ptrichisb vectors  (Addgene inc)

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM–DNA2-dependent 5′-end resection

doi: 10.1093/nar/gkae642

Figure Lengend Snippet: TRF2–RAP1 inhibits RAD51-mediated telomere D-loop formation. ( A ) Schematic of the nuclear extract D-loop assay. 32 P-labeled telomere ssDNA was incubated with nuclear extract isolated from cells and telomere plasmids containing (TTAGGG) 17 repeats. Successful invasion of the ssDNA into the plasmid forms the telomere D-loop structure. Asterisk (*) denotes the 32 P-labeled ssDNA end. ( B ) Cells expressing the TRF2 ΔB, L288R mutant accumulate RAD51-induced D-loops on telomeres. Nuclear extract from U2OS cells expressing empty vector, shTRF2, and shTRF2-depleted cells expressing TRF2 ΔB or TRF2 ΔB, L288R mutants were examined for telomere D-loop formation. The ssDNA and generated D-loops were separated on 1% agarose gel. ( C ) Quantification of telomere D-loop formation described in (B). D-loops relative to empty vectors (lane 2) from three independent experiments were shown as the mean ± S.D. Statistical evaluation was performed via one-way ANOVA. * P = 0.0126; **** P < 0.0001. ( D ) Schematic of the RAD51 or/and TRF2-mediated telomere D-loop assay. 32 P-labeled telomere ssDNA was first incubated with purified RAD51 protein to form RAD51–ssDNA filament, and then mixed with TRF2 and/or RAP1-preincubated telomere plasmid. The RAD51–ssDNA strand invasion into the protein-bound plasmid formed telomere D-loops. Asterisk indicates the labeled ssDNA end. ( E ) TRF2 only slightly inhibited RAD51-mediated telomere D-loop formation. RAD51, TRF2, TRF2 ΔB , or TRF2 ΔB, L288R and the indicated protein combinations were tested for their ability to form telomere D-loops. ( F ) D-loop formation relative to control (RAD51 alone, line 2) from three independent experiments were analyzed as shown in (E). Mean values ± S.D. were plotted. Data were evaluated by one-way ANOVA analysis. ‘ns’ indicates no significant differences ( P > 0.05); ** P = 0.0033 and 0.0057; **** P < 0.0001. ( G ) TRF2 and RAP1 synergized to promote RAD51-dependent telomere D-loop inhibition. TRF2, RAP1 and the indicated protein combinations (400 nM of TRF2 and 200, 400, 800 nM of RAP1) were tested for their ability to affect RAD51-mediated telomere D-loop formation. The reaction without ATP was used as negative control (lane 3). ( H ) Quantification of D-loops generated in Figure compared to RAD51 alone (lane 2) are analyzed. Data from three independent experiments were shown as the mean ± S.D. in the bottom panel. Statistical evaluation was performed by one-way ANOVA analysis. ns, non-significant ( P > 0.05); **** P < 0.0001.

Article Snippet: WT hTRF2 and TRF2 ΔB in pTricHisB vectors (Addgene #50 488 and #53 208) were transformed into Escherichia coli BL21(DE3) cells.

Techniques: Labeling, Incubation, Isolation, Plasmid Preparation, Expressing, Mutagenesis, Generated, Agarose Gel Electrophoresis, Purification, Control, Inhibition, Negative Control

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM–DNA2-dependent 5′-end resection

doi: 10.1093/nar/gkae642

Figure Lengend Snippet: TRF2–RAP1 blocks RAD51-mediated homology search on telomeres. ( A ) Schematic of the assay used to examine the effect of TRF2–RAP1 on the stability of RAD51-coated telomere ssDNA filaments. RAD51 protein was used to assemble filaments on 5′-biotinylated telomere ssDNA that was immobilized on streptavidin resin. TRF2 or/and RAP1 was incubated with the RAD51 coated telomere ssDNA filaments and then 10 × excess of non-biotinylated telomere ssDNA was added in the reaction. RAD51 protein levels in both the beads and the supernatant fractions were analyzed by SDS-PAGE and Coomassie Blue-staining. ( B ) TRF2–RAP1 does not affect RAD51 filament stabilization on telomere ssDNA. TRF2 and RAP1(200 or 400 nM) either individually or in combination were tested for RAD51 telomere ssDNA filament stabilization. The bead fractions containing proteins associated with the biotinylated ssDNA and the supernatant fractions containing proteins trapped on the excess ssDNA were analyzed by SDS-PAGE with Coomassie Blue staining. ( C ) The percentage of RAD51 protein in the supernatant and bead fractions were plotted as mean ± S.D. from two independent experiments. Statistical evaluation was performed by unpaired t test analysis. ns, non-significant ( P > 0.05). ( D ) Schematic of the telomere dsDNA capture assay by the RAD51 filament. RAD51 was used to assemble filaments on 5′-biotinylated telomere ssDNA that was immobilized on streptavidin resin, and then these filaments were incubated with 32 P-labled telomere dsDNA either alone or bound to TRF2–RAP1. Radiolabeled dsDNA on the bead and supernatant fractions were resolved by electrophoresis in 10% native polyacrylamide gels. ( E ) TRF2–RAP1 inhibits the ability of RAD51-bound telomere ssDNA filaments to capture telomere dsDNA. TRF2, RAP1 (200 or 400 nM) either singly or in combination were incubated with RAD51 coated telomere ssDNA filaments, and their ability to capture telomere dsDNA was analyzed. ATP was omitted from the reaction in lane 6. DNA-binding protein UAF1 was used as a negative control (lane 3). ( F ) The percentages of captured telomere dsDNA in the bead fractions are shown. The error bars represent mean values ± S.D. of data from three independent experiments. Statistical evaluation was performed by ANOVA. ns: non-significant ( P > 0.05); **** P < 0.0001.

Article Snippet: WT hTRF2 and TRF2 ΔB in pTricHisB vectors (Addgene #50 488 and #53 208) were transformed into Escherichia coli BL21(DE3) cells.

Techniques: Incubation, SDS Page, Staining, Electrophoresis, Binding Assay, Negative Control

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM–DNA2-dependent 5′-end resection

doi: 10.1093/nar/gkae642

Figure Lengend Snippet: TRF2–RAP1 promotes BLM-mediated unwinding of telomere D-loops. ( A ) Schematic of the oligo-based telomere D-loop unwinding assay. Telomere D-loop DNA substrates were generated by hybridizing three telomere DNA fragments (TD1, 32 P-labeled TD2 and TD3). TRF2 and/or RAP1 were pre-incubated with the D-loop and then BLM was added to the reaction to detect D-loop unwinding. Displacement of the invading telomere DNA strand from the D-loop indicates D-loop unwinding. ( B ) The TRF2–RAP1 promotes BLM-mediated unwinding of telomere D-loops. The effect of TRF2 and indicated mutants either singly or in combination with RAP1 on BLM-mediated telomere D-loop unwinding was examined. The sizes of 32 P-labled telomere ssDNA, D-loops, and dsDNA without the invading telomere strand are shown in lanes 1–3. ( C ) Quantification of BLM-mediated unwinding reactions displayed in Figure . The percentages of unwound D-loop are shown as mean values ± S.D. from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P > 0.05); * P = 0.0186; **** P < 0.0001. ( D ) Schematic of the assay used to measure the ability of BLM to unwind RAD51-generated telomeric D-loops. Telomeric D-loops were generated by incubating RAD51, 32 P-labled telomeric ssDNA and plasmid containing telomere repeats together as described in Figure . Native plasmid-sized telomere D-loops were obtained after deproteinization and gel purification. BLM without or with TRF2–RAP1 was then incubated with these D-loops and their unwinding was analyzed by 1% agarose gel. ( E ) BLM-mediated unwinding of RAD51-genearted telomeric D-loops was enhanced by TRF2–RAP1. WT TRF2 or various TRF2 mutants complexed with RAP1 were incubated with D-loops and tested for their effects on the ability of BLM to unwind D-loops. ( F ) The amount of D-loops formed relative to the negative control (no protein, lane 2) were quantified and plotted as mean ± S.D. from three independent experiments. Statistical evaluation was performed by ANOVA test. ns indicates non-significant ( P > 0.05); **** P < 0.0001.

Article Snippet: WT hTRF2 and TRF2 ΔB in pTricHisB vectors (Addgene #50 488 and #53 208) were transformed into Escherichia coli BL21(DE3) cells.

Techniques: Generated, Labeling, Incubation, Plasmid Preparation, Gel Purification, Agarose Gel Electrophoresis, Negative Control

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM–DNA2-dependent 5′-end resection

doi: 10.1093/nar/gkae642

Figure Lengend Snippet: TRF2–BLM interaction is required to promote BLM-mediated unwinding of telomere D-loops. ( A ) Diagram showing that the TRF2 TRFH domain interacts with specific residues in BLM helicase domain containing the conserved TRFH binding motif FILMP (amino acids 686–690). Other functional domains in TRF2 are also illustrated: N-terminal basic domain, RAP1-binding motif, TIN2-binding motif and C-terminal Myb domain. The BLM N-terminal domain, winged-helix motif (RQC); helicase/RNase D C-terminal domains (HRDC) and nuclear targeting signal domain are also illustrated. ( B ) BLM pulldown using its Maltose Binding Protein tag by amylose-resin revealed robust interaction between BLM and TRF2. His-tagged TRF2 and its mutants (TRF2 ΔB or TRF2 L288R ) and Flag-tagged RAP1 were incubated with MBP-tagged BLM and BLM mutants (3A or P690L). The protein complexes were captured on amylose-resin and the fractions were resolved on SDS-PAGE gel and visualized with Coomassie Blue staining. S, supernatant with unbound proteins; E, SDS eluate of the amylose-resin. ( C ) Co-IP analysis was used to examine BLM interaction with TRF2 in U2OS cells. Lysates from cells expressing WT Flag-BLM, mutant Flag-BLM (3A or P690L) and Myc-TRF2 were subject to co-IP analysis using anti-FLAG M2 agarose resin. Proteins were detected by Western blotting using anti-Flag or anti-Myc antibodies. ( D ) TRF2–RAP1 does not promote unwinding of telomere-D-loops by BLM mutants exhibiting reduced binding to TRF2. The effect of TRF2 and RAP1 on the ability of WT and mutant BLM (3A or P690L) to unwind telomeric D-loops were tested as in Figure . 32 P-labled ssDNA, D-loop, and dsDNA without the invading strand were loaded as size markers (lanes 1–3). ( E ) The percentages of unwound D-loops are shown as mean ± S.D. from three independent experiments. Statistical difference was evaluated via ANOVA test. Not significant: P > 0.05; **** P < 0.0001. ( F ) TRF2–RAP1 failed to promote BLM 3A mutant on unwinding of RAD51-generated D-loops. The effects of TRF2–RAP1 on BLM wild type or 3A, P690L mutants on D-loop unwinding were tested as in Figure . ( G ) The relative D-loop to the control without protein (lane 2) in the bottom panel is shown as mean ± S.D. from three independent experiments. ANOVA test was used to evaluate statistical difference. ns: non-significant ( P > 0.05); **** P < 0.0001.

Article Snippet: WT hTRF2 and TRF2 ΔB in pTricHisB vectors (Addgene #50 488 and #53 208) were transformed into Escherichia coli BL21(DE3) cells.

Techniques: Binding Assay, Functional Assay, Incubation, SDS Page, Staining, Co-Immunoprecipitation Assay, Expressing, Mutagenesis, Western Blot, Generated, Control

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM–DNA2-dependent 5′-end resection

doi: 10.1093/nar/gkae642

Figure Lengend Snippet: Both RAP1 and BLM binding to TRF2 are required to promote BLM helicase unwinding activity on the telomere 3′ overhang. ( A ) Schematic of the telomere 3′ overhang unwinding assay. A telomere dsDNA with a 39-nt 3′ overhang (GGGTTA) 6 GGG was generated by annealing a telomeric ssDNA containing (TTAGGG) 8 repeats (T3O1) with another shorter telomere ssDNA oligo containing (TAACCC) 2 repeats (T3O2). TRF2–RAP1 was first pre-incubated with the telomere dsDNA substrate and then BLM was added to detect unwinding of the 3′ overhang. ( B ) TRF2–RAP1 promotes BLM unwinding on the telomere 3′-overhang. The effect of TRF2 either alone or in combination with RAP1 on the ability of BLM to unwind the telomere 3′-overhang was tested. Heat denatured (HD) 32 P-labled telomere ssDNA (lane 1) and 3′-overhang dsDNA (lane 2) were loaded as markers. ( C ) Quantification of unwound telomere 3′-overhangs shown in Figure . The percentages of unwound telomere 3′-overhang are shown as mean ± S.D. from three independent experiments. ANOVA test was used to evaluate statistical difference. non-significant: P > 0.05; **** P < 0.0001. ( D ) TRF2-BLM binding is required to promote BLM-mediated unwinding of the telomere 3′-overhang. The effect of TRF2–RAP1 on the ability of WT BLM, BLM 3A or BLM P690L mutants to unwind telomere 3′-overhangs were tested as in Figure . ( E ) Quantification of BLM-mediated unwinding reactions shown in Figure . The percentages of unwound telomere 3′-overhang from three independent experiments are shown as mean ± S.D. Statistical evaluation was performed by ANOVA test. non-significant: P > 0.05; **** P < 0.0001.

Article Snippet: WT hTRF2 and TRF2 ΔB in pTricHisB vectors (Addgene #50 488 and #53 208) were transformed into Escherichia coli BL21(DE3) cells.

Techniques: Binding Assay, Activity Assay, Generated, Incubation

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM–DNA2-dependent 5′-end resection

doi: 10.1093/nar/gkae642

Figure Lengend Snippet: TRF2–RAP1 inhibits BLM–DNA2-mediated telomere 5′ end resection. ( A ) Schematic of the telomere 3′ overhang end resection assay. Telomere dsDNA with a 39-nt 3′overhang was pre-incubated with TRF2–RAP1 and then BLM/RPA/DNA2 added to examine telomere dsDNA end resection. ( B ) TRF2–RAP1 inhibits BLM–DNA2-mediated telomere DNA end resection. WT TRF2, TRF2 mutants (TRF2 ΔB or TRF2 ΔB, L288R ) and RAP1 individually or in combination were pre-incubated with telomere dsDNA containing a 3′-overhang. DNA end resection was examined after the BLM/RPA/DNA2 mixture were added to the reaction. ( C ) Quantification of telomere DNA end resection in Figure . The percentages of digested 5′-labeled telomere ssDNA were plotted. The error bars represent mean values ± S.D. of data from three independent experiments. Statistical evaluation was performed by ANOVA test. Non-significant: P > 0.05; **** P < 0.0001. ( D ) TRF2 binding to BLM contributes to the inhibition of telomere dsDNA end resection. WT BLM or BLM mutants (3A and P690L) were incubated with RPA and DNA2. The effects of TRF2–RAP1 on the end resection were examined as in Figure . ( E ) Quantification of percentage inhibition of telomeric end resection examined in Figure . The percentages of digested telomere 5′-ends are shown as mean ± S.D. from three independent experiments. Statistical analysis was performed by ANOVA. ** P = 0.0016; **** P < 0.0001.

Article Snippet: WT hTRF2 and TRF2 ΔB in pTricHisB vectors (Addgene #50 488 and #53 208) were transformed into Escherichia coli BL21(DE3) cells.

Techniques: Resection Assay, Incubation, Labeling, Binding Assay, Inhibition

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 represses RAD51-dependent homology-directed telomere repair by promoting BLM-mediated D-loop unwinding and inhibiting BLM–DNA2-dependent 5′-end resection

doi: 10.1093/nar/gkae642

Figure Lengend Snippet: TRF2–RAP1-BLM protects telomeres from engaging in HDR-mediated D-loop and ultrabright telomere formation in U2OS cells. ( A ) Interaction of BLM with TRF2–RAP1 is required to prevent the accumulation of telomere D-loops. Telomere D-loop formation was examined using nuclear extracts isolated from U2OS cells expressing empty vector, shBLM#1, shBLM#2, and in BLM depleted cells expressing WT BLM or BLM mutants (3A and P690L). ( B ) The amount of D-loops formed relative to the empty vector from three independent experiments are quantified and shown as mean ± S.D. ANOVA test was performed in statistical analysis. ‘ns’ indicates non-significant ( P = 0.7987); **** P < 0.0001. ( C ) Immunofluorescence-FISH analysis of BLM depleted U2OS cells expressing TRF2 ΔB; L288R revealed that BLM depletion promotes the generation of UTs. BLM was detected with anti-BLM antibody (green), telomeres visualized with the TelC-Cy3 (CCCTAA) 3 PNA telomere probe (red) and DAPI stained nuclei (blue). Scale bars: 5 μm. Yellow arrows: co-localization of BLM on telomeres; white arrows: UTs. ( D ) Quantification of percentage of U2OS cells possessing UTs with or without treatment with BLM shRNA. Data represents the mean of two independent experiments ± SD from a minimum of 250 nucleus analyzed per experiment. Statistical analysis was performed by one-way ANOVA. non-significant: P = 0.93; ** P = 0.0019. ( E ) Immunofluorescence-FISH analysis for HA-tagged WT BLM and indicated BLM mutants (green) co-localizing with telomeres (red) in U2OS cells expressing TRF2 ΔB; L288R . Scale bars: 5 μm. Yellow arrows: co-localization of BLM with telomeres; white arrows: UTs. ( F ) Quantification of percent UTs observed in sh BLM treated U2OS cells reconstituted with sh BLM resistant WT BLM and the indicated BLM mutants in TRF2 ΔB; L288R expressing U2OS cells. Data represents the mean of two independent experiments ± SD from a minimum 200 nucleus analyzed per experiment. Statistical analysis was performed by ANOVA. * P = 0.0265; ** P = 0.0066; **** P < 0.0001. ( G ) Model showing three distinct mechanisms of how TRF2–RAP1 prevents aberrant telomere HDR. First, RAP1 cooperates with TRF2 to inhibit telomere D-loop formation by blocking RAD51-mediated homology search on telomere dsDNA. Second, TRF2–RAP1 interaction with BLM promotes BLM-mediated telomeric D-loop unwinding. Third, TRF2–RAP1 represses BLM–DNA2-mediated telomere 5′-end DNA resection. In cells expressing TRF2 ΔB, L288R or BLM 3A , uncontrolled BLM–DNA2-mediated 5′ end resection of telomere dsDNA generates extensive ssDNA, enabling RAD51 nucleoprotein filament assembly and invasion into telomere dsDNA, forming telomere D-loops, resulting in massive telomere HDR and formation of UTs.

Article Snippet: WT hTRF2 and TRF2 ΔB in pTricHisB vectors (Addgene #50 488 and #53 208) were transformed into Escherichia coli BL21(DE3) cells.

Techniques: Isolation, Expressing, Plasmid Preparation, Immunofluorescence, Staining, shRNA, Blocking Assay