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Results: 9

1.
FIGURE 9.

FIGURE 9. From: Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins .

Model for processing of DSB ends by MRX-Sae2 complex. Each horizontal line represents a DNA strand with polarity as indicated. Our findings support the idea that Mre11 or its nuclease deficient mutant protein (oval), by itself, binds (potentially in a cooperative manner) to the DSB ends and catalyzes unwinding of duplex DNA leading to the separation of complementary strands, a process that is stimulated by Rad50 (light gray triangle), Xrs2 (pear shape), and Sae2 (pac man shape). See text for details.

Indrajeet Ghodke, et al. J Biol Chem. 2013 April 19;288(16):11273-11286.
2.
FIGURE 8.

FIGURE 8. From: Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins .

S. cerevisiae Sae2 binds directly to its cognate Mre11. a, far Western analysis of interactions between ScMre11 and ScSae2. Increasing concentrations of homogeneous preparation of Mre11 or BSA (25–200 nm) were spotted (from left to right) on nitrocellulose membranes. After blocking with nonfat milk, the membranes were incubated with ScMre11 or ScSae2; probed with anti-Mre11, anti-Xrs2, or anti-Sae2 antibodies; and developed as described under “Experimental Procedures.” b–d, sensorgrams showing homotypic and heterotypic interactions between Mre11, Xrs2, and Sae2 proteins. b, sensorgrams showing homotypic association of ScMre11; c, sensorgrams showing heterotypic interactions between ScMre11 and Sae2; d, sensorgrams showing heterotypic interactions between ScMre11 and Xrs2. The arrows (from left to right) indicate the time points used for injection of protein and buffer, respectively.

Indrajeet Ghodke, et al. J Biol Chem. 2013 April 19;288(16):11273-11286.
3.
FIGURE 1.

FIGURE 1. From: Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins .

Proteins used in this study. a and b, schematic linear representation of Mre11 domain organization. Alignment of S. cerevisiae Mre11 phosphoesterase motifs I (a) and II (b) with E. coli SbcD and human Mre11. An arrow indicates the site of point mutations mre11D16A (a) and mre11D56N (b) within the conserved phosphoesterase motifs. Purified protein preparations were analyzed by electrophoresis through 7.5% polyacrylamide gel and Coomassie Blue staining. c, wild-type ScMre11 and mre11D16A and mre11D56N mutant proteins. d, ScSae2. Lanes indicated by Marker contain molecular size markers. BPB, bromophenol blue.

Indrajeet Ghodke, et al. J Biol Chem. 2013 April 19;288(16):11273-11286.
4.
FIGURE 3.

FIGURE 3. From: Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins .

ScMre11 promotes bridging between linear DNA molecules to form concatemers. The assay was performed as described under “Experimental Procedures. ” Lane 1, 1-kb DNA ladder; lanes 2 and 7, linear double-stranded DNA alone; lane 3, ScMre11 incubated with linear dsDNA in the absence of MgCl2 and ATP; lane 4, linear dsDNA in the presence of DNA ligase; lanes 5, 6, 8, and 9, incubated with 400 and 800 nm ScMre11, respectively, and then with T4 DNA ligase. Lanes 8 and 9, reaction mixtures were digested by ExoIII. The arrows on the right-hand side indicate products formed by bridging between linear DNA molecules. The asterisk denotes a product generated by ScMre11 nuclease activity in the presence of MgCl2.

Indrajeet Ghodke, et al. J Biol Chem. 2013 April 19;288(16):11273-11286.
5.
FIGURE 7.

FIGURE 7. From: Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins .

Rad50, Xrs2, and Sae2 proteins stimulate DNA unwinding activity of ScMre11. Radiolabeled 80-bp duplex DNA possessing 20-mer ssDNA overhang at the 3′ end (3 nm) (depicted in the gel image on the top) was incubated in a buffer containing 20 mm Tris-HCl (pH 7.5), 1 mm DTT, 100 μg/ml BSA, and 0.1 mm EDTA, and the reaction products were separated and visualized as described under “Experimental Procedures.” Lane 1, DNA substrate alone; lane 2, heat-denatured DNA substrate. The remaining lanes contained 300 nm ScMre11 alone (lane 3), 300 nm ScRad50 alone (lane 4), 300 nm ScRad50 + 300 nm ScMre11 (lane 5), 300 nm ScMre11 + 300 nm ScRad50 + 5 mm ATP (lane 6), 700 nm ScXrs2 alone (lane 7), 300 nm ScMre11 + 300 nm ScXrs2 (lane 8), 300 nm ScMre11 + 700 nm ScXrs2 (lane 9), 700 nm ScSae2 (lane 10), 300 nm ScMre11 + 300 nm ScSae2 (lane 11), and 300 nm ScMre11 and 700 nm ScSae2 (lane 12), respectively.

Indrajeet Ghodke, et al. J Biol Chem. 2013 April 19;288(16):11273-11286.
6.
FIGURE 5.

FIGURE 5. From: Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins .

ScMre11 catalyzes unwinding of flayed duplex DNA and Holliday junction containing 3′ ssDNA overhangs. The indicated 32P-labeled DNA substrate possessing 20-mer ssDNA overhangs (3 nm) either at the 3′ or 5′ ends (depicted in the gel image at the top) was incubated in a buffer containing 20 mm Tris-HCl (pH 7.5), 1 mm DTT, 100 μg/ml BSA, 0.1 mm EDTA, and the reaction products were separated and visualized as described under “Experimental Procedures. ” Lane 1 (marked C), DNA substrate alone; lane 2 (marked Δ), heat-denatured DNA substrate; lanes 3–9, reaction mixtures contained 100, 200, 300, 400, 500, 600, and 700 nm ScMre11, respectively. The filled triangle on top of the gel image denotes the increasing concentration of ScMre11. Reaction products were separated and visualized as described under “Experimental Procedures.” a, flayed duplex DNA with 20-mer 5′ ssDNA overhang; b, flayed duplex with 20-mer 3′ ssDNA overhang; c, Holliday junction with 20-mer 5′ ssDNA overhang; d, Holliday junction with 20-mer 3′ ssDNA overhang.

Indrajeet Ghodke, et al. J Biol Chem. 2013 April 19;288(16):11273-11286.
7.
FIGURE 6.

FIGURE 6. From: Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins .

ScMre11 nuclease deficient mutant proteins bind ssDNA and promote unwinding of duplex DNA. The indicated 32P-labeled 30-mer ssDNA fragment or partial DNA duplex possessing 20-mer 3′ ssDNA overhang (3 nm) of the indicated length (depicted at the top of gel images) were incubated in a buffer containing 20 mm Tris-HCl (pH 7.5), 1 mm DTT, 100 μg/ml BSA, and 0.1 mm EDTA, and the reaction products were separated and visualized as described under “Experimental Procedures.” a and b, ssDNA binding activity of mutant Mre11 proteins. Lane 1 (marked C), DNA substrate alone; lanes 2–9, reaction mixtures contained 25, 50, 100, 125, 150,175, 200, and 250 nm of mre11D16A (a) or mre11D56N (b), respectively. c and d, DNA unwinding activity of mutant ScMre11 proteins. Lane 1 (marked C), DNA substrate alone; lane 2 (marked Δ), heat-denatured DNA; lanes 3–9, reaction mixtures contained 100, 200, 300, 400, 500, 600, and 700 nm of mre11D16A (c) or mre11D56N (d), respectively. The filled triangle on top of the gel image denotes the increasing concentration of ScMre11 nuclease deficient mutant protein.

Indrajeet Ghodke, et al. J Biol Chem. 2013 April 19;288(16):11273-11286.
8.
FIGURE 4.

FIGURE 4. From: Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins .

ScMre11 catalyzes DNA unwinding and separation of complementary single strands. The indicated 32P-labeled DNA substrate (3 nm) containing duplex regions of different lengths and ends possessing 20-mer 3′ overhangs (depicted at the top of each gel image) were incubated in a buffer containing 20 mm Tris-HCl (pH 7.5), 1 mm DTT, 100 μg/ml BSA, 0.1 mm EDTA, and the reaction products were separated and visualized as described under “Experimental Procedures. ” Lane 1 (marked C), DNA substrate alone; lane 2 (marked Δ), heat-denatured DNA substrate; lanes 3–9, reaction mixtures contained 100, 200, 300, 400, 500, 600, and 700 nm ScMre11, respectively. The filled triangle on top of the gel image denotes the increasing concentration of ScMre11. a, 20-bp duplex DNA with 20-mer 3′ ssDNA overhang; b, 30-bp duplex DNA with 20-mer 3′ ssDNA overhang; c, 40 bp duplex DNA with 20-mer 3′ ssDNA overhang; d, 50-bp duplex DNA with 20-mer 3′ ssDNA overhang; e, 60-bp duplex DNA with 20-mer 3′ ssDNA overhang; f, 70-bp duplex DNA with 20-mer 3′ ssDNA overhang; g, 80-bp duplex DNA with 20-mer 3′ ssDNA overhang; h, graphical representation of percentage of displaced single strand with increasing concentration ScMre11. Each data point in the graph is the average of three independent experiments. The error bars indicate S.E. The data were subjected to nonlinear regression analysis in GraphPad PRISM (version 5.00), using the equation for one site-specific binding with Hill slope.

Indrajeet Ghodke, et al. J Biol Chem. 2013 April 19;288(16):11273-11286.
9.
FIGURE 2.

FIGURE 2. From: Processing of DNA Double-stranded Breaks and Intermediates of Recombination and Repair by Saccharomyces cerevisiae Mre11 and Its Stimulation by Rad50, Xrs2, and Sae2 Proteins .

ScMre11 binds to DSB ends, promotes end bridging, and catalyzes unwinding of duplex DNA. Radiolabeled DNA substrate (3 nm) (depicted at the top of each gel image) was incubated in a buffer containing 20 mm Tris-HCl (pH 7.5), 1 mm DTT, 100 μg/ml BSA,0.1 mm EDTA, and the reaction products were separated and visualized as described under “Experimental Procedures.” Lane 1 (marked C), DNA substrate alone; lane 2 (marked Δ), heat-denatured DNA substrate; lanes 3–9, reaction mixtures contained 100, 200, 300, 400, 500, 600, and 700 nm ScMre11, respectively. The filled triangle on top of the gel image denotes the increasing concentration of ScMre11. Panel (i), 20-bp blunt duplex. Panel (ii), 20-bp duplex with 20-mer 5′ ssDNA overhang. Panel (iii), 20-bp duplex with 20-mer 3′ ssDNA overhang. Panel (iv), frame a, a typical AFM image of linear plasmid DNA (5.5 kb) in the absence of ScMre11. Frames b–i, linear plasmid DNA (10 μg/ml) was incubated with 2 nm ScMre11 as described under “Experimental Procedures.” Representative AFM images showing ScMre11 binding to both ends of linear dsDNA. Panel (v), frames a–d, same conditions as in panel (iv). Shown are randomly selected AFM images showing the coexistence of ScMre11 promoted end bridging of linear dsDNA molecules and ScMre11 binding to both the ends. Frame e, an AFM image of a linear dsDNA molecule apparently completely bound by ScMre11. Here, we incubated DNA under conditions as in panel (iv), but in the presence of 14 nm ScMre11. Green arrows indicate ScMre11 binding, and naked DNA is indicated by red arrows. DNA fragment lengths were measured as described previously (62). Blue arrows indicate unbound ScMre11.

Indrajeet Ghodke, et al. J Biol Chem. 2013 April 19;288(16):11273-11286.

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