Global chromatin mobility induced by a DSB is dictated by chromosomal conformation and defines the HR outcome.

Repair of DNA double-strand breaks (DSBs) is crucial for genome integrity. A conserved response to DSBs is an increase in chromatin mobility that can be local, at the site of the DSB, or global, at undamaged regions of the genome. Here, we address the function of global chromatin mobility during homologous recombination (HR) of a single, targeted, controlled DSB. We set up a system that tracks HR in vivo over time and show that two types of DSB-induced global chromatin mobility are involved in HR, depending on the position of the DSB. Close to the centromere, a DSB induces global mobility that depends solely on H2A(X) phosphorylation and accelerates repair kinetics, but is not essential. In contrast, the global mobility induced by a DSB away from the centromere becomes essential for HR repair and is triggered by homology search through a mechanism that depends on H2A(X) phosphorylation, checkpoint progression, and Rad51. Our data demonstrate that global mobility is governed by chromosomal conformation and differentially coordinates repair by HR.