Summary Protein kinases are essential for signal transduction and control of most cellular processes, including metabolism, membrane transport, motility, and cell cycle. Despite the critical role of kinases in cells and their strong association with diseases, good coverage of their interactions is available for only a fraction of the 535 human kinases. Here, we present a comprehensive mass-spectrometry-based analysis of a human kinase interaction network covering more than 300 kinases. The interaction dataset is a high-quality resource with more than 5,000 previously unreported interactions. We extensively characterized the obtained network and were able to identify previously described, as well as predict new, kinase functional associations, including those of the less well-studied kinases PIM3 and protein O-mannose kinase (POMK). Importantly, the presented interaction map is a valuable resource for assisting biomedical studies. We uncover dozens of kinase-disease associations spanning from genetic disorders to complex diseases, including cancer.

Protein recruitment to DNA break sites is an integral part of the DNA damage response (DDR). Elucidation of the hierarchy and temporal order with which DNA damage sensors as well as repair and signaling factors assemble around chromosome breaks has painted a complex picture of tightly regulated macromolecular interactions that build specialized compartments to facilitate repair and maintenance of genome integrity. While many of the underlying interactions, e.g . between repair factors and damage-induced histone marks, can be explained by lock-and-key or induced fit binding models assuming fixed stoichiometries, structurally less well defined interactions, such as the highly dynamic multivalent interactions implicated in phase separation, also participate in the formation of multi-protein assemblies in response to genotoxic stress. Although much remains to be learned about these types of cooperative and highly dynamic interactions and their functional roles, the rapidly growing interest in material properties of biomolecular condensates and in concepts from polymer chemistry and soft matter physics to understand biological processes at different scales holds great promises. Here, we discuss nuclear condensates in the context of genome integrity maintenance, highlighting the cooperative potential between clustered stoichiometric binding and phase separation. Rather than viewing them as opposing scenarios, their combined effects can balance structural specificity with favorable physicochemical properties relevant for the regulation and function of multilayered nuclear condensates.

RPA has been shown to protect single-stranded DNA (ssDNA) intermediates from instability and breakage. RPA binds ssDNA with sub-nanomolar affinity, yet dynamic turnover is required for downstream ssDNA transactions. How ultrahigh-affinity binding and dynamic turnover are achieved simultaneously is not well understood. Here we reveal that RPA has a strong propensity to assemble into dynamic condensates. In solution, purified RPA phase separates into liquid droplets with fusion and surface wetting behavior. Phase separation is stimulated by sub-stoichiometric amounts of ssDNA, but not RNA or double-stranded DNA, and ssDNA gets selectively enriched in RPA condensates. We find the RPA2 subunit required for condensation and multi-site phosphorylation of the RPA2 N-terminal intrinsically disordered region to regulate RPA self-interaction. Functionally, quantitative proximity proteomics links RPA condensation to telomere clustering and integrity in cancer cells. Collectively, our results suggest that RPA-coated ssDNA is contained in dynamic RPA condensates whose properties are important for genome organization and stability.

DNA double-strand breaks can be repaired by non-homologous end-joining or homologous recombination. Which pathway is used depends on the balance between the tumor suppressors 53BP1 and BRCA1 and on the availability of an undamaged template DNA for homology-directed repair. How cells switch from a 53BP1-dominated to a BRCA1-governed homologous recombination response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells. Our results substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1–BARD1–dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1’s binding mark H4K20me2 functionally cooperates with BRCA1–BARD1–mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, for example, to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.