Nanofluidics highlights importance of CtIP's tetrameric structure for DNA repair

Other conference contribution, 2024

The initial steps of DNA double-strand break (DSB) repair in human cells involve the MRE11-RAD50-NBS1 (MRN) complex and its cofactor, phosphorylated CtIP. The activity of these proteins in nucleolytic DSB resection that commits the repair to homologous recombination (HR) are well studied, but their role in tethering and bridging the DNA ends is less understood. We have previously shown, on the single molecule level, how phosphorylated CtIP can bridge the ends of single long (∼50 kb) DNA molecules using nanofluidic channels. The study revealed that the way which CtIP bridges DNA is dependent on its oligomeric state. The tetrameric wild-type CtIP almost exclusively generated circular products as an effect of intramolecular bridging while the tetramerization-deficient CtIPL27E mutant showed results in line with intermolecular bridging. Additionally, truncated mutants showed that the bridging by CtIP is promoted by domains distinct from those that stimulate the nuclease activity of MRN. In this study we use nanofluidics to build on these results and characterize how CtIP bridges long DNA molecules with ends of different design. The method is based on the confinement of long DNA molecules in nanochannels. In contrast to tethering one or both ends to a substrate, as in most common single DNA molecule methods, the DNA is completely free in the nanochannels, meaning that reaction on the DNA ends can be conveniently studied. By utilizing customizable long DNA substrates in conjunction with a set of truncated mutants we seek to investigate CtIP’s ability to bridge DNA with differing modifications and the importance of CtIP’s oligomeric and phosphorylated state to further understand the mechanism behind DNA bridging by CtIP.