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1.
FIGURE 1.

FIGURE 1. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

Sld3 binds to origin ssDNA (ARS1). A, schematic of the origin ARS1. B, 5 pmol of GST-Sld3 or GST was incubated with varying amounts (0.09, 0.28, 0.90, or 2.8 pmol) of radiolabeled ssDNA or dsDNA from ARS1 as described under “Experimental Procedures.” The DNA sequences are in Table 1. C, results from experiments shown in B were quantified, averaged, and plotted. The data are mean ± S.E.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.
2.
FIGURE 4.

FIGURE 4. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

Mutation of the seven 5′-thymines of ssARS1–2 abolishes binding by Sld3. A–C, 5 pmol of GST-Sld3 or GST was incubated with radiolabeled ssDNA as described under “Experimental Procedures.” The DNA sequences are written above the figure. Wild-type ssARS1-2 and the mutations shown were examined for binding to GST-Sld3. WT represents the wild-type ssARS1-2 sequence. Mutated nucleotides are shown in boldface type. The data were quantified, averaged, and plotted as shown. The data are mean ± S.E.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.
3.
FIGURE 2.

FIGURE 2. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

CDK-phosphorylated Sld3 binds to origin ssDNA. A, Sld3 containing an N-terminal PKA tag was radiolabeled with either PKA or CDK. PKA-Sld3 and CDK-Sld3 were then matched for radioactive counts and total Sld3 concentration. Varying amounts of the proteins (0.1, 0.3, 1.0, or 3.0 pmol) were then incubated with 5 pmol of biotinylated ssARS1-2, ssARS1-4, dsARS1-12, or biotin. The bound radioactive Sld3 was analyzed by SDS-PAGE followed by phosphorimaging. B, results from experiments shown in A were quantified, averaged, and plotted. The data are mean ± S.E.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.
4.
FIGURE 5.

FIGURE 5. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

Sld3 binds to origin ssDNA (ssARS305–1). A, 5 pmol of GST-Sld3 or GST was incubated with radiolabeled ssDNA or dsDNA from ARS305 as described under “Experimental Procedures.” The DNA sequences are in Table 1. B, results from experiments shown in A were quantified, averaged, and plotted. C, 2 pmol of GST-Sld3 was incubated with 2 pmol of radiolabeled ssARS1-2 in the presence of varying amounts of unlabeled ssARS305-1 (0, 1.4, 2, 4, or 8 pmol). D, results from experiments shown in C were quantified, averaged, and plotted. The data are mean ± S.E.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.
5.
FIGURE 8.

FIGURE 8. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

Origin single-stranded DNA (ssARS1–2) releases Sld3 from Mcm2–7, allowing GINS to bind Mcm2–7. A, D, and G, 100 pmol of 32P-labeled Sld3 was incubated in the absence or presence of 100 pmol of GINS, 100 pmol of Mcm2–7, or 200 pmol of ssARS1-2. The mixture was then subjected to size-exclusion chromatography as described under “Experimental Procedures.” The radioactive counts in each fraction were used to calculate the pmol of Sld3 in each fraction. Sld3 (pmol) was then plotted versus the elution of molecular weight standards. B, E, and H, 100 pmol of 32P-labeled GINS was incubated in the absence or presence of 100 pmol of Sld3, 100 pmol of Mcm2–7, or 200 pmol of ssARS1-2. The mixture was then subjected to size-exclusion chromatography as described under “Experimental Procedures.” C, F, and I, 100 pmol of 32P-labeled Mcm2–7 was incubated in the absence or presence or 100 pmol of Sld3, 100 pmol of GINS, or 200 of pmol ssARS1-2. The mixture was then subjected to size-exclusion chromatography as described under “Experimental Procedures.” The data are mean ± S.E.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.
6.
FIGURE 9.

FIGURE 9. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

Origin single-stranded DNA (ssARS1–2) promotes the formation of the CMG complex. A, 100 pmol of 32P-labeled Sld3 was incubated with 100 pmol of Cdc45 and 100 pmol of Mcm2–7 in the absence or presence of 100 pmol of GINS, and/or 200 pmol of ssARS1-2. The mixture was then subjected to size-exclusion chromatography as described under “Experimental Procedures.” The radioactive counts in each fraction were used to calculate the pmol of Sld3 in each fraction. Sld3 (pmol) was then plotted versus the elution of molecular weight standards. B, 100 pmol of 32P-labeled Cdc45 was incubated with 100 pmol of Sld3 and 100 pmol of Mcm2–7 in the absence or presence of 100 pmol of GINS, and/or 200 pmol of ssARS1-2. The mixture was then subjected to size-exclusion chromatography as described under “Experimental Procedures.” C, 100 pmol of 32P-labeled Mcm2–7 was incubated with 100 pmol of Sld3 and 100 pmol of Cdc45 in the absence or presence of 100 pmol of GINS, and/or 200 pmol of ssARS1-2. The mixture was then subjected to size-exclusion chromatography as described under “Experimental Procedures.” D, 100 pmol of 32P-labeled GINS was incubated with 100 pmol of Cdc45 and 100 pmol of Mcm2–7 in the absence or presence of 100 pmol of Sld3, and/or 200 pmol of ssARS1-2. The mixture was then subjected to size-exclusion chromatography as described under “Experimental Procedures.” The data are mean ± S.E.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.
7.
FIGURE 3.

FIGURE 3. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

Native Sld3 binds to origin ssDNA with an apparent Kd of 53 nm. 10 nm fluorescently labeled (5-FAM) ssARS1-2, ssARS1-4, or dsARS1-12 was mixed with varying concentrations of native Sld3 and incubated for 20 min at room temperature. Polarized fluorescence intensities were measured at excitation and emission wavelengths of 495 and 538 nm, respectively, at room temperature. The data were plotted as the change in anisotropy versus protein concentration in nm, and the curve was fit with the equation, y = m0/(m0 + m1). The table underneath the graph shows the apparent Kd for Sld3 interaction with ssARS1-2, ssARS1-4, or dsARS1-12. The data are mean ± S.E.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.
8.
FIGURE 10.

FIGURE 10. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

Model for the initiation of DNA replication. Two sister Mcm2–7 complexes are loaded onto an early replication origin in late M phase and G1. Sld3-Cdc45 binds to an early replication origin in G1 phase. The interaction between Sld3 and a replication origin may be mediated by a direct interaction between Sld3 and Mcm2–7. During S phase, the CDK and DDK kinases are active at the replication origin. GINS and Sld2, as part of the preloading complex (composed of Sld2, GINS, Dpb11, and polymerase ϵ) are recruited to an origin by a CDK-mediated event. The DNA melts in the region adjacent to the origin recognition complex, allowing Sld3 and Sld2 to bind to the single strands of the origin. Binding of Sld3 to single-stranded DNA at an origin releases Sld3 from Mcm2–7, allowing Mcm2–7 to bind to GINS. The Cdc45-Mcm2–7-GINS (CMG) complexes separate and move bidirectionally to initiate bidirectional DNA unwinding. Sld3 and Sld2 do not migrate with the replication fork.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.
9.
FIGURE 7.

FIGURE 7. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

Origin single-stranded DNA (ssARS1–2) promotes the association between GINS and Mcm2–7 in the presence of Sld3. A, ssARS1-2 does not disrupt the interaction between GINS and Mcm2–7. 2 pmol of GST-Mcm2–7 was incubated with 2 pmol of radiolabeled PKA-GINS and varying amounts of ssARS1-2 (0, 2, 6, 20, or 60 pmol) in the absence or presence of 5 mm ATP. Bound radioactive GINS was analyzed by SDS-PAGE. B, results from experiments shown in A were quantified, averaged, and plotted. C, 2 pmol of GST-Mcm2–7 was incubated with 2 pmol of radiolabeled PKA-GINS, the presence or absence of 10 pmol of Sld3, and varying amounts of ssARS1-2 (0, 5, 20, or 50 pmol) in the absence or presence of 5 mm ATP. Bound radioactive GINS was analyzed by SDS-PAGE. D, results from experiments shown in C were quantified, averaged, and plotted. E, 2 pmol of GST-Mcm2–7 was incubated with 2 pmol of radiolabeled PKA-GINS, 10 pmol of Cdc45, 10 pmol of Sld3, and varying amounts of ssARS1-2 (0, 5, 20, or 50 pmol) in the absence or presence of 5 mm ATP. Bound radioactive GINS was analyzed by SDS-PAGE. F, results from experiments shown in E were quantified, averaged, and plotted. The data are mean ± S.E.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.
10.
FIGURE 6.

FIGURE 6. From: Origin Single-stranded DNA Releases Sld3 Protein from the Mcm2-7 Complex, Allowing the GINS Tetramer to Bind the Mcm2-7 Complex.

Origin ssDNA (ssARS1–2) dislodges Sld3 from Mcm2–7. ssARS1-2 does not disrupt the interaction between Sld3 and Dpb11 or between Sld3 and Cdc45. A, 2 pmol of CDK-treated or mock-treated (i.e. no kinase, buffer added only) GST-Sld3 or GST was incubated with varying amounts of PKA-radiolabeled Dpb11 (0.06, 0.2, 0.6, or 2 pmol). Bound radioactive Dpb11 was analyzed by SDS-PAGE. B, results from experiments shown in A were quantified, averaged, and plotted. C, 2 pmol of CDK-treated GST-Sld3 was incubated with 2 pmol of radiolabeled Dpb11 and varying amounts of ssARS1-2 (0, 6, or 20 pmol). Bound radioactive Dpb11 was analyzed by SDS-PAGE. D, results from experiments shown in C were quantified, averaged, and plotted. E, Sld3 containing an N-terminal PKA tag was radiolabeled with either PKA or CDK. PKA-Sld3 and CDK-Sld3 were then matched for radioactive counts and total Sld3 concentration. 2 pmol of PKA-Sld3 or CDK-Sld3 were then incubated with 2 pmol of GST-Cdc45 and varying concentrations of ssARS1-2 (0, 2, 6, or 20 pmol), and the bound radioactive Sld3 was analyzed by SDS-PAGE. F, results from experiments shown in E were quantified, averaged, and plotted. G, 2 pmol of radiolabeled Mcm2–7 in the presence or absence of 5 mm ATP or ATP-γS was incubated with 2 pmol of GST-Sld3 and varying concentrations of ssARS1-2 (0, 1, 2, 4, or 8 pmol). H, results from experiments shown in G were quantified, averaged, and plotted. The data are mean ± S.E.

Irina Bruck, et al. J Biol Chem. 2011 May 27;286(21):18602-18613.

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