A key role of chromatin kinases is to phosphorylate histone tails during mitosis to spatiotemporally regulate cell division. Vaccinia-related kinase 1 (VRK1) is a serine–threonine kinase that phosphorylates histone H3 threonine 3 (H3T3) along with other chromatin-based targets. While structural studies have defined how several classes of histone-modifying enzymes bind to and function on nucleosomes, the mechanism of chromatin engagement by kinases is largely unclear. Here, we paired cryo-electron microscopy with biochemical and cellular assays to demonstrate that VRK1 interacts with both linker DNA and the nucleosome acidic patch to phosphorylate H3T3. Acidic patch binding by VRK1 is mediated by an arginine-rich flexible C-terminal tail. Homozygous missense and nonsense mutations of this acidic patch recognition motif in VRK1 are causative in rare adult-onset distal spinal muscular atrophy. We show that these VRK1 mutations interfere with nucleosome acidic patch binding, leading to mislocalization of VRK1 during mitosis, thus providing a potential new molecular mechanism for pathogenesis.

No abstract

No abstract

Clinical genetic testing readily detects germline genetic variants. Yet, the evidence available for variant classification as benign or pathogenic is often limited by the rarity of individual variants, leading to many “variant of uncertain significance” (VUS) classifications. VUS cannot guide clinical decisions, complicating counseling and management. Laboratory assays can potentially aid reclassification, but require benchmarking against variants with definitive interpretations to have sufficient predictive power for clinical use. Of all clinically identified germline variants in hereditary breast cancer gene PALB2 (Partner and Localizer of BRCA2), ~50% are VUS and ~90% of VUS are missense. Truncating PALB2 variants have homologous recombination (HR) defects and instead rely on error-prone non-homologous end-joining (NHEJ) for DNA damage repair (DDR). Recent reports show some missense PALB2 variants may also be damaging, but thus far functional studies have lacked benchmarking controls. Using the Traffic Light Reporter (TLR) to quantify cellular HR and NHEJ using fluorescent markers, we assessed variant-level DDR capacity in hereditary breast cancer genes. We first determined the TLR’s dynamic range using BRCA2 missense variants of known significance as benchmarks for normal/abnormal HR function. We then tested 37 PALB2 variants, generating functional data for germline PALB2 variants at a moderate level of evidence for a pathogenic interpretation (PS3_moderate) for 8 variants, or a supporting level of evidence in favor of a benign interpretation (BS3_supporting) for 13 variants, based on the ability of the assay to correctly classify PALB2 validation controls. This new data can be applied in subsequent variant interpretations for direct clinical benefit.