Abstract
The resolution of joint molecules that link recombining sister chromatids is essential for chromosome segregation. Here, we determine the fate of unresolved recombination intermediates that arise in GEN1-/- knock-out cells depleted for MUS81, the two nucleases required for resolution. We find that intermediates persist until mitosis where they form a distinct class of anaphase bridges, which we term homologous recombination ultra-fine bridges, or HR-UFBs. The HR-UFBs are distinct from replication stress-associated UFBs, that arise at common fragile sites, and from centromeric UFBs. HR-UFBs are processed by BLM helicase to generate single-stranded RPA-coated bridges that are broken at mitosis. In the next cell cycle, DNA breaks activate the DNA damage checkpoint and chromosome fusions arise by non-homologous end joining. Consequently, the cells undergo a cell cycle delay and massive cell death. These results lead us to present a model detailing how unresolved recombination intermediates can promote DNA damage and chromosomal instability.
Highlights
The resolution of joint molecules that link recombining sister chromatids is essential for chromosome segregation
Three major types of ultra-fine DNA bridges (UFBs) have been described[7]: (i) The most common originate in centromeric regions (C-UFBs), and involve double-stranded catenanes that are resolved by topoisomerase II8, 9; (ii) UFBs can arise from late replication intermediates at common fragile sites (CFSs) following replication stress (FS-UFBs), and are characterized by the presence of twin FANCD2 repair foci[7, 10,11,12,13]; and (iii) telomeric UFBs (T-UFBs) in which replication stalling or fusion events occur at telomeric sequences[14,15,16]
To determine the consequences of aberrant mitosis caused by unresolved recombination intermediates, we established a ‘resolvase-deficient’ experimental system in cultured human cells by siRNA-depleting MUS81 from a GEN1-/- k/o cell line generated from 293 cells using CRISPR/Cas[934] (Fig. 1a)
Summary
The resolution of joint molecules that link recombining sister chromatids is essential for chromosome segregation. The cells undergo a cell cycle delay and massive cell death These results lead us to present a model detailing how unresolved recombination intermediates can promote DNA damage and chromosomal instability. Proper chromosome segregation depends on the removal of all physical connections between sister chromatids prior to anaphase These connections can be proteinaceous, such as cohesin linkages[1], or might be mediated through DNA bridges that are a potential source of genome instability[2]. Intermediates of homologous recombination (HR), provide a covalent link between sister chromatids and cause chromosome segregation defects if not removed before anaphase[17]. BLM helicase activity is required for the conversion of the recombination intermediates into RPA-coated single-stranded bridges that are broken upon cell division, leading to chromosome aberrations and activation of the DNA damage checkpoint in the cell cycle
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