buffer b  (Cell Signaling Technology Inc)

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. ( A, B ) TRF2 binds 32 P-TERRA and telomere R-loops. His-tagged TRF2 protein (0–80 nM) was incubated with 5 nM radiolabeled TERRA (A) or telomere R-loops (B). The mobility shifts of the TRF2–RNA complex were analyzed by 10% native polyacrylamide gel electrophoresis. ( C ) Quantification of the binding data in panels (A, B). The error bars represent mean values ± SD of data from three independent experiments. ( D ) TRF2–RAP1 interaction enhances TRF2’s binding to telomere R-loops. Purified WT TRF2, mutants TRF2 ΔB , TRF2 L288R , TRF2 ΔB,L288R , and WT RAP1 alone or in the indicated combinations were tested for telomere R-loop binding. The mobility shift of the TRF2–RNA complexes was analyzed by 10% polyacrylamide gels. ( E, F ) Quantification of the R-loop binding data in panel (D). Error bars represent mean values ± SD of data from three independent experiments. ( G ) TRF2 (50, 100, 150, 200, and 250 nM) without or with RAP1 (100 nM) was incubated with telomere dsDNA and R-loops (10 nM each) to determine relative binding affinities. The ability of TRF2 or TRF2–RAP1 to bind to these nucleic acid substrates was analyzed by 10% polyacrylamide gels. ( H, I ) The R-loop and dsDNA binding data in panel (G) were quantified and plotted. Error bars represent mean values ± SD of data from three independent experiments.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Binding Assay, Incubation, Polyacrylamide Gel Electrophoresis, Purification, Mobility Shift

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: TRF2–RAP1 promotes BLM-mediated unwinding of telomere R-loops. ( A ) (Top) Schematic of the oligo-based telomere R-loop unwinding assay. Telomere R-loop substrates were generated by hybridizing 32 P-labeled TERRA and two telomere DNA fragments (TDR2 and TDR3). TRF2 and/or RAP1 were pre-incubated with the R-loops and then BLM was added to the reaction, and the complex was resolved by 10% native polyacrylamide gel electrophoresis to monitor for R-loop unwinding. Displacement of the invading radiolabeled TERRA from R-loops indicates that R-loop unwinding. (Bottom) The TRF2–RAP1 complex promotes BLM-mediated unwinding of telomere R-loops. The effects of TRF2 alone (40, 80 nM) or in combination with RAP1 (20, 40, 80 nM) on the ability of BLM (20 nM) to unwind telomere R-loops were examined. 32 P-labeled TERRA and R-loops were resolved by native-PAGE and shown in lanes 1 and 2. ( B ) Quantification of BLM-mediated R-loop unwinding reactions in panel (A). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .9485); **** P < .0001. ( C ) The TRF2 basic domain is required for efficient unwinding of telomere R-loops. The effect of WT TRF2, TRF2 ΔB , TRF2 ΔB,L288R , and RAP1 to enhance BLM-mediated telomere R-loop unwinding was examined. The sizes of 32 P-labeled TERRA and R-loops were resolved by native-PAGE, as shown in lanes 1 and 2. ( D ) Quantification of BLM-mediated R-loop unwinding reactions in panel (C). The percentages of unwound R-loops are shown as mean values ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. **** P < .0001. ( E ) The TRF2–BLM interaction enhances telomere R-loop unwinding. The effect of TRF2–RAP1 on the ability of WT and mutant BLM (3A or P690L) to unwind telomere R-loops was tested as in Fig. . 32 P-labeled TERRA and R-loops were loaded as size markers (lanes 1 and 2) and resolved by native-PAGE. ( F ) Quantification of the percentages of unwound R-loops in panel (E) as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. ns: non-significant ( P = .98); **** P < .0001.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Generated, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Clear Native PAGE, Mutagenesis

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: BLM preferentially releases TERRA over ssDNA from telomere D/R-loops. ( A ) Schematic of the assay used to measure how TRF2–RAP1 promotes BLM-mediated unwinding of RAD51/ssDNA and RAD51AP1/TERRA-generated telomeric D/R-loops. Telomere D/R-loops were generated by incubating RAD51 with IRDye-700-labeled telomere ssDNA (red), RAD51AP1 with IRDye-800-labeled TERRA (green), and telomere plasmids together as described in Fig. . Native plasmid-sized telomere D/R-loops were obtained after deproteinization and column purification. BLM with or without TRF2–RAP1 was then incubated with these D/R-loops, and ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. ( B ) BLM preferentially releases TERRA over ssDNA from telomere D/R-loop. BLM (20, 40, 80 nM) was tested for its ability to unwind telomere D/R-loops or TRF2–RAP1-bound D/R-loops. ssDNA, TERRA release, or D/R-loop unwinding was analyzed by 1% agarose gels. The unwinding of telomere D/R-loops by BLM was enhanced by TRF2–RAP1. ( C ) Quantification of the amount of D- and R-loops relative to the negative control (no proteins, lane 1). Data were plotted as mean ± SD from three independent experiments. Statistical evaluation was performed by ANOVA test. * P = .02282; ** P = .001278; *** P = .0007284; **** P < .0001. ( D ) The effects of TRF2–RAP1 on WT BLM, the helicase-dead BLM K695R or BLM mutants on D/R-loop unwinding were tested as in panel (B). In contrast to WT BLM, TRF2–RAP1 cannot enhance BLM ’s ability to unwind telomere D/R-loops. D/R-loop unwinding was analyzed by 1% agarose gels. ( E ) Quantification of the relative amounts of D-loops or R-loops to the control without proteins (lane 1) is shown as mean ± SD from three independent experiments. ANOVA test was used to evaluate statistical differences. ns: non-significant ( P = .15; .4147; .8026); ** P = .001193; *** P = .000158.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Generated, Labeling, Plasmid Preparation, Purification, Incubation, Negative Control, Control

Journal: Nucleic Acids Research

Article Title: TRF2–RAP1 inhibits homology-directed repair of telomeres by promoting BLM-mediated removal of telomere R-loops

doi: 10.1093/nar/gkag272

Figure Lengend Snippet: TRF2–RAP1–BLM is required to resolve telomere R-loops in U2OS cells. ( A ) U2OS cells expressing TRF2 ΔB, L288R were treated with shControl, shBLM, or shTRF2. Immunofluorescence-FISH analysis of cells containing UTs (PNA telomere probe, red) co-localized with R-loops (S9.6 antibody, green) and DAPI-stained nuclei (blue). White arrow: co-localization of R-loops on UTs. U2OS cells expressing shBLM-resistant WT BLM cDNA and indicated BLM mutants were treated with shBLM, shTRF2, and TRF2 ΔB, L288R . IF-FISH analysis was performed to detect UT/R-loop co-localization. White arrow: co-localization of R-loops on UTs. ( C ) Quantification of data from Fig. and , showing the number of UT/R-loop colocalizations per U2OS cell. Data from three independent experiments is shown as mean ± SEM from minimum 200 nuclei per experiment. Statistical evaluation was performed by one-way ANOVA test. ns: non-significant ( P > .9999); ** P = .0032; .0035; .0062; .0092; .0052; **** P < .0001. ( D ) Model showing that TRF2–RAP1 inhibits telomere HDR by promoting BLM-mediated telomere R-loop removal. RAD51AP1 and TERRA-dependent R-loops promote RAD51-mediated telomere D-loop formation. The TRF2–RAP1 complex and TRF2–BLM interaction are required to promote BLM helicase-mediated unwinding of telomere R-loops and then D-loops. The RAP1–TRF2–BLM complex represses HDR on telomeres by removing R-loops to inhibit telomere D-loop formation.

Article Snippet: The deproteinized reaction mixtures by SDS and proteinase K were passed through Micro Bio-Spin 6 Column (Bio-Rad), equilibrated with buffer B. TRF2–RAP1 (50 nM) was pre-incubated with the D/R-loop substrate (2.5 nM) on ice for 10 min. Then BLM (20–80 nM) was added and incubated at 37°C for 20 min.

Techniques: Expressing, Immunofluorescence, Staining