Monitoring Repair of UV-Induced 6-4-Photoproducts with a Purified DDB2 Protein Complex

Because cells are constantly subjected to DNA damaging insults, DNA repair pathways are critical for genome integrity [1]. DNA damage recognition protein complexes (DRCs) recognize DNA damage and initiate DNA repair. The DNA-Damage Binding protein 2 (DDB2) complex is a DRC that initiates nucleotide excision repair (NER) of DNA damage caused by ultraviolet light (UV) [2]-[4]. Using a purified DDB2 DRC, we created a probe ("DDB2 proteo-probe") that hybridizes to nuclei of cells irradiated with UV and not to cells exposed to other genotoxins. The DDB2 proteo-probe recognized UV-irradiated DNA in classical laboratory assays, including cyto- and histo-chemistry, flow cytometry, and slot-blotting. When immobilized, the proteo-probe also bound soluble UV-irradiated DNA in ELISA-like and DNA pull-down assays. In vitro, the DDB2 proteo-probe preferentially bound 6-4-photoproducts [(6-4)PPs] rather than cyclobutane pyrimidine dimers (CPDs). We followed UV-damage repair by cyto-chemistry in cells fixed at different time after UV irradiation, using either the DDB2 proteo-probe or antibodies against CPDs, or (6-4)PPs. The signals obtained with the DDB2 proteo-probe and with the antibody against (6-4)PPs decreased in a nearly identical manner. Since (6-4)PPs are repaired only by nucleotide excision repair (NER), our results strongly suggest the DDB2 proteo-probe hybridizes to DNA containing (6-4)PPs and allows monitoring of their removal during NER. We discuss the general use of purified DRCs as probes, in lieu of antibodies, to recognize and monitor DNA damage and repair

A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics

Under physiological conditions, protein oxidation and misfolding occur with very low probability and on long times scales. Single-molecule techniques provide the ability to distinguish between properly folded and damaged proteins that are otherwise masked in ensemble measurements. However, at physiological conditions these rare events occur with a time constant of several hours, inaccessible to current single-molecule approaches. Here we present a magnetic-tweezers-based technique that allows, for the first time, the study of folding of single proteins during week-long experiments. This technique combines HaloTag anchoring, sub-micrometer positioning of magnets, and an active correction of the focal drift. Using this technique and protein L as a molecular template, we generate a magnet law by correlating the distance between the magnet and the measuring paramagnetic bead with unfolding/folding steps. We demonstrate that, using this magnet law, we can accurately measure the dynamics of proteins over a wide range of forces, with minimal dispersion from bead to bead. We also show that the force calibration remains invariant over week-long experiments applied to the same single proteins. The approach demonstrated in this Article opens new, exciting ways to examine proteins on the “human” time scale and establishes magnetic tweezers as a valuable technique to study low-probability events that occur during protein folding under force

The decrease of DDB2 proteo-probe and 6-4 PP signals over time are nearly identical.

<p>(<b>A</b>) Typical signals after UV damage observed <i>in situ</i> with the DDB2 proteo-probe, an anti-CPD antibody, or an anti-(6-4)PP antibody. Nuclei are delineated based on DAPI staining and using CellProfiler. (<b>B</b>) The DDB2 proteo-probe signal decreases exponentially with time. Average signal per nucleus normalized to signal at 5 minutes. Red dashed curve: one phase exponential decay fit calculated with a non-linear least square method (R² = 0.86). (<b>C</b>) The anti-(6-4)PP signal decreases exponentially with time. Average signal per nucleus normalized to signal at 5 minutes. Blue dashed curve: one phase exponential decay fit calculated with a non-linear least square method (R² = 0.83). (<b>D</b>) The anti-CPD signal remains constant over a two hour period. Average signal per nucleus normalized to signal at 5 minutes. Black dashed line: linear fit on the α-CPD signal (R² = 0.18). (<b>B</b>), (<b>C</b>), and (<b>D</b>): cells were irradiated with UV-C (10 J/m²). The average of three replicas is shown. Each replica represents an average of at least 60 cells. Error bars: s.e.m. (<b>E</b>) A single one phase exponential decay model summarizes the kinetic of (6-4)PPs removal <i>in situ</i>. The single model is based on the decay fits obtained with DDB2 proteo-probe and anti-(6-4)PP data. The grey band represents the area enclosing the true decay curve with 99% confidence. The dotted line indicates the predicted half-life (<i>t</i>_1/2) of (6-4)PPs <i>in situ</i> after UV irradiation.</p

The DDB2 proteo-probe recognizes 6-4-photoproducts <i>in vitro</i>.

<p>(<b>A</b>) The DDB2 proteo-probe signal increases linearly with fluence (J/m²). Fibroblasts were irradiated with different doses of UV-C. Each point is an average of three replicas. Each replica represents an average of at least 60 cells. Dashed line: linear fit (R² = 0.94). Error bars: s.e.m. (<b>B</b>) The DDB2 proteo-probe signal is DNA-dependent. Fibroblasts were irradiated with UV-C (10 J/m²), and untreated or treated with DNase. Nuclei are visualized by DAPI staining. (<b>C</b>) The DDB2 proteo-probe signal can be competed with UV-treated plasmid DNA. Fibroblasts and plasmid DNA were irradiated with UV-C (10 J/m² and 300 J/m², respectively). The DDB2 proteo-probe was incubated with plasmid DNA prior to hybridization onto irradiated fibroblasts. Dashed line: no plasmid control proteo-probe signal level. Each point is an average of three replicas. Each replica represents an average of at least 400 cells. Error bars: s.e.m. (<b>D</b>) The DDB2 proteo-probe binds preferentially to 6-4-photoproducts [(6-4)PP] over cyclobutane pyrimidine dimers (CPD). The DDB2 proteo-probe was immobilized on agarose beads, and incubated with the DNA restriction fragments of a plasmid containing, or not, a unique lesion [(6-4)PP or CPD]. The average ratio of the amount of lesion-containing over lesion-free DNA fragments bound to the proteo-probe is shown (<i>n</i> = 3). Error bars: s.e.m.</p

A purified DDB2 protein complex can be used to detect UV-induced DNA damage.

<p>(<b>A</b>) Experimental strategy to prepare the DDB2 proteo-probe. (<b>B</b>) Signal obtained by hybridization of the DDB2 proteo-probe onto fibroblasts with or without damaging treatments. Hybridized DDB2 proteo-probe is revealed by anti-HA immunofluorescence. Nuclei are visualized by DAPI staining. Nuclei are delineated based on DAPI staining and using CellProfiler <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085896#pone.0085896-Carpenter1" target="_blank">[26]</a>.</p