The Removal of Histones from Ultrafine Anaphase Bridges Studied by Optical Tweezers

Abstract

During mitosis, duplicated DNA has to be faithfully segregated into two daughter cells. In anaphase, regions of entangled double-stranded DNA between segregating sister chromatids can give rise to DNA threads known as ultrafine anaphase bridges (UFBs). Failure to resolve UFBs can result in genome instabilities. It has been proposed that a key step in UFB resolution is the removal of histones, and that this is facilitated by the SNF2-family protein PICH. The mechanism of nucleosome remodeling by PICH has, however, remained elusive. Here, we present a single-molecule assay that recapitulates chromatin re-organization during UFB resolution. In vitro chromatin constructs consisting of 4 to 12 high affinity histone binding sites (601 sequences) within an 8.5 kbp DNA molecule were prepared using a combination of restriction-enzyme based cloning and Gibson assembly. After reconstitution with human histones, these constructs were manipulated within a multi-channel microfluidic flow cell using dual-trap optical tweezers. This approach allows us to measure tension-induced nucleosome unwrapping in the presence or absence of PICH. Our results suggest that, under moderate tension (10 pN), PICH facilitates the unwrapping of nucleosomes. In further experiments, we aim to correlate nucleosome stability with the dynamics of single fluorescently-labelled PICH proteins on DNA. Taken together, our single-molecule approach provides new insights into the mechanisms by which PICH is involved in anaphase UFB resolution.