Study and visualize DNA replication processes at the nanoscale

Step 3: Easy-to-use data analysis provides insights into the steps, kinetics, and potential variations of the molecular process.

Step 2: Single-molecule fluorescence imaging allows the identification and real-time tracking of multiple types of proteins on the DNA substrate.

Step 1: Proteins are expressed in cells and nuclear extracts are loaded into the C-Trap where they interact with well-defined DNA substrates.

DNA lesion search dynamics. One-dimensional diffusion constants (D) on DNA plotted over the lifetimes of complexes on the DNA for ATL. The kymographs and data show a different diffusion mode compared to AGT on the left image. Images from: Tessmer et al. Int. J. Mol. Sci. 2024

DNA lesion search dynamics. One-dimensional diffusion constants (D) on DNA plotted over the lifetimes of complexes on the DNA for AGT. The insets show representative kymographs (green traces) obtained by fluorescence microscopy-coupled dual trap optical tweezers, in which the y direction corresponds to the positions on the DNA tether (shown schematically between two beads held in the two optical traps), and the x direction to time.

Representative florescence images of mEGFP-tagged PARP1(WT), PARP1(ΔC), and PARP2(WT) localization on tethered λ DNA, which was nicked on both strands by CAS9(D10A) with a 60 bp gap between the nicks. Only PARP1(WT) shows condensation at the damage site. Scale bar, 2 μm. Image source: Chappidi et al. Cell 2024

Force measurement as a function of distance from tethered λ DNA extended in the presence of mEGFP-tagged PARP1(WT) or PARP1(ΔC) or PARP2(WT) (1 μM). Thick line represents the mean value. Image source: Chappidi et al. Cell 2024

Distance measurement as a function of time from force clamped λ DNA or λ DNA harboring a PARP1 condensate. Thick line represents mean value. Image source: Chappidi et al. Cell 2024

Workflow of the optical tweezer force clamp and force-extension experiments. Image source: Chappidi et al. Cell 2024

A representative kymograph showing diffusion-driven delivery of BRCA2-RAD51 complexes to ssDNA in the vicinity of the ds-ssDNA junction. Static BRCA2-eGFP molecules bound directly to the ssDNA gap. Mobile BRCA2-eGFP molecules diffuse along dsDNA. 25 nM RAD51-A466 (red) was incubated with DNA in the presence of 5 nM BRCA2-eGFP (blue) and 1.25 nM RPA in the presence of 5 nM SYTOX Orange. Position of the ssDNA gap is indicated by dashed lines.

Schematic of RAD51 filament binding experiment, in which λ DNA was pre-incubated with a 1:1 mixture of labeled and unlabeled RAD51 and then moved to a channel containing BRCA2-eGFP to monitor BRCA2 binding.