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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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UvrBC binding to DNA. (A) Imaging of dual color Qdot-labeled UvrBC shows the presence of both UvrB (green Qdot) and UvrC (purple Qdot) on DNA tightropes (unlabeled). The bright object on the lower right is the corner of a silica pedestal bead. (B) UvrBC diffusion is shown as movement on an unlabeled DNA tightrope over successive frames temporally separated by 1.03 s. (C) The percentage of motile complexes are shown to be unaffected by labeling strategy. Mean (±SE) values were plotted: 34.3% (±5.9, n = 58 molecules), 33.6% (±4.3, n = 130 molecules) and 32.2% (±5.8, n = 45 molecules) for dual labeled, labeled B or labeled C, respectively. AFM images of 458-bp dsDNA incubated with UvrB–Qdot conjugates (D) in the absence and (E) presence of in vivo biotinylated UvrC-avi. The image size is at 1 × 1 µm, and the Z scale is at 2 nm. (F) and (G) are zoomed 3D displays of the marked areas in (D) and (E).
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gkt177-F3: UvrBC binding to DNA. (A) Imaging of dual color Qdot-labeled UvrBC shows the presence of both UvrB (green Qdot) and UvrC (purple Qdot) on DNA tightropes (unlabeled). The bright object on the lower right is the corner of a silica pedestal bead. (B) UvrBC diffusion is shown as movement on an unlabeled DNA tightrope over successive frames temporally separated by 1.03 s. (C) The percentage of motile complexes are shown to be unaffected by labeling strategy. Mean (±SE) values were plotted: 34.3% (±5.9, n = 58 molecules), 33.6% (±4.3, n = 130 molecules) and 32.2% (±5.8, n = 45 molecules) for dual labeled, labeled B or labeled C, respectively. AFM images of 458-bp dsDNA incubated with UvrB–Qdot conjugates (D) in the absence and (E) presence of in vivo biotinylated UvrC-avi. The image size is at 1 × 1 µm, and the Z scale is at 2 nm. (F) and (G) are zoomed 3D displays of the marked areas in (D) and (E).

Mentions: Previous studies have only been able to suggest the potential presence of UvrBC complexes on a specific Y-substrate DNA (18,43). To investigate the possible formation of UvrBC complexes on normal dsDNA, we differentially labeled UvrB and UvrC for direct fluorescence imaging. We used UvrB–Qdot conjugates, which were formed using HA-tagged UvrB, primary HA antibody and secondary antibody-coated Qdots (39), in combination with avi-tagged UvrC. UvrB and UvrC were pre-incubated at a 2:1 ratio (UvrB:UvrC) before introduction to DNA tightropes in the flow chamber. We observed clear and abundant binding of UvrB–Qdot on DNA only in the presence of UvrC, under the conditions where only the UvrB was labeled or when both proteins were differentially color labeled with Qdots (Figure 3; Supplementary Movies S3 and S4). No difference in diffusion constant, diffusive exponent and percentage of motile complexes was seen between labeling strategies (Figure 3 and Supplementary Table S2). To confirm our imaging data, AFM was used to demonstrate that UvrB only bound to dsDNA in the presence of UvrC (Figure 3). Together, these data represent the first direct demonstration of UvrBC complexes on dsDNA in the absence of UvrA or a pre-formed bubble or DNA flap (18,43). Importantly, when UvrC–Qdots were pre-loaded onto DNA before addition of UvrB–Qdots (with a wash in between to remove free UvrC and UvrC–Qdots in solution), no loading of UvrB–Qdots on DNA was observed. This result is consistent with previous observations that UvrB does not independently bind to dsDNA (4,6) and implies that the UvrB-interacting domain on UvrC was inaccessible for UvrB once UvrC was loaded onto DNA.Figure 3.


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)

UvrBC binding to DNA. (A) Imaging of dual color Qdot-labeled UvrBC shows the presence of both UvrB (green Qdot) and UvrC (purple Qdot) on DNA tightropes (unlabeled). The bright object on the lower right is the corner of a silica pedestal bead. (B) UvrBC diffusion is shown as movement on an unlabeled DNA tightrope over successive frames temporally separated by 1.03 s. (C) The percentage of motile complexes are shown to be unaffected by labeling strategy. Mean (±SE) values were plotted: 34.3% (±5.9, n = 58 molecules), 33.6% (±4.3, n = 130 molecules) and 32.2% (±5.8, n = 45 molecules) for dual labeled, labeled B or labeled C, respectively. AFM images of 458-bp dsDNA incubated with UvrB–Qdot conjugates (D) in the absence and (E) presence of in vivo biotinylated UvrC-avi. The image size is at 1 × 1 µm, and the Z scale is at 2 nm. (F) and (G) are zoomed 3D displays of the marked areas in (D) and (E).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt177-F3: UvrBC binding to DNA. (A) Imaging of dual color Qdot-labeled UvrBC shows the presence of both UvrB (green Qdot) and UvrC (purple Qdot) on DNA tightropes (unlabeled). The bright object on the lower right is the corner of a silica pedestal bead. (B) UvrBC diffusion is shown as movement on an unlabeled DNA tightrope over successive frames temporally separated by 1.03 s. (C) The percentage of motile complexes are shown to be unaffected by labeling strategy. Mean (±SE) values were plotted: 34.3% (±5.9, n = 58 molecules), 33.6% (±4.3, n = 130 molecules) and 32.2% (±5.8, n = 45 molecules) for dual labeled, labeled B or labeled C, respectively. AFM images of 458-bp dsDNA incubated with UvrB–Qdot conjugates (D) in the absence and (E) presence of in vivo biotinylated UvrC-avi. The image size is at 1 × 1 µm, and the Z scale is at 2 nm. (F) and (G) are zoomed 3D displays of the marked areas in (D) and (E).
Mentions: Previous studies have only been able to suggest the potential presence of UvrBC complexes on a specific Y-substrate DNA (18,43). To investigate the possible formation of UvrBC complexes on normal dsDNA, we differentially labeled UvrB and UvrC for direct fluorescence imaging. We used UvrB–Qdot conjugates, which were formed using HA-tagged UvrB, primary HA antibody and secondary antibody-coated Qdots (39), in combination with avi-tagged UvrC. UvrB and UvrC were pre-incubated at a 2:1 ratio (UvrB:UvrC) before introduction to DNA tightropes in the flow chamber. We observed clear and abundant binding of UvrB–Qdot on DNA only in the presence of UvrC, under the conditions where only the UvrB was labeled or when both proteins were differentially color labeled with Qdots (Figure 3; Supplementary Movies S3 and S4). No difference in diffusion constant, diffusive exponent and percentage of motile complexes was seen between labeling strategies (Figure 3 and Supplementary Table S2). To confirm our imaging data, AFM was used to demonstrate that UvrB only bound to dsDNA in the presence of UvrC (Figure 3). Together, these data represent the first direct demonstration of UvrBC complexes on dsDNA in the absence of UvrA or a pre-formed bubble or DNA flap (18,43). Importantly, when UvrC–Qdots were pre-loaded onto DNA before addition of UvrB–Qdots (with a wash in between to remove free UvrC and UvrC–Qdots in solution), no loading of UvrB–Qdots on DNA was observed. This result is consistent with previous observations that UvrB does not independently bind to dsDNA (4,6) and implies that the UvrB-interacting domain on UvrC was inaccessible for UvrB once UvrC was loaded onto DNA.Figure 3.

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