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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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UvrC–Qdots interact with DNA tightropes. (A) AFM image of a UvrC–Qdot conjugate bound to DNA. (B) An illustration of DNA tightrope showing three Qdot-labeled protein complexes bound to a single tightrope of DNA suspended between two 5 -µm silica beads. (C) UvrC–Qdots (purple) bound to individual λ-DNA strands labeled with YOYO-1 (green).
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gkt177-F1: UvrC–Qdots interact with DNA tightropes. (A) AFM image of a UvrC–Qdot conjugate bound to DNA. (B) An illustration of DNA tightrope showing three Qdot-labeled protein complexes bound to a single tightrope of DNA suspended between two 5 -µm silica beads. (C) UvrC–Qdots (purple) bound to individual λ-DNA strands labeled with YOYO-1 (green).

Mentions: AFM imaging was used to assess the conjugation of biotinylated UvrC to streptavidin-coated Qdots and the ability of UvrC–Qdot conjugates to bind dsDNA. This approach revealed that UvrC–Qdots could bind to 458-bp dsDNA fragment (Figure 1A).Figure 1.


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)

UvrC–Qdots interact with DNA tightropes. (A) AFM image of a UvrC–Qdot conjugate bound to DNA. (B) An illustration of DNA tightrope showing three Qdot-labeled protein complexes bound to a single tightrope of DNA suspended between two 5 -µm silica beads. (C) UvrC–Qdots (purple) bound to individual λ-DNA strands labeled with YOYO-1 (green).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt177-F1: UvrC–Qdots interact with DNA tightropes. (A) AFM image of a UvrC–Qdot conjugate bound to DNA. (B) An illustration of DNA tightrope showing three Qdot-labeled protein complexes bound to a single tightrope of DNA suspended between two 5 -µm silica beads. (C) UvrC–Qdots (purple) bound to individual λ-DNA strands labeled with YOYO-1 (green).
Mentions: AFM imaging was used to assess the conjugation of biotinylated UvrC to streptavidin-coated Qdots and the ability of UvrC–Qdot conjugates to bind dsDNA. This approach revealed that UvrC–Qdots could bind to 458-bp dsDNA fragment (Figure 1A).Figure 1.

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