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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 UvrBC. (A) Mean percentage (±SE, where n refers to experiments repeated on different days) of moving UvrBC in the presence of ATP (light gray bars). Values were 47.9% (±4.8, n = 7), 46.9% (±4.5, n = 5), 49.9% (±2.9, n = 5) at 50, 100 and 150 mM KCl, respectively. In the absence of ATP (dark gray bars), values were 64.1% (±4.2, n = 7), 58.5% (±1.2, n = 6) and 42.7% (±4.5, n = 5) at 50, 100 and 150 mM KCl, respectively. Cumulative number of molecules examined was 1256; 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 UvrBC versus salt concentration in the presence of ATP. The coloring is a percentage scale relative to the maximum bin size. N was 49, 48 and 72 in increasing order of salt concentration. See Supplementary Figure S6 for original data with errors and representative kymographs.
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gkt177-F4: The motile characteristics of UvrBC. (A) Mean percentage (±SE, where n refers to experiments repeated on different days) of moving UvrBC in the presence of ATP (light gray bars). Values were 47.9% (±4.8, n = 7), 46.9% (±4.5, n = 5), 49.9% (±2.9, n = 5) at 50, 100 and 150 mM KCl, respectively. In the absence of ATP (dark gray bars), values were 64.1% (±4.2, n = 7), 58.5% (±1.2, n = 6) and 42.7% (±4.5, n = 5) at 50, 100 and 150 mM KCl, respectively. Cumulative number of molecules examined was 1256; 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 UvrBC versus salt concentration in the presence of ATP. The coloring is a percentage scale relative to the maximum bin size. N was 49, 48 and 72 in increasing order of salt concentration. See Supplementary Figure S6 for original data with errors and representative kymographs.

Mentions: In the absence of nucleotide cofactors, ∼20% of all the motile UvrBC proteins observed were of sufficient duration and clarity to be analyzed for MSD (total observed = 1256, MSD analyzed = 287). The percentage of motile molecules decreased as the salt concentration was raised (Figure 4A). There was a small increase in diffusion constant as the concentration of KCl was increased from 100 to 150 mM (Table 2 and Figure 4B) that is not predicted for a sliding molecule (37). It may be possible that some of the electrostatic contacts with DNA are impaired either through direct shielding of protein–DNA contacts or through a salt-induced conformational change in the protein. The salt dependency of the diffusive exponent for UvrBC (+ATP or −ATP) was similar to that observed with UvrC alone; at low salt, sub-diffusion was observed [α = 0.57 (+ATP) at 50 mM and 0.69 (+ATP) at 100 mM KCl]; at 150 mM KCl, the diffusive exponent increased [α = 0.87 (+ATP)], indicating free diffusion along a smoother diffusive landscape.Figure 4.


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 UvrBC. (A) Mean percentage (±SE, where n refers to experiments repeated on different days) of moving UvrBC in the presence of ATP (light gray bars). Values were 47.9% (±4.8, n = 7), 46.9% (±4.5, n = 5), 49.9% (±2.9, n = 5) at 50, 100 and 150 mM KCl, respectively. In the absence of ATP (dark gray bars), values were 64.1% (±4.2, n = 7), 58.5% (±1.2, n = 6) and 42.7% (±4.5, n = 5) at 50, 100 and 150 mM KCl, respectively. Cumulative number of molecules examined was 1256; 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 UvrBC versus salt concentration in the presence of ATP. The coloring is a percentage scale relative to the maximum bin size. N was 49, 48 and 72 in increasing order of salt concentration. See Supplementary Figure S6 for original data with errors and representative kymographs.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt177-F4: The motile characteristics of UvrBC. (A) Mean percentage (±SE, where n refers to experiments repeated on different days) of moving UvrBC in the presence of ATP (light gray bars). Values were 47.9% (±4.8, n = 7), 46.9% (±4.5, n = 5), 49.9% (±2.9, n = 5) at 50, 100 and 150 mM KCl, respectively. In the absence of ATP (dark gray bars), values were 64.1% (±4.2, n = 7), 58.5% (±1.2, n = 6) and 42.7% (±4.5, n = 5) at 50, 100 and 150 mM KCl, respectively. Cumulative number of molecules examined was 1256; 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 UvrBC versus salt concentration in the presence of ATP. The coloring is a percentage scale relative to the maximum bin size. N was 49, 48 and 72 in increasing order of salt concentration. See Supplementary Figure S6 for original data with errors and representative kymographs.
Mentions: In the absence of nucleotide cofactors, ∼20% of all the motile UvrBC proteins observed were of sufficient duration and clarity to be analyzed for MSD (total observed = 1256, MSD analyzed = 287). The percentage of motile molecules decreased as the salt concentration was raised (Figure 4A). There was a small increase in diffusion constant as the concentration of KCl was increased from 100 to 150 mM (Table 2 and Figure 4B) that is not predicted for a sliding molecule (37). It may be possible that some of the electrostatic contacts with DNA are impaired either through direct shielding of protein–DNA contacts or through a salt-induced conformational change in the protein. The salt dependency of the diffusive exponent for UvrBC (+ATP or −ATP) was similar to that observed with UvrC alone; at low salt, sub-diffusion was observed [α = 0.57 (+ATP) at 50 mM and 0.69 (+ATP) at 100 mM KCl]; at 150 mM KCl, the diffusive exponent increased [α = 0.87 (+ATP)], indicating free diffusion along a smoother diffusive landscape.Figure 4.

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