a Division of Oncogenomics , The Netherlands Cancer Institute , Plesmanlaan 121, 1066 CX Amsterdam , The Netherlands.
b Proteomics Facility , The Netherlands Cancer Institute , Plesmanlaan 121, 1066 CX Amsterdam , The Netherlands.
Cell Cycle. 2018;17(1):124-136. doi: 10.1080/15384101.2017.1404210. Epub 2018 Jan 2.
The main pathways for the repair of DNA double strand breaks (DSBs) are non-homologous end-joining (NHEJ) and homologous recombination directed repair (HDR). These operate mutually exclusive and are activated by 53BP1 and BRCA1, respectively. As HDR can only succeed in the presence of an intact copy of replicated DNA, cells employ several mechanisms to inactivate HDR in the G1 phase of cell cycle. As cells enter S-phase, these inhibitory mechanisms are released and HDR becomes active. However, during DNA replication, NHEJ and HDR pathways are both functional and non-replicated and replicated DNA regions co-exist, with the risk of aberrant HDR activity at DSBs in non-replicated DNA. It has become clear that DNA repair pathway choice depends on inhibition of DNA end-resection by 53BP1 and its downstream factors RIF1 and MAD2L2. However, it is unknown how MAD2L2 accumulates at DSBs to participate in DNA repair pathway control and how the NHEJ and HDR repair pathways are appropriately activated at DSBs with respect to the replication status of the DNA, such that NHEJ acts at DSBs in pre-replicative DNA and HDR acts on DSBs in post-replicative DNA. Here we show that MAD2L2 is recruited to DSBs in H4K20 dimethylated chromatin by forming a protein complex with 53BP1 and RIF1 and that MAD2L2, similar to 53BP1 and RIF1, suppresses DSB accumulation of BRCA1. Furthermore, we show that the replication status of the DNA locally ensures the engagement of the correct DNA repair pathway, through epigenetics. In non-replicated DNA, saturating levels of the 53BP1 binding site, di-methylated lysine 20 of histone 4 (H4K20me2), lead to robust 53BP1-RIF1-MAD2L2 recruitment at DSBs, with consequent exclusion of BRCA1. Conversely, replication-associated 2-fold dilution of H4K20me2 promotes the release of the 53BP1-RIF1-MAD2L2 complex and favours the access of BRCA1. Thus, the differential H4K20 methylation status between pre-replicative and post-replicative DNA represents an intrinsic mechanism that locally ensures appropriate recruitment of the 53BP1-RIF1-MAD2L2 complex at DNA DSBs, to engage the correct DNA repair pathway.
DNA 双链断裂 (DSBs) 的主要修复途径是非同源末端连接 (NHEJ) 和同源重组修复 (HDR)。这两种途径相互排斥，分别由 53BP1 和 BRCA1 激活。由于 HDR 只能在复制 DNA 的完整拷贝存在的情况下成功，因此细胞采用几种机制在细胞周期的 G1 期使 HDR 失活。当细胞进入 S 期时，这些抑制机制被释放，HDR 变得活跃。然而，在 DNA 复制过程中，NHEJ 和 HDR 途径都是功能性的，非复制的，并且复制和非复制的 DNA 区域共存，在非复制的 DNA 中的 DSB 处存在异常 HDR 活性的风险。现在已经清楚，DNA 修复途径的选择取决于 53BP1 及其下游因子 RIF1 和 MAD2L2 对 DNA 末端切除的抑制。然而，尚不清楚 MAD2L2 如何在 DSB 处积累以参与 DNA 修复途径的控制，以及 NHEJ 和 HDR 修复途径如何在 DNA 复制状态的情况下在 DSB 处被适当地激活，使得 NHEJ 在预复制的 DNA 中的 DSB 处起作用，并且 HDR 在复制后的 DNA 中的 DSB 处起作用。在这里，我们显示 MAD2L2 通过与 53BP1 和 RIF1 形成蛋白质复合物被募集到 H4K20 二甲基化染色质中的 DSB 处，并且 MAD2L2 与 53BP1 和 RIF1 相似，抑制 BRCA1 的 DSB 积累。此外，我们显示 DNA 的复制状态通过表观遗传学在局部确保正确的 DNA 修复途径的参与。在非复制的 DNA 中，53BP1 结合位点的饱和水平，即组蛋白 4 (H4) 的赖氨酸 20 二甲基化 (H4K20me2)，导致 DSB 处的 53BP1-RIF1-MAD2L2 募集增强，从而导致 BRCA1 的排斥。相反，与复制相关的 2 倍稀释的 H4K20me2 促进 53BP1-RIF1-MAD2L2 复合物的释放，并有利于 BRCA1 的进入。因此，在预复制和复制后的 DNA 之间的差异 H4K20 甲基化状态代表一种内在机制，该机制在局部确保 53BP1-RIF1-MAD2L2 复合物在 DNA DSB 处的适当募集，以参与正确的 DNA 修复途径。
