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Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes.

Hughes CD, Wang H, Ghodke H, Simons M, Towheed A, Peng Y, Van Houten B, Kad NM - Nucleic Acids Res. (2013)

Bottom Line: This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion.These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC.Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.

ABSTRACT
Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA-D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB-DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC-DNA complex, we investigated three UvrB mutants: Y96A, a β-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.

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The motile characteristics of UvrC. (A) Percentage of moving UvrC versus salt concentration. Values for mean percentage (±SE, where n refers to experiments repeated on different days) moving were 14.9% (±3.7, n = 13), 29.8% (±5.8, n = 10) and 43.3% (±3.5, n = 10) for 50, 100 and 150 mM KCl, respectively. Cumulative number of molecules examined was 1949; the number of molecules examined per condition is shown in the figure. (B) 3D density plots of the diffusion constant versus the α factor (slope of log–log MSD versus time plot) of UvrC under different salt concentrations. The coloring is a percentage scale relative to the maximum bin size. N was 44, 44 and 45 in increasing order of the respective salt condition. See Supplementary Figure S5 for original data with errors and representative kymographs.
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gkt177-F2: The motile characteristics of UvrC. (A) Percentage of moving UvrC versus salt concentration. Values for mean percentage (±SE, where n refers to experiments repeated on different days) moving were 14.9% (±3.7, n = 13), 29.8% (±5.8, n = 10) and 43.3% (±3.5, n = 10) for 50, 100 and 150 mM KCl, respectively. Cumulative number of molecules examined was 1949; the number of molecules examined per condition is shown in the figure. (B) 3D density plots of the diffusion constant versus the α factor (slope of log–log MSD versus time plot) of UvrC under different salt concentrations. The coloring is a percentage scale relative to the maximum bin size. N was 44, 44 and 45 in increasing order of the respective salt condition. See Supplementary Figure S5 for original data with errors and representative kymographs.

Mentions: At 50 mM KCl, UvrC was observed to have a low incidence (∼15%) of motility. However, at elevated KCl concentrations, a significant increase in the number of motile UvrC molecules was observed, from 15 to 30% at 100 mM KCl to 43% at 150 mM KCl (P < 0.01) (Figure 2A and Supplementary Movie S2). As expected, 1 mM ATP induced no significant difference in protein motility at any KCl concentrations (data not shown). Of the moving proteins (n = 1949), ∼90% moved continuously without visibly pausing during the recording. Approximately 95% of these molecules were fit to Equation (2) for diffusional or sub-diffusional motion; the small remainder could not be confidently characterized. The diffusion constant of UvrC decreased from 36.0 (±6.7) ×10−3 µm2 s−1 at 50 mM KCl to 7.0 (±1.6) ×10−3 µm2 s−1 at 100 mM KCl and 9.3 (±1.4) ×10−3 µm2 s−1 at 150 mM KCl (Table 1 and Figure 2B). Further insight into how UvrC diffuses on the DNA was provided by characterization of its diffusive exponents. For all salt concentrations studied, the average exponent value was <1; such values suggest a stop–start motion, which indicates searching followed by interrogation of the DNA (30,42). However, as the salt concentration was raised, a clear trend toward a value of 1 was seen (Table 1 and Figure 2B), suggesting reduced sub-diffusive behavior (see ‘Materials and Methods’ section for explanation of α values).Figure 2.


Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes.

Hughes CD, Wang H, Ghodke H, Simons M, Towheed A, Peng Y, Van Houten B, Kad NM - Nucleic Acids Res. (2013)

The motile characteristics of UvrC. (A) Percentage of moving UvrC versus salt concentration. Values for mean percentage (±SE, where n refers to experiments repeated on different days) moving were 14.9% (±3.7, n = 13), 29.8% (±5.8, n = 10) and 43.3% (±3.5, n = 10) for 50, 100 and 150 mM KCl, respectively. Cumulative number of molecules examined was 1949; the number of molecules examined per condition is shown in the figure. (B) 3D density plots of the diffusion constant versus the α factor (slope of log–log MSD versus time plot) of UvrC under different salt concentrations. The coloring is a percentage scale relative to the maximum bin size. N was 44, 44 and 45 in increasing order of the respective salt condition. See Supplementary Figure S5 for original data with errors and representative kymographs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643590&req=5

gkt177-F2: The motile characteristics of UvrC. (A) Percentage of moving UvrC versus salt concentration. Values for mean percentage (±SE, where n refers to experiments repeated on different days) moving were 14.9% (±3.7, n = 13), 29.8% (±5.8, n = 10) and 43.3% (±3.5, n = 10) for 50, 100 and 150 mM KCl, respectively. Cumulative number of molecules examined was 1949; the number of molecules examined per condition is shown in the figure. (B) 3D density plots of the diffusion constant versus the α factor (slope of log–log MSD versus time plot) of UvrC under different salt concentrations. The coloring is a percentage scale relative to the maximum bin size. N was 44, 44 and 45 in increasing order of the respective salt condition. See Supplementary Figure S5 for original data with errors and representative kymographs.
Mentions: At 50 mM KCl, UvrC was observed to have a low incidence (∼15%) of motility. However, at elevated KCl concentrations, a significant increase in the number of motile UvrC molecules was observed, from 15 to 30% at 100 mM KCl to 43% at 150 mM KCl (P < 0.01) (Figure 2A and Supplementary Movie S2). As expected, 1 mM ATP induced no significant difference in protein motility at any KCl concentrations (data not shown). Of the moving proteins (n = 1949), ∼90% moved continuously without visibly pausing during the recording. Approximately 95% of these molecules were fit to Equation (2) for diffusional or sub-diffusional motion; the small remainder could not be confidently characterized. The diffusion constant of UvrC decreased from 36.0 (±6.7) ×10−3 µm2 s−1 at 50 mM KCl to 7.0 (±1.6) ×10−3 µm2 s−1 at 100 mM KCl and 9.3 (±1.4) ×10−3 µm2 s−1 at 150 mM KCl (Table 1 and Figure 2B). Further insight into how UvrC diffuses on the DNA was provided by characterization of its diffusive exponents. For all salt concentrations studied, the average exponent value was <1; such values suggest a stop–start motion, which indicates searching followed by interrogation of the DNA (30,42). However, as the salt concentration was raised, a clear trend toward a value of 1 was seen (Table 1 and Figure 2B), suggesting reduced sub-diffusive behavior (see ‘Materials and Methods’ section for explanation of α values).Figure 2.

Bottom Line: This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion.These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC.Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK.

ABSTRACT
Nucleotide excision DNA repair is mechanistically conserved across all kingdoms of life. In prokaryotes, this multi-enzyme process requires six proteins: UvrA-D, DNA polymerase I and DNA ligase. To examine how UvrC locates the UvrB-DNA pre-incision complex at a site of damage, we have labeled UvrB and UvrC with different colored quantum dots and quantitatively observed their interactions with DNA tightropes under a variety of solution conditions using oblique angle fluorescence imaging. Alone, UvrC predominantly interacts statically with DNA at low salt. Surprisingly, however, UvrC and UvrB together in solution bind to form the previously unseen UvrBC complex on duplex DNA. This UvrBC complex is highly motile and engages in unbiased one-dimensional diffusion. To test whether UvrB makes direct contact with the DNA in the UvrBC-DNA complex, we investigated three UvrB mutants: Y96A, a β-hairpin deletion and D338N. These mutants affected the motile properties of the UvrBC complex, indicating that UvrB is in intimate contact with the DNA when bound to UvrC. Given the in vivo excess of UvrB and the abundance of UvrBC in our experiments, this newly identified complex is likely to be the predominant form of UvrC in the cell.

Show MeSH
Related in: MedlinePlus