Reconstitution of anaphase DNA bridge recognition and disjunction

Abstract

Faithful chromosome segregation requires that the sister chromatids be disjoined completely. Defective disjunction can lead to the persistence of histone-free threads of DNA known as ultra-fine bridges (UFBs) that connect the separating sister DNA molecules during anaphase. UFBs arise at specific genomic loci and can only be visualized by detection of associated proteins such as PICH, BLM, topoisomerase IIIα, and RPA. However, it remains unknown how these proteins work together to promote UFB processing. We used a combination of ensemble biochemistry and new single-molecule assays to reconstitute key steps of UFB recognition and processing by these human proteins in vitro. We discovered characteristic patterns of hierarchical recruitment and coordinated biochemical activities that were specific for DNA structures modeling UFBs arising at either centromeres or common fragile sites. Our results describe a mechanistic model for how unresolved DNA replication structures are processed by DNA-structure-specific binding factors in mitosis to prevent pathological chromosome nondisjunction.

RPA phosphorylation regulates DNA resection

“…Phosphorylation of RPA abrogates or changes this interaction and decreases BLM's ability to initiate at ss/ds junctions, in addition to promoting the intrinsic strandswitching activity of BLM. Regulation of BLM strand switching may be important during the later stages of HR (i.e., joint molecule dissolution), Holliday junction migration, resolution of ultra-fine bridges, and at stalled replication forks (Bachrati and Hickson, 2008;Bizard and Hickson, 2014;Croteau et al, 2014;Liu et al, 2014;Machwe et al, 2006;Ralf et al, 2006;Sarlós et al, 2018;Wu and Hickson, 2003). In the context of DNA resection, one of the two ssDNA strands are degraded by a nuclease, forcing BLM to slow down or stall.…”

Histone H2A phosphorylation recruits topoisomerase II α to centromeres to safeguard genomic stability

“…Defects in this process can lead to the persistence of DNA threads known as ultra-fine bridges (UFBs) between separating sister chromatids during anaphase (Biebricher et al, 2013;Liu et al, 2014;Sarlos et al, 2018). Timely disjunction of sister chromatids is critical for faithful chromosome segregation.…”

“…Timely disjunction of sister chromatids is critical for faithful chromosome segregation. Defects in this process can lead to the persistence of DNA threads known as ultra-fine bridges (UFBs) between separating sister chromatids during anaphase (Biebricher et al, 2013;Liu et al, 2014;Sarlos et al, 2018). UFBs are devoid of histones and usually cannot be stained with conventional DNA dyes, but can be visualized by detection of associated proteins such as the SNF2 family DNA translocase PICH (Baumann et al, 2007) and the Bloom syndrome helicase BLM Ke et al, 2011).…”

Identification of hemicatenane-specific binding proteins by fractionation of Hela nuclei extracts

“…Devoid of catalytic activity, the RMI1 might stabilize the open form of the Topoisomerase IIIα to favor strand passage. Finally, one family of ultra fine bridges has been shown to be decorated by the BTRR complex and it is possible that the decatenase activity of Topoisomerase IIIα be used to resolve their inter-strand links [14,15].…”

“…Species were resolved by electrophoresis under native conditions. Three DNAs were tested for their binding to SND1-64: dsMC09 (lanes 1-5); dsMC10 (lane 6-10); HC (lanes [11][12][13][14][15]. Concentrations of protein were as indicated (lanes 1, 6, 11: 0; lanes 2, 7, 12: 0.1 µM; lanes 3, 8, 13: 0.3 µM; lanes 4, 9, 14: 0.9 µM; lanes 5, 10, 15: 2.7 µM).…”