Replication-Coupled Dilution of H4K20me2 Guides 53BP1 to Pre-replicative Chromatin.

The bivalent histone modification reader 53BP1 accumulates around DNA double-strand breaks (DSBs), where it dictates repair pathway choice decisions by limiting DNA end resection. How this function is regulated locally and across the cell cycle to channel repair reactions toward non-homologous end joining (NHEJ) in G1 and promote homology-directed repair (HDR) in S/G2 is insufficiently understood. Here, we show that the ability of 53BP1 to accumulate around DSBs declines as cells progress through S phase and reveal that the inverse relationship between 53BP1 recruitment and replicated chromatin is linked to the replication-coupled dilution of 53BP1's target mark H4K20me2. Consistently, premature maturation of post-replicative chromatin restores H4K20me2 and rescues 53BP1 accumulation on replicated chromatin. The H4K20me2-mediated chromatin association of 53BP1 thus represents an inbuilt mechanism to distinguish DSBs in pre- versus post-replicative chromatin, allowing for localized repair pathway choice decisions based on the availability of replication-generated template strands for HDR.

Pellegrino S ，Michelena J ，Teloni F ，Imhof R ，Altmeyer M ... -  《Cell Reports》

H4K20me2 distinguishes pre-replicative from post-replicative chromatin to appropriately direct DNA repair pathway choice by 53BP1-RIF1-MAD2L2.

Simonetta M ，de Krijger I ，Serrat J ，Moatti N ，Fortunato D ，Hoekman L ，Bleijerveld OB ，Altelaar AFM ，Jacobs JJL ... -  《-》

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

DNA double-strand breaks can be repaired by non-homologous end-joining or homologous recombination. Which pathway is used depends on the balance between the tumor suppressors 53BP1 and BRCA1 and on the availability of an undamaged template DNA for homology-directed repair. How cells switch from a 53BP1-dominated to a BRCA1-governed homologous recombination 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. Our results 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-BARD1-mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, for example, to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

Michelena J ，Pellegrino S ，Spegg V ，Altmeyer M ... -  《Life Science Alliance》

Roles for 53BP1 in the repair of radiation-induced DNA double strand breaks.

In mammalian cells, the mediator protein, 53BP1, exerts distinct impacts on the repair of DNA double strand breaks (DSBs) depending on the setting, for example whether the DSBs arise at telomeres or during replication or class switch recombination. Here, we focus on two roles of 53BP1 in response to ionising radiation (IR)-induced DSBs (IR-DSBs). Canonical DNA non-homologous end-joining (c-NHEJ) is the major DSB repair pathway with homologous recombination (HR) contributing to DSB repair in S/G2 phase. ATM signalling promotes histone modifications and protein assembly in the DSB vicinity, which can be visualised as irradiation induced foci (IRIF). 53BP1 assembles at DSBs in a complex manner involving the formation of nano-domains. In G1 and G2 phase, X- or gamma-ray induced DSBs are repaired with biphasic kinetics. 70-80 % of DSBs are repaired with fast kinetics in both cell cycle phases by c-NHEJ; the remaining DSBs are repaired with slower kinetics in G2 phase via HR and in G1 by a specialised form of c-NHEJ termed Artemis and resection-dependent c-NHEJ, due to a specific requirement for the nuclease, Artemis and resection factors. 53BP1 is essential for the repair of DSBs rejoined with slow kinetics in G1 and G2 phase. This 53BP1 function requires its tandem BRCT domain and interaction with NBS1. As a distinct function, 53BP1 suppresses resection during both HR and Artemis and resection-dependent c-NHEJ. This latter role requires RIF1 and is counteracted by BRCA1. 53BP1 appears to be dispensable for the rejoining of the fast c-NHEJ repair process.

Shibata A ，Jeggo PA 《-》

Roles for the DNA-PK complex and 53BP1 in protecting ends from resection during DNA double-strand break repair.

p53-binding protein 1 (53BP1) exerts distinct impacts in different situations involving DNA double-strand break (DSB) rejoining. Here we focus on how 53BP1 impacts upon the repair of ionising radiation-induced DSBs (IR-DSBs) and how it interfaces with Ku, the DNA end-binding component of canonical non-homologous end-joining (c-NHEJ), the major DSB repair pathway in mammalian cells. We delineate three forms of IR-DSB repair: resection-independent c-NHEJ, which rejoins most IR-DSBs with fast kinetics in G1 and G2, and Artemis and resection-dependent c-NHEJ and homologous recombination (HR), which repair IR-DSBs with slow kinetics in G1 and G2 phase, respectively. The fast component of DSB repair after X-ray exposure occurs via c-NHEJ with normal kinetics in the absence of 53BP1. Ku is highly abundant and has avid DNA end-binding capacity which restricts DNA end-resection and promotes resection-independent c-NHEJ at most IR-DSBs. Thus, 53BP1 is largely dispensable for resection-independent c-NHEJ. In contrast, 53BP1 is essential for the process of rejoining IR-DSBs with slow kinetics. This role requires 53BP1's breast cancer susceptibility gene I (BRCA1) C-terminal (BRCT) 2 domain, persistent ataxia telangiectasia mutated (ATM) activation and potentially relaxation of compacted chromatin at heterochromatic-DSBs. In distinction, 53BP1 inhibits resection-dependent IR-DSB repair in G1 and G2, and this resection-inhibitory function can be counteracted by BRCA1. We discuss a model whereby most IR-DSBs are rapidly repaired by 53BP1-independent and resection-independent c-NHEJ due to the ability of Ku to inhibit resection, but, if delayed, then resection in the presence of Ku is triggered, the 53BP1 barrier comes into force and BRCA1 counteraction is required for resection.

Shibata A ，Jeggo PA 《-》