Abstract
Although kinetochores normally play a key role in sister chromatid separation and segregation, chromosome fragments lacking kinetochores (acentrics) can in some cases separate and segregate successfully. In Drosophila neuroblasts, acentric chromosomes undergo delayed, but otherwise normal sister separation, revealing the existence of kinetochore- independent mechanisms driving sister chromosome separation. Bulk cohesin removal from the acentric is not delayed, suggesting factors other than cohesin are responsible for the delay in acentric sister separation. In contrast to intact kinetochore-bearing chromosomes, we discovered that acentrics align parallel as well as perpendicular to the mitotic spindle. In addition, sister acentrics undergo unconventional patterns of separation. For example, rather than the simultaneous separation of sisters, acentrics oriented parallel to the spindle often slide past one another toward opposing poles. To identify the mechanisms driving acentric separation, we screened 117 RNAi gene knockdowns for synthetic lethality with acentric chromosome fragments. In addition to well-established DNA repair and checkpoint mutants, this candidate screen identified synthetic lethality with X-chromosome-derived acentric fragments in knockdowns of Greatwall (cell cycle kinase), EB1 (microtubule plus-end tracking protein), and Map205 (microtubule-stabilizing protein). Additional image-based screening revealed that reductions in Topoisomerase II levels disrupted sister acentric separation. Intriguingly, live imaging revealed that knockdowns of EB1, Map205, and Greatwall preferentially disrupted the sliding mode of sister acentric separation. Based on our analysis of EB1 localization and knockdown phenotypes, we propose that in the absence of a kinetochore, microtubule plus-end dynamics provide the force to resolve DNA catenations required for sister separation.
Highlights
Eukaryotic cells have evolved mechanisms to detect and protect against genomic insults
Previous studies demonstrated that sister chromosomes lacking kinetochores often undergo separation, segregation and transmission
We show that during anaphase, acentric sister separation is achieved through Topoisomerase activity, an enzyme that resolves these DNA linkages, as well as forces generated on the acentrics by the growing ends of highly dynamic microtubule polymers
Summary
Eukaryotic cells have evolved mechanisms to detect and protect against genomic insults. Studies demonstrate that DNA damage persisting through metaphase delays anaphase onset This delay is mediated both by the DNA damage and spindle assembly checkpoint pathways [2,3]. The persistence of unrepaired double-strand breaks (DSBs) at metaphase is problematic due to the formation of chromosome fragments, one of which lacks a telomere and the other lacking a kinetochore and a telomere. The latter type are known as acentrics and are incapable of forming canonical microtubule-kinetochore attachments that drive sister chromosome separation and segregation. Proposed mechanisms of acentric segregation include neo-centromere formation [14] and direct association of the acentric chromosome with microtubules [15] or a kinetochore-bearing chromosome [16,17,18]
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