Figure 2.

PARP1 specifically binds to abasic site arrays embedded in long DNA substrates. (A) Schematic of the DNA tightrope assay. Long DNA substrates with AP sites at defined positions are suspended between 5 μm poly-L-lysine coated silica beads. These lesion sites can be labeled by a streptavidin (SA) Qdot (red dots) through an adjacent biotin or recognized by labeled DNA repair proteins (green dots). Co-localization of protein and lesion is shown as a yellow glow around closely positioned red and green dots. (B) Labeling of His-MBP-TEV-PARP1 by antibody sandwich (see Supplementary Information). Domains of PARP1 are colored as follows: Zinc Fingers 1, 2 and 3 in green, light gray and blue, respectively; BRCT in black; WGR in red; HD in wheat; and ART in dark yellow. (C) Still video frame and kymograph of PARP1 protein array on long DNA substrates containing defined AP sites. Positions of the silica beads are outlined by white circles. Asterisks mark the dissociating particle. Horizontal and vertical scale bars represent 5 s and 2 kp, respectively. (D) Still video frame and kymographs of Qdot605-mHisAB-PARP1 (red) binding to SA-Qdot655 labeled AP-BiodT sites (green). Color-coded red, green, and yellow arrows highlight these binding events where appropriate. Horizontal and vertical scale bars represent 50 s and 2 kb, respectively. (E) Co-localization (yellow) Venn diagrams of Qdot-labeled AP-BiodT sites (red) with Qdot-labeled PARP1 (green) on AP-BiodT DNA tightropes in the dual-color assay (N = 208). (F) Distributions of pair-wise distances between labeled AP-BiodT sites in DNA (white, N = 231, top), labeled PARP1 on AP DNA (orange, N = 26, middle), and labeled PARP1 on AP-BiodT DNA (blue, N = 28, bottom). Solid black curve represents Gaussian fitting to the distance distribution for labeled AP-BiodT sites.