Investigating the Interaction of Single UvrA Dimers with DNA Using a Combination of Fluorescent Microscopy and Optical Tweezers

Abstract

Nucleotide excision repair (NER) is a DNA repair mechanism responsible for replacement of base pairs damaged e.g. by exposure to UV light. In E. coli, NER is initiated by the UvrA-dimer, which searches for and localizes damaged nucleotides to which then the UvrB-dimer is recruited. We study the damage search of UvrA by visualizing single, fluorescently labeled UvrA-dimers interacting with a DNA molecule manipulated by double optical tweezers. This approach enables us to observe UvrA binding in real time to DNA under different tensions.We find that, in the presence of ATP, the UvrA-dimer displays static, non-specific DNA binding of long duration (>10s), in accord with a recent publication [1]. In addition, our results reveal that non-specific UvrA binding is tension dependent: Binding events are prolonged on DNA under tension, indicating that the dissociation rate decreases while the association rate remains rather constant, thus supporting a model in which UvrA binding unwinds DNA [2].Finally, we observe that the duration of binding events decreases by several orders of magnitude if ADP instead of ATP is present in the buffer (<<1s). Binding is sensitive to tension, even more so than in the ATP experiment, resulting in a more than tenfold increase of the duration of binding events when DNA tension is increased from 5 to 65pN. In addition, our results demonstrate that UvrA has a higher binding affinity for ADP than for ATP. We are currently investigating whether ADP binding plays an important role in accelerating the DNA damage search in vivo.[1] Kad, N. M., Wang, H., Kennedey, G. G., Warshaw, D. M., van Houten, B. (2010). Mol. Cell 37, 702–713.[2] Oh, E., Grossman, L. (1986). Nucleic Acids Res. 14, 8557–8571.