Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly
Ubiquitinated-PCNA protects replication forks from DNA2-mediated degradation by regulating Okazaki fragment maturation and chromatin assembly
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DOI：
10.1038/s41467-020-16096-w
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发表时间：
2020-05-01
影响因子：
16.6
通讯作者：
Moldovan, George-Lucian
中科院分区：
文献类型：
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作者：
Thakar, Tanay;Leung, Wendy;Moldovan, George-Lucian
在遗传毒性应激时，增殖细胞核抗原泛素化允许通过招募病变旁路DNA聚合酶来复制受损的DNA。然而，在正常的S期进展中，增殖细胞核抗原也是普遍存在的。通过使用由CRISPR/Cas9基因编辑产生的293T和RPE1细胞，我们证明了这种修饰促进了细胞的增殖，并抑制了正常生长条件下的基因组不稳定性。失去增殖细胞核抗原泛素化导致DNA2依赖但不依赖于Mre11的新生DNA在停滞的复制叉处的核溶解降解。这种降解与复制分叉后的缺陷缝隙填充和冈崎片段的不完全成熟有关，这干扰了ATAD5有效地卸载增殖细胞核抗原和随后CAF-1的核小体沉积。此外，伴随着增殖细胞核抗原泛素化和BRCA途径的丧失，导致新生DNA降解和PARP抑制剂敏感性增加。总之，我们表明，通过确保有效的Okazaki片段成熟，增殖细胞核抗原泛素化保护分叉的完整性，并促进BRCA缺陷细胞对PARP抑制剂的抗性。增殖细胞核抗原是DNA复制和细胞增殖所必需的。在这里，作者揭示了增殖细胞核抗原泛素化通过调节Okazaki片段成熟和染色质组装来保护停滞的复制叉子免受DNA2介导的降解。
Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors. PCNA is essential for DNA replication and cellular proliferation. Here, the authors reveal that PCNA ubiquitination protects stalled replication forks from DNA2-mediated degradation via regulation of Okazaki fragment maturation and chromatin assembly.