of V ( D ) J recombination signal sequences ( RSS ) or I-Sce1 sites are separated by the red fluorescent protein ( RFP ) coding sequence ( Figure 10 , top panels ) . These substrates provide an internal control ( RFP expression ) for plasmid uptake and nuclear localization in the transfected cells . Additionally , they include the SV40 origin of replication , and are thus efficiently replicated episomally in 293 T cells . If recombination occurs , the RFP gene is deleted , juxtaposing the CFP gene with the promoter allowing CFP expression as a reporter of end joining activity that can be quantified by flow cytometry . 10 . 7554/eLife . 22900 . 028Figure 10 . XRCC4 and XLF phosphorylation site mutation does not affect end joining of episomal substrates . Fluorescent substrates ( depicted in top panels ) were utilized to detect V ( D ) J coding and signal joints in XRCC4/XLF-deficient 293 T cells transiently expressing full-length RAG1 , RAG2 , and WT , Ala , or Asp mutants of XRCC4 and XLF ( A ) , or joining of I-Sce1 induced DSBs ( B ) . Bottom panels show percent recombination of episomal fluorescent substrates . Error bars indicate SEM from five independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 028 Although XLF is dispensable for V ( D ) J recombination in murine lymphocytes , XLF is required for V ( D ) J recombination in human lymphocytes , and patients with mutations in XLF exhibit a SCID phenotype ( Buck et al . , 2006; Woodbine et al . , 2014 ) . Thus , in 293 T cells lacking both XRCC4 and XLF , transfection of the XRCC4 only encoding construct does not reconstitute either coding or signal joining; in contrast , substantial complementation is observed for both coding and signal end joining when both XRCC4 and XLF are expressed ( Figure 10A ) . As can be seen , the expression constructs encoding XRCC4-XLF with 14 phosphorylation sites substituted to either Ala or Asp fully reconstitute both V ( D ) J coding and signal end joining . We conclude that phospho-mimetic or -blocking mutations in the disordered C-terminal tails of XRCC4 and XLF do not affect joining of RAG-induced DSBs of episomal substrates . In contrast to RAG induced DSBs that are exclusively dependent on c-NHEJ for repair , DSBs introduced by restriction enzymes can be repaired by a-NHEJ , although this repair is slower , less efficient , and requires terminal short sequence homology ( Deriano and Roth , 2013 ) . As can be seen in Figure 10B , some end joining is observed in cells lacking both XRCC4 and XLF , or in cells complemented with only XRCC4; this reflects a-NHEJ activity . However , end joining is much more efficient when both XRCC4 and XLF are expressed . This is true whether I-Sce1 alone initiates the DSBs , or whether a fusion protein of I-Sce1 and the trex exonuclease is used to initiate DSBs . As with RAG-induced DSBs , blocking or phospho-mimicking the 14 sites does not affect the efficiency of end joining . All together from these data , we conclude that stable XRCC4/XLF DNA bridging is not required for efficient end joining of proximal I-Sce1 or RAG induced DSBs on episomal substrates . XRCC4 is required not only to stimulate LIG4 , but is also required for LIG4 stability . An additional construct was prepared in which three point mutations were introduced into XRCC4 that disrupt XRCC4's interaction with LIG4 [F180D +I181D+A183V] ( Modesti et al . , 2003 ) . XRCC4-XLF expressed from this construct should be capable of forming complexes , but would not stabilize or stimulate LIG4 . This construct was tested for its ability to promote end joining of the I-Sce1 substrate . As can be seen ( Figure 11 ) , the LIG4 mutant XRCC4-XLF construct does not enhance end joining . These data suggest that DNA bridging by XRCC4-XLF complexes does not promote a-NHEJ . 10 . 7554/eLife . 22900 . 029Figure 11 . XRCC4 and XLF complexes do not promote a-NHEJ . The fluorescent substrate ( depicted in the top panel ) was utilized to detect joining of I-Sce1 induced DSBs . Bottom panel shows percent recombination of episomal fluorescent I-Sce1 end joining substrate in XRCC4/XLF-deficient 293 T cells transiently expressing I-Sce1 , XRCC4 alone , WT XRCC4 and XLF , or an XRCC4 mutant that is defective in LIG4 interaction and WT XLF as indicated . Error bars indicate SEM from three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 029 To further assess the concomitant role of XRCC4 and XLF C-terminal disordered tails in the response to radiomimetic drugs , we generated stable 293 T cell clones expressing WT , Ala , Asp , or vector control by integrating the IRES co-expression constructs described above . Two clones of each construct were analyzed , but only one clone presented . As can be seen , stably reconstituted XRCC4/XLF -/- cells express levels of XRCC4 and XLF ( WT and variants ) similar to endogenous levels ( Figure 12A ) . The complemented XRCC4 and XLF proteins have slightly slower electrophoretic mobility because of their C-terminal myc tags . The Asp mutant has an even more substantial change in electrophoretic mobility because of the charge difference that results from S/T to Asp substitution10 . 7554/eLife . 22900 . 030Figure 12 . XRCC4 and XLF phosphorylation site mutants do not fully reverse sensitivity to radio-mimetic drugs . ( A ) Immunoblots of stable expression of WT , Ala , or Asp mutants of XRCC4 and XLF in 293 T cells that lack both XRCC4 and XLF . * non-specific species . ( B ) 293 T cells expressing WT , Ala , Asp , or no XRCC4 or XLF were exposed to Zeocin ( 500 μg/ml for 1 hr ) or not and analyzed after 1 hr or 24 hr by immunoblotting for XRCC4 , β-actin , or γ-H2AX as indicated . Zeocin ( C ) and Neocarzinostatin ( D ) sensitivity of 293T XRCC4/XLF double deficient cell strains complemented by stable expression of equivalent levels of WT , Ala , or Asp mutants of XRCC4 and XLF . Error bars indicate SEM from at least three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 03010 . 7554/eLife . 22900 . 031Figure 12—figure supplement 1 . XRCC4 and XLF phosphorylation site mutants do not fully reverse sensitivity to radio-mimetic drugs . ( Left ) Immunoblot showing comparable to endogenous , stable expression of WT , Ala , or Asp mutants of XRCC4 and XLF in previously described XRCC4 deficient HCT116 cells that were deleted for XLF . Parental cell strains , as well as XLF deficient cells that were generated by AAV targeting are also depicted . ( Right ) Neocarzinostatin sensitivity of the indicated cell strains stably expressing WT , Ala , or Asp mutants of XRCC4 and XLF . Error bars indicate SEM from at least three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 03110 . 7554/eLife . 22900 . 032Figure 12—figure supplement 2 . XRCC4 and XLF phosphorylation site mutants do not fully reverse sensitivity to radio-mimetic drugs . ( Left ) Immunoblot showing comparable to endogenous , stable expression of WT , Ala , or Asp mutants of XRCC4 and XLF in XRCC4-deficient hamster XR-1 cells deleted for XLF . ( Right ) Neocarzinostatin sensitivity of the indicated cell strains stably expressing WT , Ala , or Asp mutants of XRCC4 and XLF . Error bars indicate SEM from at least three independent experiments . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 032 The substantial phosphorylation of XRCC4 induced by DNA damage results in a dramatic change in electrophoretic mobility . To determine whether the phospho-blocking mutant substantially blocks damage-induced phosphorylation , XRCC4 phosphorylation was assessed by immunoblotting . As can be seen ( Figure 12B ) , a marked shift in XRCC4's electrophoretic mobility is induced by zeocin ( at 1 or 24 hr post treatment ) in cells expressing WT but not Ala XRCC4 and XLF . The shift is equivalent to the change in mobility observed in the XRCC4 Asp mutant . The radiomimetic antibiotics zeocin ( a bleomycin analog ) and neocarzinostatin induce DSBs by free-radical attack of sugar residues in both DNA strands . The DSBs that result from either drug have complex DNA ends , with the complexity of damage being more dramatic with neocarzinostatin as compared to zeocin ( reviewed in Povirk , 2012 ) . As can be seen ( Figure 12C and D ) , WT XRCC4 and XLF substantially reverse both zeocin and neocarzinostatin sensitivity of 293T XRCC4/XLF deficient cells . In 293 T cells , blocking all 14 phosphorylation sites with alanine substitutions reduces cell survival after exposure to either zeocin or neocarzinostatin compared to cells expressing WT controls . In contrast , the Asp variants combination does not reverse either zeocin or neocarzinostatin sensitivity in XRCC4/XLF deficient 293 T cells . These data suggest that in 293 T cells , XRCC4-XLF stable DNA bridging ( abrogated in the Asp mutants combination ) is required for repair of zeocin and neocarzinostatin induced DSBs . Moreover , the fact that the Ala variants combination is also impaired suggests that disassembly of XRCC4-XLF complexes is also required to facilitate repair . To further extend our analysis in other cell lines , we used CRISPR/Cas9 to disrupt XLF in XRCC4 deficient HCT116 cells ( Figure 12—figure supplement 1 , left panel ) that were generated by AAV gene targeting ( Fattah et al . , 2014 ) . In HCT116 cells , both the Ala and Asp variants fully reconstitute zeocin resistance ( not shown ) . In contrast , cells expressing either Asp or Ala mutants are more sensitive to neocarzinostatin ( the drug inducing more dramatic end aberrations ) than WT controls ( Figure 12—figure supplement 1 , right panel ) . Thus , as we and others have shown previously ( Li et al . , 2008; Roy et al . , 2015 ) , different organisms , and different cell types have variable dependence on XLF . To corroborate these findings , we used again CRISPR/Cas9 to disrupt XLF in the XRCC4 deficient CHO cell strain XR-1 ( Figure 12—figure supplement 2 , left panel ) . XR-1 XRCC4/XLF double deficient cells were transfected with the XRCC4-XLF expression constructs . Similar , to 293 T cells , whereas WT XRCC4 and XLF substantially reverse neocarzinostatin hypersensitivity , complementation is progressively less substantial with the Ala and Asp variant constructs ( Figure 12—figure supplement 2 , right panel ) . The in vitro analyses of XRCC4/XLF phospho-mimetic complexes reported above demonstrated that phospho-mimicking ablates DNA bridging but does not alter XRCC4/XLF’s ability to promote LIG4 activity . We have also shown previously ( Roy et al . , 2015 ) that a separation of function XLF mutant , proficient in stimulating LIG4 but deficient in DNA bridging , is functional in supporting episomal V ( D ) J joining , consistently with the phospho-mimetic mutants presented here , which are also proficient in stimulating LIG4 but deficient in DNA bridging . We next addressed whether joining of chromosomal DSBs is affected by disrupting XRCC4/XLF mediated DNA bridging . To assess chromosomal end joining in isogenic cell strains expressing WT , Ala , or Asp versions of XRCC4 and XLF , we exploited CRISPR/Cas9 targeting of the FANCG gene on chromosome 9 , which we found to be remarkably efficient in 293 T cells . We reasoned that DNA bridging might be required specially to join distant DSBs , and targeting reagents for three sites within ~4 kb on chromosome nine were prepared ( Figure 13A ) . Joining of two separate deletions was assessed , one that results in ~300 bp deletion , and one that results in ~4 kb deletion . Although this strategy does not allow quantification of joining , the quality of joining can be assessed by sequencing . 293 T cells expressing WT XRCC4 and XLF , 14 X Ala , 14 X Asp , or no XRCC4/XLF were transfected with various combinations of gRNA/Cas9 plasmids . Cells were harvested and DNA prepared 72 hr later and PCR performed to detect chromosomal deletions . As can be seen , chromosomal deletions are readily detected with both gRNA pairs in cells that have been reconstituted with XRCC4 and XLF . Although no joints are observed in the vector control cell strain with one gRNA pair , deletional joints are observed with the other suggesting that certain DSBs may be more readily joined by a-NHEJ than other DSBs . By electrophoretic mobility , the deletional joints using gRNAs 2 and 3 ( that induce the small 300 bp deletion ) appear remarkably similar in cells expressing WT , Ala , and Asp versions of XRCC4 and XLF ( Figure 13B ) . Indeed , sequence analysis revealed no significant differences ( data not shown ) . In contrast , the 4 kb deletions generated in the four cell strains appear much more heterogeneous . By sequence analyses , joints recovered from cells expressing WT or alanine substituted XRCC4 and XLF are very similar . As expected , joints recovered from cells lacking XRCC4 and XLF display increased nucleotide loss . Although joints from all four cell strains predominately occur at regions of short end terminal homology ( SSH ) , the length of SSH is increased in cells lacking XRCC4 and XLF ( Figure 13C ) . Joints recovered from cells expressing aspartate substituted XRCC4 and XLF show an intermediate increase in nucleotide loss , and moderate increase in the length of SSH utilized in joining ( Figure 13D ) . We conclude that DNA bridging facilitates accurate repair of certain chromosomal DNA DSBs . 10 . 7554/eLife . 22900 . 033Figure 13 . Phospho-mimicking XRCC4 and XLF alters repair of chromosomal DNA DSBs . ( A ) Diagram of region on chromosome nine targeted by three different gRNAs and position of primers utilized to detect chromosomal deletions induced by Cas9 and indicated gRNAs . ( B ) Ethidium bromide staining of PCR amplifications of DNA isolated from cells expressing WT , Ala , Asp , or no XRCC4 and XLF and transfected with the different combinations of gRNAs as indicated . A no gRNA control DNA of parental 293 T cells is also included to help define products induced by Cas9 induced deletions . ( C ) Summary of sequenced joints utilizing gRNAs 1 and 3 that result in a 4 kb chromosomal deletion . ( D ) Graphs represent the percentage of joints deleting increasing numbers of nucleotides from predicted double strand break site . DOI: http://dx . doi . org/10 . 7554/eLife . 22900 . 033
How could the disordered C-terminal tails of XRCC4 and XLF affect the behavior of XRCC4-XLF complexes at the molecular level ? Structural and docking studies have revealed that XRCC4 and XLF can form alternating filamentous structures that can further assemble into bundles with a central channel ( Andres et al . , 2012; Hammel et al . , 2011; Ropars et al . , 2011; Wu et al . , 2011 ) . One model proposes that when interacting with DNA , XRCC4-XLF complexes would wrap around DNA , trapping it in the central channel forming a tube-like structure that can diffuse along the DNA with very little friction ( Brouwer et al . , 2016 ) . DNA tethering by these bundles would be promoted by the ability of XRCC4 dimers to tetramerize by interdigitation of their α-helical stalks ( Andres et al . , 2012 ) . However , this model doesn’t take into account the positions of the disordered C-terminal tails of XRCC4 and XLF because the crystal structures were obtained with truncated forms of XRCC4 and XLF lacking the C-terminal regions . Yet , building upon this model and others ( Hammel et al . , 2011; Ropars et al . , 2011; Wu et al . , 2011 ) , there are several potential mechanisms by which the disordered C-terminal tails of XRCC4 and XLF could stabilize the proteins/DNA interaction . First , the tails could promote stabilization of the XRCC4-XLF homodimer-homodimer interactions during filament assembly and concurrently favor DNA binding since the DNA binding domain of XLF resides at the extreme C-terminus ( Figure 1B , Andres et al . , 2007 ) . Second , the tails could be involved in stabilization of filament bundles ( Andres et al . , 2012; Wu et al . , 2011 ) that could correspond to the sleeve-like structure sliding on DNA that were observed at the single molecule level ( Brouwer et al . , 2016 ) . Finally , the tails could also contribute to the robust DNA tethering observed for XRCC4-XLF complexes ( Brouwer et al . , 2016 ) . Of note , in ITC experiments the thermodynamic parameters that contribute to the free energy of the XRCC4-XLF interaction ( enthalpy and entropy ) are distributed differently for the XRCC4-Asp-XLF-Asp complex as compared to either the WT-WT and Ala-Ala combinations . These data suggest that the tails may adopt a variety of conformations; deciphering these will require additional experimental strategies [nuclear magnetic resonance spectroscopy , electron paramagnetic resonance , FRET , and molecular dynamics simulations] . In vitro , XRCC4 and XLF independently bind to DNA with greater stability as the DNA substrate increases in size ( Hentges et al . , 2006; Lu et al . , 2007; Modesti et al . , 1999 ) . Clearly , XRCC4-XLF complexes bind to and bridge DNA with much higher affinity as compared to that of either homodimer alone ( Andres et al . , 2012 ) . Here , we find that XLF alone is able to bridge DNA when the size of the DNA molecule is increased ( 2 . 7 kb instead of 0 . 5 kb , Figure 7 ) . Given that XLF is a dimer where each monomer has a single DNA binding domain located within the very last ~20 residues of the C-terminus ( Andres et al . , 2007 ) , we propose that a single XLF dimer interacts with , thus bridges , two independent DNA molecules . The XLF DNA binding domain is akin to the ‘AT-hook’ DNA binding motifs present in High Mobility Group A proteins; these AT-hook motifs are all similar to XLF’s C-terminal tail in that they are intrinsically disordered DNA binding protein domains ( Aravind and Landsman , 1998; Fonfría-Subirós et al . , 2012 ) . The AT-hook motif is a weak DNA binding module interacting with the minor groove of DNA preferentially with AT rich regions . If XLF shares this mode of DNA binding , this would provide a good explanation for the observed concentration and DNA size dependence that is requisite for stable DNA binding and bridging by XLF . In addition , we find the XLF-Asp phospho-mimetic variant , in which all the ‘phosphorylated’ residues are located in the disordered tails but away from the very C-terminal DNA binding domain , exhibits normal DNA binding by EMSA but has a reduced ability to bridge DNA ( Figures 3 and 7 ) . A possible explanation for this behavior is that one of the two XLF ‘phosphorylated’ tails in a homodimer masks the DNA binding domain of the other tail in the XLF dimer with sufficient affinity to confer , DNA binding , but not to facilitate bridging . Alternatively , they could attach and detach fast from DNA so that , overall , they are bound to DNA but not stably enough to bridge two DNA molecules . Several levels of phosphorylation-dependent regulation of XRCC4 and XLF have been reported that affect function . Phosphorylation of XRCC4 on T233 by CDK2 has been implicated in recruitment of PNK , Aprataxin , and APLF through interaction with the FHA domain present in these DNA processing enzymes , thus affecting the scaffold function of XRCC4 ( Cherry et al . , 2015; Clements et al . , 2004; Iles et al . , 2007; Koch et al . , 2004; Macrae et al . , 2008 ) . Phosphorylation of XLF by Akt on T181 triggers its dissociation from the LIG4/XRCC4 complex and its sequestration in the cytoplasm ( Liu et al . , 2015 ) . However , a functional impact for either of these phosphorylations on the actual efficacy of c-NHEJ has not been reported . The functional relevance of DNA-PK/ATM phosphorylations of either XRCC4 or XLF is a long-standing question . Although DNA-PK phosphorylation of XRCC4 in vitro reduces its affinity for DNA ( Modesti et al . , 1999 ) , neither XRCC4 nor XLF DNA-PK/ATM dependent phosphorylations are required for c-NHEJ as measured by complementation of radiosensitivity or V ( D ) J joining assays in living cells ( Lee et al . , 2004; Yu et al . , 2003 , 2008 ) . We have reported previously that DNA-PK autophosphorylation sites exist as a functional cluster; in these studies , ablation of one or two sites had no functional impact . The functional impact of phosphorylation could only be appreciated when most or all of the sites in the cluster were abrogated ( Cui et al . , 2005; Ding et al . , 2003; Neal et al . , 2014 ) . Thus , if the C-terminal tails of XLF and XRCC4 function together , the impact of phosphorylation might not be apparent if each is studied in isolation , as in the approach utilized in previous studies to assess the functional relevance of XLF and XRCC4 DNA-PK/ATM mediated phosphorylations ( Yu et al . , 2003 , 2008 ) . Thanks to the advent of CRISPR/Cas9 genome-editing techniques , the potential functional redundancy or the need for concomitant phosphorylation in multi-protein complexes can be more easily assessed . In fact , cells deficient in multiple factors can readily generated , paving the way to study the impact of phosphorylation of the C-termini of both XRCC4 and XLF in concert . Here , we show that simultaneously phospho-mimicking sites in both the C-terminal tails of XRCC4 and XLF , completely abrogates DNA tethering in vitro , suggesting no functional redundancy between these phosphorylations , and that phosphorylation of both is required to efficiently disrupt XRCC4-XLF complexes bound to DNA . Phospho-mimicking mutants are severely compromised in DNA bridging in vitro . Consistent with a functional role in DNA bridging of chromosomal DSBs , co-expression of XRCC4 and XLF phospho-mimetic variants in XRCC4/XLF double deficient 293 T cells , HCT116 cells , or XR-1 cells failed to fully rescue cellular sensitivity to neocarzinostatin — even though these complexes can stimulate LIG4/XRCC4 activity in vitro and join both RAG or I-Sce1-induced DSBs on episomal substrates in living cells . We conclude that DNA bridging is required to repair neocarzinostatin induced chromosomal DNA DSBs . This conclusion is bolstered by results presented in Figure 13 demonstrating that rejoined chromosomal DSBs from cells that cannot form XRCC4/XLF filaments ( because of phospho-mimicking substitutions ) display increased nucleotide loss , and a higher dependence on short sequence homology at the DNA termini . Moreover , co-expression of XRCC4 and XLF phospho-blocking mutants in XRCC4/XLF double deficient cells only partially reversed sensitivity to neocarzinostatin providing evidence that phosphorylation-dependent disruption of XRCC4 and XLF complexes is also very important for repair . We suggest that dismantle of XRCC4-XLF-DNA complexes ( after repair is complete ) is a crucial step in c-NHEJ , perhaps analogously to the recently established requisite release of the Ku ring after completion of repair ( Brown et al . , 2015; Postow , 2011; Postow et al . , 2008; van den Boom et al . , 2016 ) , and that is tightly regulated by phosphorylation of the disordered C-terminal tails of XRCC4 and XLF , possibly by DNA-PK/ATM .
Expression plasmids and purification procedures for XRCC4-WT , XRCC4 ( 1–157 ) , XLF-WT , XLF ( 1-224 ) , XLF-L115A , XLF-L115D have been described ( Andres et al . , 2012 , 2007 ) . The expression constructs for the XRCC4 and XLF Ala and Asp variants were generated by site-directed mutagenesis of the vectors expressing the WT proteins as indicated in Figure 1 , and purified by the methods indicated above . The LIG4/XRCC4 complex was produced as described ( Cottarel et al . , 2013 ) . The LIG4/XRCC4-eGFP variants were produced by co-transformation of pCDFduet-1-LIG4-His6 and pRSFduet-1-XRCC4-eGFP ( WT , Ala or Asp variants ) in Rosetta 2 pLysS cells ( Novagen ) , expressed and purified as indicated above for the LIG4/XRCC4 complex . All proteins batches were dialyzed against 150 mM KCl , 20 HEPES pH8 , 1 mM EDTA , 2 mM DTT and 10% ( v/v ) glycerol , flash frozen in liquid nitrogen and stored at −80°C . T4 DNA ligase was obtained from New England Biolabs . Binding reactions ( 10 μl ) contained 200 ng of linearized pUC19 plasmid DNA , 10 mM HEPES pH 8 , 0 . 5 mM EDTA , 0 . 5 mM DTT , 5% ( v/v ) glycerol , and 75 mM KCl after addition of proteins at the indicated concentrations . Reactions were incubated at room temperature for 30 min and directly resolved by 0 . 6% agarose gel eletrophoresis in standard Tris-Borate-EDTA buffer . Gels were stained in Tris-Borate-EDTA buffer supplemented with 0 . 5 μg/mL ethidium bromide , destained in deionized water and documented using a UV light trans-illuminator and a CCD camera . DNA ligation assays were performed in a volume of 10 μL containing 200 ng of linearized pUC19 plasmid DNA ( XbaI for cohesive ends and SmaI for blunt ended ends ) , 1 mM ATP , 2 mM MgCl2 , 10 mM HEPES pH 8 , 0 . 5 mM EDTA , 1 mM DTT , 5% ( v/v ) glycerol , and 75 mM KCl after addition of proteins at the indicated concentrations . Reactions were incubated at room temperature for 15 min at room temperature before addition of 40 cohesive end units of T4 DNA ligase , or LIG4/XRCC4 ( and XLF ) at the indicated concentrations and further incubated for 30 min at room temperature . At the end of the incubation Sarkosyl ( Sigma-Aldrich ) and Pronase ( Roche ) were added at , respectively , 2% ( w/v ) and 1 mg/mL final concentrations and incubated for 30 min at 55°C . Samples were next supplemented with 1/5 vol of a solution containing 50 mM EDTA , 50 mM Tris pH 7 . 5 , 60% ( v/v ) glycerol and tracking dyes , and resolved by 0 . 8% agarose gel electrophoresis in standard Tris-Borate-EDTA buffer . Gels were stained in Tris-Borate-EDTA buffer supplemented with 0 . 5 μg/mL ethidium bromide , destained in deionized water , and documented using a UV light trans-illuminator and a CCD camera . The one-end biotinylated 1000 bp DNA substrate was prepared by PCR using Phire Hot Start DNA Polymerase ( New England Biolabs ) , primers 5’-Biotin-GAGTTTTATCGCTTCCATGACG and 5’-AATTTATCCTCAAGTAAGGGGC and ϕX174 DNA as template . The blunt ended 500 bp DNA fragment was similarly prepared but using primers 5’-GAGTTTTATCGCTTCCATGACG and 5’-CAGAAAATCGAAATCATCTTCG . The blunt ended 2 . 7 kb DNA substrate was prepared by digesting of pUC19 DNA with SmaI , gel purified , and stored in 10 mM Tris pH 8 , 1 mM EDTA . For the DNA bridging assay , 20 μL of streptavidin-coated bead suspension ( Dynabeads M-280 Streptavidin , Invitrogen ) were washed twice with 100 μL of DNA binding buffer ( DBB ) containing 150 mM KCl , 20 mM HEPES pH 8 , 1 mM EDTA , 1 mM DTT , 10% ( v/v ) glycerol , 0 . 5 mg/mL of Acetylated BSA ( Ambion ) and 0 . 2% ( v/v ) TWEEN 20 . Beads were resuspended in 20 μl DBB containing 1 μg of 1000 bp biotin-labeled DNA and incubated 10 min at 4°C to allow attachment . After washing twice with 100 μL DBB , beads were resuspended in 40 μL of DBB containing 1 μg of 500 bp ( or 2 . 7 kb ) DNA and the proteins at the indicated concentrations and incubated for 15 min at RT . Beads were next washed twice with 100 μL DBB and resuspended in 20 μL DBB without BSA but containing pronase at 1 mg/mL and 2% ( w/v ) Sarkosyl , and incubated at 55°C for 30 min . The samples were fractionated by 0 . 8% agarose gel electrophoresis in standard Tris-Borate-EDTA buffer . Gels were stained in Tris-Borate-EDTA buffer supplemented with 0 . 5 μg/mL ethidium bromide , destained in deionized water , and documented using a UV light trans-illuminator and a CCD camera . XRCC4 and XLF were mixed each at 20 μM in 20 μL of 150 mM KCl , 20 HEPES pH 8 , 1 mM EDTA , 2 mM DTT and 10% ( v/v ) glycerol incubated for 20 min at 4°C before addition of 20 μL of a stock of BS3 at 12 . 5 mM in water ( ThermoFisher Scientific ) . Reactions were incubated at room temperature for 1 hr and terminated by addition of 8 μL of 10% ( w/v ) SDS , 10 mM 2-mercaptoethanol , 20% ( v/v ) glycerol , 0 . 2 M Tris pH 6 . 8% and 0 . 05% ( w/v ) bromophenol blue . Samples were boiled for 3 min , fractionated by denaturing SDS polyacrylamide gel electrophoresis and the gels stained with Coomassie Brilliant Blue R250 before image capture . Purified XRCC4 and XLF ( WT , Ala and Asp variants ) were dialysed overnight and diluted in a buffer consisting of 20 mM Tris pH 8 and 150 mM NaCl . Titration experiments were carried out on a MicroCal ITC200 micro-calorimeter ( GE Healthcare , Piscataway , NJ ) . Experiments consisted of 13 injections of 3 μl of the titrant ( XLF ) into the analyte ( XRCC4 ) , using a titrant concentration set to be in the order of 10 or 13 times the analyte concentration . A first small injection ( 0 . 4 μl ) was included in the titration protocol in order to remove air bubbles trapped in the syringe prior to the titration . Raw data were adjusted setting the zero to the titration saturation heat value . Integrated raw ITC data were fitted using a one site non-linear least squares fit model using the MicroCal plugin as available in Origin 9 . 1 ( OriginLab Corp . , RRID:SCR_002815 ) . Kd , ΔG0 , and –TΔS0 values were calculated from the fitted ΔH0 and Ka values using the relations:Kd=1/KaΔG0=−R⋅T⋅ln⁡ ( Ka ) ΔG0=ΔH0−TΔS0 where R is the ideal gas constant ( 1 . 987 cal · K−1 · mol−1 ) and T the absolute temperature Surface Plasmon Resonance experiments have been conducted on a Biacore T200 instrument ( GE Healthcare ) using a four channels streptavidin-coated sensor chip ( BR-1005–31 , GE Healthcare ) . The chip was activated before DNA capture by a rapid wash with 1 M NaCl and 50 mM NaOH in MilliQ water and then in 50% ( v/v ) Isopropanol . The 400 bp-long dsDNA molecules have been generated by PCR using ϕX174 DNA as template and primers 5’-Biotin-GAGTTTTATCGCTTCCATGACG and 5’-ACTTGACTCATGATTTCTTACC , gel purified , diluted to a concentration of 22 . 5 ng/mL in 10 mM Tris pH 8 , 100 mM KCl , and 1 mM EDTA , and flushed in channels 2 , 3 , and four until the instrument response reached respectively 258 RU , 252 RU , and 511 RU . Channel one was not flushed with the DNA and used as reference channel during data analysis . All proteins ( WT and variants ) have been diluted in 75 mM KCl , 20 mM HEPES pH 8 , 10% ( v/v ) glycerol , 1 mM DTT , 1 mM EDTA to the desired final concentrations . The same buffer with the addition of 0 . 05% ( v/v ) TWEEN 20 was used as running buffer during experiments . Protein concentration was adjusted at about 10 μM , re-inspected by NanoDrop to double check the initial concentration , and finally diluted to the desired final concentration . In kinetics experiments requiring protein titrations , subsequent dilutions were done using the same pipetting volume and a single pipette tip for each different protein dilution serie . Protein samples were injected in the chip channels and let interact with the DNA on the surface typically for 240 or 300 s . Dissociation was observed during 480 s before the channels were washed and the chip regenerated with a 30 s-long flush of a 2 M KCl aqueous solution . A SDS-PAGE gel of the protein samples used for SPR experiments is shown in Figure 1—figure supplement 1 . In all cases with the exception of XRCC4 alone , a simple kinetics model of first order reactions failed to properly describe the data . We thus used equilibrium affinity and biphasic dissociation rates to quantify and compare the results . 293 T cells ( RRID:CVCL_0063 ) were the generous gift of Dr . Kefei Yu who purchased the cell strain from ATCC . XR-1 cells ( RRID:CVCL_K253 ) were the generous gift of Dr . Tom Stamato . HCT116 cells ( RRID:CVCL_0291 ) and the single XRCC4 ( RRID:CVCL_HE00 ) and XLF ( RRID: CVCL_HD86 ) knockout cells were the generous gift of Dr . Eric Hendrickson . Cells were regularly checked for mycoplasma contamination using the Mycoplasma Plus PCR primer set ( Stratagene Cat# 302008 ) . Antibodies used are mouse monoclonal anti-β-actin ( sigma A5441 , RRID:AB_476744 ) ; mouse monoclonal anti-γ-H2AX ( Millipore 05–636 RRID:AB_309864 ) ; rabbit polyclonal anti-XLF ( Abcam ab33499 , RRID:AB_778945 ) ; rabbit polyclonal anti-XRCC4 ( Serotec AHP387 , RRID:AB_2218607 ) . Cas9-targeted gene disruption was performed using methods similar to those reported by ( Mali et al . , 2013 ) . Briefly , gRNAs specific for XLF , XRCC4 or ATM were synthesized as 455 bp fragments ( Integrated DNA Technologies ) . The synthesized fragments were cloned into pCR2 . 1 using a TOPO TA cloning kit according to the manufacturers’ instructions ( Life Technologies ) . Cells were transfected with 1 µg gRNA plasmid and 1 µg Cas9 expression plasmid ( Addgene , RRID:SCR_002037 ) . In some cases , cells were co-transfected with 0 . 2 µg of pcDNA6 ( Life Technologies ) or pSuper-Puro to confer blasticidin or puromycin resistance . With the two human cell strains , western blotting was used to identify clones with deletions in each of these factors; in all cases , deletion was also confirmed by PCR amplification that revealed deletions at the target site . PCR amplification of the hamster XLF gene was used to define frameshift mutations in the XR-1 cell strain . The 19-mers specific for each factor synthesized into the 455 bp fragments are as follows: ATM: TCTTTCTGTGAGAAAATAC XRCC4: CCTGCAGAAAGAAAATGAA XLF: GGCCTGTTGATGCAGCCAT . The expression constructs providing for co-expression of WT , Ala , and Asp XRCC4 and XLF were sequentially assembled into pMSCV-puro ( Clontech ) , adding C-terminal Myc tags to each protein , and an IRES sequence between XRCC4 and XLF . Phosphorylation site substitutions are indicated in Figure 1 . RAG1 and RAG2 expression plasmids were a gift from Dr . David Roth . Construction of the I-SceI expression plasmid was described ( Neal et al . , 2016 ) . The I-Sce1-trex2 fusion expression plasmid was obtained from Dr . Jeremy Stark . Fluorescent V ( D ) J substrate plasmids have been described ( Neal et al . , 2016 ) . Briefly , the RFP coding sequence was inserted between the 12RSS and 23RSS , replacing the oop transcription terminator in both pJH290 and pJH289 . The recombination cassette was cloned upstream of the CFP coding sequence in peCFP-N1 ( which includes the SV40 origin of replication ) . The fluorescent V ( D ) J substrates ( diagramed in Figure 10 , top panels ) and V ( D ) J assays have recently been described ( Neal et al . , 2016 ) . These substrates ( derived from pECFP-N1 , Clontech ) contain the SV40 origin of replication , and are thus efficiently replicated episomally in all primate cell strains . Briefly , extrachromosomal fluorescent V ( D ) J assays were performed on cells plated at 20–40% confluency into 24-well plates in complete medium . Cells were transfected with 0 . 125 µg substrate , 0 . 25 µg RAG1 and 0 . 25 µg RAG2 per well using polyethylenimine ( PEI , Polysciences ) using 2 µL of PEI at 1 μg/mL per 1 µg of DNA . In experiments with additional expression plasmids , 0 . 25 µg of the expression plasmid or vector control was included . Cells were harvested 72 hr after transfection and analyzed for CFP and RFP expression by flow cytometry . The percentage of recombination was calculated as the percentage of cells expressing CFP divided by the percentage expressing RFP . Data presented represent at least three independent experiments , which each includes triplicate transfections . For chromosomal joining assays , the plasmid pSpCas9 ( BB ) −2A-Puro ( Addgene , RRID:SCR_002037 ) which provides co-expression of a gRNA , Cas9 , and the puromycin resistance gene was utilized . The target region 20-mers specific for three sites on chromosome nine are as follows: Target 1: GCTCCAGCTGCCTGGACCTG Target 2: GGGCCAGGCCTGGGTTCAAC Target 3: GACTTAAGAGAAAGGGACTG . 293 T cells expressing WT , Ala , Asp , or no XRCC4 or XLF were transfected with combinations of gRNAs as indicated and plated on puromycin after 24 hr . Cells were harvested 72 hr after transfection and genomic DNA extracted using DNAzol ( Thermo-Fisher ) according to the manufacturer's recommendations . PCR amplification was performed using Go-Taq ( Thermo-Fisher ) with the following amplification primers: Primer 1: CAGAAGTGCCCAGGCCCCTCC Primer 2: CCCAAGATGTCCCGGCTGTGGG Primer 3: CCATGGGCCTCTCTGTCCTTGCAC . Clonogenic survival assays were performed for HCT116 cells and XR-1 cells . Briefly , a hundred cells were plated for each transfection into complete medium containing the indicated dose of zeocin or neocarzinostatin in 60 mm diameter tissue culture dishes . After 7 to 10 days , cell colonies were stained with 1% ( w/v ) crystal violet in ethanol to measure relative survival . MTT staining was performed to assess cell viability for 293 T cells . 30 , 000 to 50 , 000 cells were plated in each well of a 24-well plate , containing medium with varying concentrations of drug . After 5 to 7 days of zeocin or neocarzinostatin treatment , cells were treated with 1 mg/mL MTT ( Sigma ) solution for 1 hr . Medium containing MTT was then removed and formazan crystals thus produced were solubilized in acidic isopropanol . Absorbance was read at 570 nm to determine relative survival .
XRCC4 and DNA Ligase 4 ( LIG4 ) form a tight complex that provides DNA ligase activity for classical non-homologous end joining ( the predominant DNA double-strand break repair pathway in higher eukaryotes ) and is stimulated by XLF . Independently of LIG4 , XLF also associates with XRCC4 to form filaments that bridge DNA . These XRCC4/XLF complexes rapidly load and connect broken DNA , thereby stimulating intermolecular ligation . XRCC4 and XLF both include disordered C-terminal tails that are functionally dispensable in isolation but are phosphorylated in response to DNA damage by DNA-PK and/or ATM . Here we concomitantly modify the tails of XRCC4 and XLF by substituting fourteen previously identified phosphorylation sites with either alanine or aspartate residues . These phospho-blocking and -mimicking mutations impact both the stability and DNA bridging capacity of XRCC4/XLF complexes , but without affecting their ability to stimulate LIG4 activity . Implicit in this finding is that phosphorylation may regulate DNA bridging by XRCC4/XLF filaments .
DNA in human and other animal cells is organised into structures called chromosomes . One of the most dangerous types of DNA damage is a double-strand break , where both strands of the DNA helix are broken in the same place . If this damage is not repaired it can be serious enough to kill the cell . If the DNA is repaired badly , part of one chromosome can become attached to another – a defect known as a chromosome translocation . Fortunately , cells are equipped with machineries that can recognise and fix these breaks . One of these processes is known as “non-homologous end joining” and it involves a set of proteins including two known as XRCC4 and XLF . These proteins work like a bandage , holding together the broken DNA until it is repaired . Both proteins have long tails , but the role of these structures was not clear . During DNA repair , the cell chemically modifies the tails of these proteins by a process called phosphorylation . However , previous studies have found that it is possible to prevent the modification of the tail of one of the proteins , or even remove the tail entirely , without affecting the repair process . Here , Normanno et al . investigated the effect of blocking the modification of the tails of both proteins at the same time . For the experiments , the tails were both altered in various places to either block or mimic the phosphorylation that normally occurs during DNA repair . Mimicking the phosphorylation of both tails affected the ability of XRCC4 and XLF to stay attached to the DNA , suggesting that the phosphorylation helps these proteins to detach from the DNA once the repair is complete . Furthermore , in human embryonic kidney cells the altered proteins were less able to repair DNA damage in response to a drug that causes double-strand breaks . These findings improve our understanding of how cells repair their DNA to maintain a complete set of genetic information . Defects in DNA repair are linked to conditions where the brain does not develop properly , whilst some cancer therapies deliberately inflict double-strand breaks to kill cancer cells . In the future , these findings may lead to improvements in radiotherapy and other treatments for human diseases .