Dynamics of chromatin factors RSF1, CENPS and CENPX at DNA damage sites.

Chromatin has a major influence on the DNA damage response (DDR). Several chromatin-related factors participate in specialised DNA packaging during the DDR including the CENPS and CENPX histone fold proteins, also known as MHF1/2, and the chromatin remodelling factor RSF1 although their contribution has remained unclear. We defined a timeline for RSF1, CENPS, and CENPX recruitment at DNA double strand breaks (DSBs) induced in live HeLa cells by microirradiation and calibrated this to published data to clarify the potential for their involvement in the DDR. CENPS, CENPX and RSF1 are recruited with a half time of ∼100 s and removed with a half time of ∼2000 s. Enrichment for cell cycle phase revealed that this recruitment occurs in G1, S and G2 phases, but that its half time in G2 appears to be delayed and stronger than in G1. Integration of these observations with timelines for other DDR factors reveals that CENPS and CENPX recruitment occurs simultaneously immediately after ATM activation and RNF8-RNF168 activity. The removal of CENPS and CENPX is at a similar time to loading of RPA and assembly of RAD51. This places RSF1, CENPS and CENPX in the vicinity of DSBs at the time when nucleosomes are being actively remodelled during the chromatin-dependent early response to DNA damage involving pathway choice and resection, and their increased abundance at DSBs in G2 correlates with extended resection for HR.