Asf1 facilitates dephosphorylation of Rad53 after DNA double-strand break repair

Abstract

To allow for sufficient time to repair DNA double-stranded breaks (DSBs), eukaryotic cells activate the DNA damage checkpoint. In budding yeast, Rad53 (mammalian Chk2) phosphorylation parallels the persistence of the unrepaired DSB and is extinguished when repair is complete in a process termed recovery or when the cells adapt to the DNA damage checkpoint. A strain containing a slowly repaired DSB does not require the histone chaperone Asf1 to resume cell cycle progression after DSB repair. When a second, rapidly repairable DSB is added to this strain, Asf1 becomes required for recovery. Recovery from two repairable DSBs also depends on the histone acetyltransferase Rtt109 and the cullin subunit Rtt101, both of which modify histone H3 that is associated with Asf1. We show that dissociation of histone H3 from Asf1 is required for efficient recovery and that Asf1 is required for complete dephosphorylation of Rad53 when the upstream DNA damage checkpoint signaling is turned off. Our data suggest that the requirements for recovery from the DNA damage checkpoint become more stringent with increased levels of damage and that Asf1 plays a histone chaperone-independent role in facilitating complete Rad53 dephosphorylation following repair.

ASF1a Promotes Non-homologous End Joining Repair by Facilitating Phosphorylation of MDC1 by ATM at Double-Strand Breaks

“…ASF1a promotes H3 acetylation at lys56 by the CBP/p300 acetyltransferase, which is required for nucleosome reassembly after DNA repair (Das et al, 2009; Groth et al, 2007). ASF1 is required for checkpoint recovery and the return to normal cell cycle after DNA damage repair (Tsabar et al, 2016), but there is no report that shows ASF1 is directly involved in DSB repair. We have discovered that ASF1a is specifically required to promote NHEJ and thus suppress HR.…”

Delineation of the role of chromatin assembly and the Rtt101Mms1 E3 ubiquitin ligase in DNA damage checkpoint recovery in budding yeast

“…The Haber group recently reported that Asf1 and Rtt101 are required for checkpoint recovery, but in their hands they only had this role when they induced two DSBs [ 58 ], not one DSB as is the case in our studies [ 24 ] Fig 3 . This may be related to the fact that the Haber lab only observed defective checkpoint recovery in response to a single DSB when they remove both Asf1 and CAF-1 at the same time, but not with the individual mutations [ 35 ] as is the case in our hands [ 24 ].…”

Mec1ATR Autophosphorylation and Ddc2ATRIP Phosphorylation Regulates DNA Damage Checkpoint Signaling

“…Based on these findings and on our Ddc2-GFP localization data, we postulate that the degradation of the Ddc2 pool, which is not localized to chromatin could be used to limit continuous Mec1-Ddc2 recruitment to the break site in the presence of a persistent DNA lesion. Conditional depletion of Ddc2 by using an auxin-inducible degron system causes rapid Rad53 dephosphorylation (Tsabar et al, 2016). Additionally, the overexpression of Ddc2, but not Mec1, is shown to cause a hyperactivation of the checkpoint activity .…”