Fig. 1: Fluorescently-tagged PARP1 engages nicked DNA substrates.
From: Nucleosome unwrapping and PARP1 allostery drive affinities for chromatin and DNA breaks
a A structural model of HaloTag-PARP1 built from aligning the alphafold3 structure with PARP1 structure PDB code 2N8A and HaloTag modeled from PDB code 6U32. Domains are colored as in the diagram beneath. b A schematic for performing these single-molecule experiments, in which streptavidin-coated beads are captured in optical traps (I), DNA (biotin = blue dots) tethered between the beads (II), the substrate is washed in a buffer channel (III), and then lastly moved into a channel with nuclear extracts to visualize overexpressed PARP1 binding events (IV). c To generate DNA nicks, Nt.BspQI digestion creates eight observable nicks distributed through the length of lambda DNA as shown. d Representative kymograph data of HaloTag-PARP1 binding nick sites (white lines are stationary events), as well as an example of dwell/gap times utilized to determine on and off rates. e A cumulative residence time distribution (CRTD) of the dwell times observed, with fit shown in red. f A cumulative gap time distribution (CGTD) determines the on rate for binding, with fit shown in green. These two rates can then be combined to determine KD values for interaction. DNA figures created in BioRender by Schaich, M. (2025). https://BioRender.com/ufjd01j.
