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

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.

Conservation and Divergence in Nucleotide Excision Repair Lesion Recognition

“…Importantly, we observed significantly enhanced DNA binding affinity for a pre-formed UvrBC complex with a K D of ϳ500 nM compared with UvrB alone (K D of ϳ4 M). These findings are consistent with observations by Kad and co-workers (20), who found the UvrBC complex to dissociate considerably less easily from undamaged DNA than UvrB alone. A possible mechanism for this enhancement may be via interactions of UvrC with domain 4 of UvrB (31) because the DNA binding affinities were comparable for UvrBC and the UvrB⌬4 variant that lacks this domain (Table 1).…”

“…S4). This ϳ10-fold increased affinity is consistent with DNA binding observed only for UvrBC and not for UvrB alone by Kad and co-workers (20) and is suggestive of a role of the UvrBC interaction in enhancing DNA binding by UvrB, potentially through repositioning of the autoinhibitory domain 4 of UvrB away from the protein core. Consistent with this interpretation, a UvrB variant that does not contain domain 4 (UvrB⌬4) showed similar DNA binding affinity (K D ϳ400 nM) as UvrBC (Table 1).…”

“…Our data suggest that this interaction may serve the further purpose of enhancing DNA binding by UvrB and/or stability of the UvrB-DNA complex. The affinity of UvrB for dsDNA and also for pre-formed DNA bubbles is low in the absence of UvrA, and it has previously been reported as below the detection limit using different experimental approaches (19,20). Our MST as well as BLI and AFM data clearly demonstrate DNA binding by UvrB with affinities in the low micromolar range (Table 1).…”

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

“…32, and single-molecule methods have been used to experimentally validate and visualize various protein search strategies (27,28,(33)(34)(35). We have previously developed a DNA tightrope assay that enables the direct visualization of dynamics of QD-conjugated proteins on DNA (27)(28)(29)(30). Briefly, in this assay, λ-DNA tightropes are strung-up between 5-μm poly-L-lysinecoated beads, which are deposited on a PEGylated coverslip ( Fig.…”

“…To better understand damage recognition by UV-DDB, we used a single-molecule DNA tightrope assay (27)(28)(29)(30) to observe the real time interactions of quantum dot (QD)-conjugated wildtype (WT) UV-DDB or UV-DDB containing the K244E mutation in DDB2, with damaged DNA substrates with high temporal and spatial resolution. Observations of individual molecules reveal the presence of short-lived intermediates and heterogeneity in molecular properties that may be lost due to bulk averaging of the properties of an unsynchronized ensemble of molecules.…”