XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.

Human chromosomes are captured along microtubule walls (lateral attachment) and then tethered to microtubule-ends (end-on attachment) through a multi-step end-on conversion process. Upstream regulators that orchestrate this remarkable change in the plane of kinetochore-microtubule attachment in human cells are not known. By tracking kinetochore movements and using kinetochore markers specific to attachment status, we reveal a spatially defined role for Aurora-B kinase in retarding the end-on conversion process. To understand how Aurora-B activity is counteracted, we compare the roles of two outer-kinetochore bound phosphatases and find that BubR1-associated PP2A, unlike KNL1-associated PP1, plays a significant role in end-on conversion. Finally, we uncover a novel role for Aurora-B regulated Astrin-SKAP complex in ensuring the correct plane of kinetochore-microtubule attachment. Thus, we identify Aurora-B as a key upstream regulator of end-on conversion in human cells and establish a late role for Astrin-SKAP complex in the end-on conversion process.

SummaryA common assumption is that human chromosomes carry equal chances of mis-segregation during compromised cell division. Human chromosomes vary in multiple parameters that might generate bias, but technological limitations have precluded a comprehensive analysis of chromosome-specific aneuploidy. Here, by imaging specific centromeres coupled with high-throughput single-cell analysis as well as single-cell sequencing, we show that aneuploidy occurs non-randomly following common treatments to elevate chromosome mis-segregation. Temporary spindle disruption leads to elevated mis-segregation and aneuploidy of a subset of chromosomes, particularly affecting chromosomes 1 and 2. Unexpectedly, we find that a period of mitotic delay weakens centromeric cohesion and promotes chromosome mis-segregation and that chromosomes 1 and 2 are particularly prone to suffer cohesion fatigue. Our findings demonstrate that inherent properties of individual chromosomes can bias chromosome mis-segregation and aneuploidy rates, with implications for studies on aneuploidy in human disease.

The microtubule polymer grows and shrinks predominantly from one of its ends called the ‘plus-end’. Plus-end regulation during interphase is well understood. However, mitotic regulation of plus-ends is only beginning to be understood in mammalian cells. During mitosis, the plus-ends are tethered to specialized microtubule capture sites. At these sites, plus-end-binding proteins are loaded and unloaded in a regulated fashion. Proper tethering of plus-ends to specialized sites is important so that the microtubule is able to translate its growth and shrinkage into pushing and pulling forces that move bulky subcellular structures. We discuss recent advances on how mitotic plus-ends are tethered to distinct subcellular sites and how plus-end-bound proteins can modulate the forces that move subcellular structures. Using end binding 1 (EB1) as a prototype plus-end-binding protein, we highlight the complex network of plus-end-binding proteins and their regulation through phosphorylation. Finally, we develop a speculative ‘moving platform’ model that illustrates the plus-end's role in distinguishing correct versus incorrect microtubule interactions.