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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 effect of UvrB mutation on the motile characteristics of UvrBC at 50 mM KCl and in the presence of ATP. (A) Percentage of moving UvrBC for WT, UvrBY96AC, UvrBΔhairpinC and UvrBD338NC complexes. Mean (±SE, where n refers to experiments repeated on different days) values were 47.9% (±4.8, n = 7), 84.1% (±2.3, n = 4), 48.4% (±9.9, n = 6) and 60.1% (±5.9, n = 3), respectively. (B) 3D density plots of the diffusion constant versus the α factor for each UvrBC mutant complex. The coloring is a percentage scale relative to the maximum bin size. N (in same order as aforementioned) was 49, 61, 41 and 48. See Supplementary Figure S7 for original data with errors and representative kymographs.
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gkt177-F5: The effect of UvrB mutation on the motile characteristics of UvrBC at 50 mM KCl and in the presence of ATP. (A) Percentage of moving UvrBC for WT, UvrBY96AC, UvrBΔhairpinC and UvrBD338NC complexes. Mean (±SE, where n refers to experiments repeated on different days) values were 47.9% (±4.8, n = 7), 84.1% (±2.3, n = 4), 48.4% (±9.9, n = 6) and 60.1% (±5.9, n = 3), respectively. (B) 3D density plots of the diffusion constant versus the α factor for each UvrBC mutant complex. The coloring is a percentage scale relative to the maximum bin size. N (in same order as aforementioned) was 49, 61, 41 and 48. See Supplementary Figure S7 for original data with errors and representative kymographs.

Mentions: To address this question directly, we studied three UvrB mutants. The first two UvrB variants, Y96A (8) and the β-hairpin deletion (8,44), affect DNA binding. The final UvrB mutant, D338N, is deficient in ATP hydrolysis (45). All of the UvrB mutants were pre-complexed with UvrC before addition to the DNA tightropes, and their motion studied in low-salt conditions where the greatest differences in behavior would be expected. The first mutant, UvrBY96A, was designed to remove a crucial residue located at the base of the β-hairpin that inserts into the DNA to detect damage (8,25,27,44). At 50 mM KCl, this mutant possessed a statistically different lifetime (36.8 s: Table 3, P < 0.001) compared with wild-type UvrBC [47.6 s (+ATP): Table 2]. Furthermore as shown in Figure 5A, the number of motile complexes for UvrBY96A was considerably higher (83%) compared with WT + ATP (48%). Additionally, UvrBY96A mutant’s diffusion constant (12.1 × 10−3 (±3.0) µm2 s−1) was significantly (P < 0.001) greater than WT + ATP [2.8 × 10−3 (±0.4) µm2 s−1] at low salt (Table 3). The diffusive exponent (α = 0.75) was also higher, suggesting free diffusion, and is almost identical to WT at 150 mM KCl (α = 0.87). All of these results suggest that UvrBY96A has affected the motion of the UvrBC complex by reducing the incidence of sub-diffusion along the DNA. Given the UvrBY96A mutant showed such a substantial change from WT, we made the more severe mutation of deleting the entire β-hairpin implicated in DNA damage recognition (27,44). Removing this β-hairpin should eliminate a large interaction between UvrB and DNA, but not affect its ability to interact with UvrC. At low salt, this deletion mutant possessed a lifetime (14.0 s) and diffusive exponent (α = 0.87) significantly different from those of the WT protein at 50 mM KCl, and in fact, more closely resembled WT UvrBC data at 150 mM KCl (Table 3 and Figure 5). In addition, the β-hairpin deletion mutant’s diffusion constant was approximately twice that of wild-type at high-salt concentrations, suggesting that UvrBΔhairpinC complex was moving unencumbered along the DNA. Importantly, these results indicate that alterations in the DNA interacting region of UvrB affect UvrBC motility.Figure 5.


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 effect of UvrB mutation on the motile characteristics of UvrBC at 50 mM KCl and in the presence of ATP. (A) Percentage of moving UvrBC for WT, UvrBY96AC, UvrBΔhairpinC and UvrBD338NC complexes. Mean (±SE, where n refers to experiments repeated on different days) values were 47.9% (±4.8, n = 7), 84.1% (±2.3, n = 4), 48.4% (±9.9, n = 6) and 60.1% (±5.9, n = 3), respectively. (B) 3D density plots of the diffusion constant versus the α factor for each UvrBC mutant complex. The coloring is a percentage scale relative to the maximum bin size. N (in same order as aforementioned) was 49, 61, 41 and 48. See Supplementary Figure S7 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-F5: The effect of UvrB mutation on the motile characteristics of UvrBC at 50 mM KCl and in the presence of ATP. (A) Percentage of moving UvrBC for WT, UvrBY96AC, UvrBΔhairpinC and UvrBD338NC complexes. Mean (±SE, where n refers to experiments repeated on different days) values were 47.9% (±4.8, n = 7), 84.1% (±2.3, n = 4), 48.4% (±9.9, n = 6) and 60.1% (±5.9, n = 3), respectively. (B) 3D density plots of the diffusion constant versus the α factor for each UvrBC mutant complex. The coloring is a percentage scale relative to the maximum bin size. N (in same order as aforementioned) was 49, 61, 41 and 48. See Supplementary Figure S7 for original data with errors and representative kymographs.
Mentions: To address this question directly, we studied three UvrB mutants. The first two UvrB variants, Y96A (8) and the β-hairpin deletion (8,44), affect DNA binding. The final UvrB mutant, D338N, is deficient in ATP hydrolysis (45). All of the UvrB mutants were pre-complexed with UvrC before addition to the DNA tightropes, and their motion studied in low-salt conditions where the greatest differences in behavior would be expected. The first mutant, UvrBY96A, was designed to remove a crucial residue located at the base of the β-hairpin that inserts into the DNA to detect damage (8,25,27,44). At 50 mM KCl, this mutant possessed a statistically different lifetime (36.8 s: Table 3, P < 0.001) compared with wild-type UvrBC [47.6 s (+ATP): Table 2]. Furthermore as shown in Figure 5A, the number of motile complexes for UvrBY96A was considerably higher (83%) compared with WT + ATP (48%). Additionally, UvrBY96A mutant’s diffusion constant (12.1 × 10−3 (±3.0) µm2 s−1) was significantly (P < 0.001) greater than WT + ATP [2.8 × 10−3 (±0.4) µm2 s−1] at low salt (Table 3). The diffusive exponent (α = 0.75) was also higher, suggesting free diffusion, and is almost identical to WT at 150 mM KCl (α = 0.87). All of these results suggest that UvrBY96A has affected the motion of the UvrBC complex by reducing the incidence of sub-diffusion along the DNA. Given the UvrBY96A mutant showed such a substantial change from WT, we made the more severe mutation of deleting the entire β-hairpin implicated in DNA damage recognition (27,44). Removing this β-hairpin should eliminate a large interaction between UvrB and DNA, but not affect its ability to interact with UvrC. At low salt, this deletion mutant possessed a lifetime (14.0 s) and diffusive exponent (α = 0.87) significantly different from those of the WT protein at 50 mM KCl, and in fact, more closely resembled WT UvrBC data at 150 mM KCl (Table 3 and Figure 5). In addition, the β-hairpin deletion mutant’s diffusion constant was approximately twice that of wild-type at high-salt concentrations, suggesting that UvrBΔhairpinC complex was moving unencumbered along the DNA. Importantly, these results indicate that alterations in the DNA interacting region of UvrB affect UvrBC motility.Figure 5.

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