DNA replication errors that escape the proofreading activity of the replicative DNA polymerase are repaired by DNA mismatch repair (MMR). The initiation of MMR in Escherichia coli involves the recognition of the mismatch by MutS, binding of MutL, and activation of the endonuclease MutH which incises DNA at a hemi-methylated GATC site. MutS exists in a dimer-tetramer equilibrium, but the function of the tetramer during MMR remains unknown. Here, we used in vitro MutH activation assays to examine the role of MutS in the reaction steps that couple mismatch recognition to daughter strand incision. To study the behavior of different MutS oligomers, we used obligate dimers and tetramers and quantified GATC site incision on circular and linear DNA substrates. Especially in the presence of free DNA ends, MutS tetramers mediate more efficient incision than MutS dimers, likely due to tetramers diffusing slower and therefore being more successful in assembling the active incision complex before dissociating from the DNA at the ends. Likewise, we observed that MutS tetramers have a higher preference for nicking the GATC site close to the mismatch than dimeric MutS. Through probabilistic modeling, we show that this increased preference is consistent with a fourfold decrease in diffusion constant for the tetramer compared to the dimer. We propose that during mismatch repair, DNA excision tracts resulting from MutS tetramer-mediated incision will be shorter than those mediated by the dimer, and that this explains the reported higher repair efficiencies of wild-type MutS compared to the dimer.

Single-cell proteomics has the potential to decipher tumor heterogeneity, and a method like single-cell proteomics by mass spectrometry (SCoPE-MS) allows profiling several tens of single cells for >1,000 proteins per cell. This method, however, cannot link the proteome of individual cells with phenotypes of interest. Here, we developed a microscopy-based functional single-cell proteomic-profiling technology, called FUNpro, to address this. FUNpro enables screening, identification, and isolation of single cells of interest in a real-time fashion, even if the phenotypes are dynamic or the cells of interest are rare. We applied FUNpro to proteomically profile a newly identified small subpopulation of U2OS osteosarcoma cells displaying an abnormal, prolonged DNA damage response (DDR) after ionizing radiation (IR). With this, we identified the PDS5A protein contributing to the abnormal DDR dynamics and helping the cells survive after IR.

Polymersomes have the potential to be applied in targeted alpha radionuclide therapy, while in addition preventing release of recoiling daughter isotopes. In this study, we investigated the cellular uptake, post uptake processing and intracellular localization of polymersomes. Methods: High-content microscopy was used to validate polymersome uptake kinetics. Confocal (live cell) microscopy was used to elucidate the uptake mechanism and DNA damage induction. Intracellular distribution of polymersomes in 3-D was determined using super-resolution microscopy. Results: We found that altering polymersome size and concentration affects the initial uptake and overall uptake capacity; uptake efficiency and eventual plateau levels varied between cell lines; and mitotic cells show increased uptake. Intracellular polymersomes were transported along microtubules in a fast and dynamic manner. Endocytic uptake of polymersomes was evidenced through co-localization with endocytic pathway components. Finally, we show the intracellular distribution of polymersomes in 3-D and DNA damage inducing capabilities of²¹³Bi labeled polymersomes. Conclusion: Polymersome size and concentration affect the uptake efficiency, which also varies for different cell types. In addition, we present advanced assays to investigate uptake characteristics in detail, a necessity for optimization of nano-carriers. Moreover, by elucidating the uptake mechanism, as well as uptake extent and geometrical distribution of radiolabeled polymersomes we provide insight on how to improve polymersome design.