A New Role for UvrB in NER? Single Molecule Imaging of the NER Complex UvrBC-DNA

Abstract

Direct observation of a multi-protein DNA repair pathway is now possible using single molecule methods. Previously we have shown that UvrA and UvrB belonging to the bacterial nucleotide excision repair pathway collaborate to locate damage. Here we report on the next phase of repair, dual incision by UvrC. However as UvrC is in such short supply in the cell, locating a pre-incision complex at a site of damage becomes the key rate limiting step of NER. By labelling UvrB and UvrC with different coloured quantum dots their interactions with DNA tightropes can be studied at the single molecule level using oblique angle fluorescence microscopy. We found UvrC interacts with DNA and performs a 1D diffusional search (7.4x10−2 μm2s−1). Surprisingly, we also found that UvrC could load UvrB onto DNA generating a previously unseen UvrBC-DNA complex. This UvrBC complex is highly mobile relative to UvrC alone (15% vs. 57% mobility) and engages in unbiased 1D diffusion with a coefficient of 6.6x10−3 μm2s−1. Ionic strength profoundly affects the motion of the proteins; at elevated salt more UvrC molecules slide, however UvrBC mobility remains constant. Furthermore, the inclusion of ATP decreases UvrBC's dwell time on DNA from 175s to 83s. Based on these and further results from the study of various mutants we deduce that UvrB switches UvrC's search mechanism from 3D distributive to 1D sliding. We propose a new chaperoning role for UvrB in NER, where it protects the genome from unwanted UvrC nuclease activity but facilitates UvrC's location of pre-incision UvrB complexes.