Department of Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
J Cell Biol. 2021 Jan 4;220(1). doi: 10.1083/jcb.202003148.
The DNA damage response (DDR) has a critical role in the maintenance of genomic integrity during chromosome replication. However, responses to replication stress evoked by tight DNA-protein complexes have not been fully elucidated. Here, we used bacterial LacI protein binding to lacO arrays to make site-specific replication fork barriers on the human chromosome. These barriers induced the accumulation of single-stranded DNA (ssDNA) and various DDR proteins at the lacO site. SLX4-XPF functioned as an upstream factor for the accumulation of DDR proteins, and consequently, ATR and FANCD2 were interdependently recruited. Moreover, LacI binding in S phase caused underreplication and abnormal mitotic segregation of the lacO arrays. Finally, we show that the SLX4-ATR axis represses the anaphase abnormality induced by LacI binding. Our results outline a long-term process by which human cells manage nucleoprotein obstacles ahead of the replication fork to prevent chromosomal instability.
DNA 损伤反应 (DDR) 在染色体复制过程中对维持基因组完整性起着关键作用。然而，对于由紧密的 DNA-蛋白质复合物引起的复制应激反应，尚未得到充分阐明。在这里，我们使用细菌 LacI 蛋白结合 lacO 阵列在人类染色体上制造特定位置的复制叉障碍。这些障碍在 lacO 位点诱导了单链 DNA（ssDNA）和各种 DDR 蛋白的积累。SLX4-XPF 作为 DDR 蛋白积累的上游因子发挥作用，随后 ATR 和 FANCD2 被相互招募。此外，LacI 在 S 期的结合导致 lacO 阵列的复制不足和异常有丝分裂分离。最后，我们表明，SLX4-ATR 轴抑制了 LacI 结合诱导的后期异常。我们的结果概述了人类细胞在复制叉之前处理核蛋白障碍以防止染色体不稳定性的长期过程。
