a Division of Biology and Biological Engineering , California Institute of Technology , Pasadena , CA , USA.
Cell Cycle. 2019 Apr;18(8):898-913. doi: 10.1080/15384101.2019.1598728. Epub 2019 Apr 12.
Besides TopBP1, ETAA1 has been identified more recently as an activator of the ATR-ATRIP complex in human cells. We have examined the role of ETAA1 in the Xenopus egg-extract system, which has been instrumental in the study of ATR-ATRIP. Depletion of ETAA1 from egg extracts did not noticeably reduce the activation of ATR-ATRIP in response to replication stress, as monitored by the ATR-dependent phosphorylation of Chk1 and RPA. Moreover, lack of ETAA1 did not appear to affect DNA replication during an unperturbed S-phase. Significantly, we find that TopBP1 is considerably more abundant than ETAA1 in egg extracts. We proceeded to show that ETAA1 could support the activation of ATR-ATRIP in response to replication stress if we increased its concentration in egg extracts by adding extra full-length recombinant ETAA1. Thus, TopBP1 appears to be the predominant activator of ATR-ATRIP in response to replication stress in this system. We have also explored the biochemical mechanism by which ETAA1 activates ATR-ATRIP. We have developed an in vitro system in which full-length recombinant ETAA1 supports activation of ATR-ATRIP in the presence of defined components. We find that binding of ETAA1 to RPA associated with single-stranded DNA (ssDNA) greatly stimulates its ability to activate ATR-ATRIP. Thus, RPA-coated ssDNA serves as a direct positive effector in the ETAA1-mediated activation of ATR-ATRIP.
除了 TopBP1，ETAA1 最近也被鉴定为人类细胞中 ATR-ATRIP 复合物的激活剂。我们已经在 Xenopus 卵提取物系统中研究了 ETAA1 的作用，该系统在 ATR-ATRIP 的研究中非常重要。用卵提取物中的 ETAA1 耗尽法处理，并没有明显减少复制应激下 ATR-ATRIP 的激活，这可以通过 ATR 依赖性 Chk1 和 RPA 的磷酸化来监测。此外，缺乏 ETAA1 似乎并不影响未受干扰的 S 期的 DNA 复制。值得注意的是，我们发现 TopBP1 在卵提取物中的丰度明显高于 ETAA1。我们继续表明，如果我们通过添加额外的全长重组 ETAA1 来增加卵提取物中 ETAA1 的浓度，ETAA1 可以支持复制应激下 ATR-ATRIP 的激活。因此，在这个系统中，TopBP1 似乎是复制应激下 ATR-ATRIP 的主要激活剂。我们还探索了 ETAA1 激活 ATR-ATRIP 的生化机制。我们开发了一种体外系统，其中全长重组 ETAA1 在存在定义成分的情况下支持 ATR-ATRIP 的激活。我们发现，ETAA1 与单链 DNA（ssDNA）结合，极大地刺激了它激活 ATR-ATRIP 的能力。因此，RPA 包裹的 ssDNA 作为 ETAA1 介导的 ATR-ATRIP 激活的直接正效应因子。
