Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

Abstract

DNA double-strand breaks can be repaired by two competing mechanisms, nonhomologous end-joining (NHEJ) and homologous recombination (HR). Whether one or the other repair pathway is favored depends on the availability of an undamaged template DNA that allows for homology-directed repair. The tumor suppressor proteins 53BP1 and BRCA1 are considered antagonistic players in this repair pathway choice, as 53BP1 restrains DNA end resection, whereas BRCA1, together with its partner protein BARD1, displaces 53BP1 from damaged replicated chromatin and promotes HR. How cells switch from a 53BP1-dominated to a BRCA1-dominated response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells, in comparison to 53BP1 accumulation at damaged unreplicated chromatin. These findings substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1's binding mark H4K20me2 functionally cooperates with BRCA1-BARD1mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, e.g. to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency

“…53BP1 functions in both unperturbed and perturbed replication (Lukas et al, 2011;Schmid et al, 2018). Dual deficiency in replication and BRCA1 expression could enhance 53BP1 in chromatin because replication dilutes its recruiting histone mark that in turn favors BRCA1 binding (Michelena et al, 2021;Pellegrino et al, 2017;Saredi et al, 2016). In BRCA1 KO cells, 53BP1 could interfere with XRCC1 recruitment or its attempts to process the lagging strand.…”

“…However, unlike PARP1, XRCC1 was aberrantly low in BRCA1 KO cells, with its reduc-tion primarily in EdU + cells (Figures 7B and 7C). BRCA1 KO cells also had a reduced EdU intensity in EdU + cells and elevated 53BP1 nuclear foci (Figures 7D, 7E, and S7A), suggesting that replication was less robust and/or underreplicated (Lukas et al, 2011;Michelena et al, 2021;Pellegrino et al, 2017;Saredi et al, 2016). These data suggest that in BRCA1 KO cells, PARP1 fails to effectively recruit XRCC1 to engage backup lagging strand synthesis (Hanzlikova and Caldecott, 2019).…”

Cell-cycle dependent inhibition of BRCA1 signaling by the lysine methyltransferase SET8

“…The SET8-induced switch from BRCA1/BARD1 to 53BP1 recruitment on replicated chromatin is independent of SUV4-20H activity on H4K20me1. A previous report has proposed that the inverse relationship between 53BP1 recruitment and the maturation of replicated chromatin is triggered by the replicationcoupled dilution of H4K20me2, which is a high-affinity binding site for 53BP1 (Michelena et al 2021;Pellegrino et al 2017). Hence, we asked whether a conversion of H4K20me1 to H4K20me2 by SUV4-20Hs was necessary for the accumulation of 53BP1 on replicated chromatin upon SET8 stabilization or whether the switch from H4K20me to H4K20me1 might be sufficient on its own since 53BP1 can also bind to H4K20me1.…”

“…As cells progress through S phase, the ability of 53BP1 to accumulate around DNA breaks decline. This has been notably attributed to the replication-coupled dilution of H4K20me2 mark on postreplicated chromatin (Michelena et al 2021;Tarsounas et Sung 2020). Yet, 53BP1 can also interact with histone H4K20 mono-methylation (H4K20me1) and trimethylation (H4K20me3) (Botuyan et al 2006;Svobodová et al 2018), thereby questioning this model.…”