Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Biol Chem. 2010 Feb 19;285(8):5974-82. doi: 10.1074/jbc.M109.076109. Epub 2009 Dec 22.
At clinically relevant doses, chemotherapeutic S(N)1 DNA methylating agents induce an ATR-mediated checkpoint response in human cells that is dependent on functional MutSalpha and MutLalpha. Deficiency of either mismatch repair activity renders cells highly resistant to this class of drug, but the mechanisms linking mismatch repair to checkpoint activation have remained elusive. In this study we have systematically examined the interactions of human MutSalpha and MutLalpha with proteins of the ATR-Chk1 pathway using both nuclear extracts and purified proteins. Using nuclear co-immunoprecipitation, we have detected interaction of MutSalpha with ATR, TopBP1, Claspin, and Chk1 and interaction of MutLalpha with TopBP1 and Claspin. We were unable to detect interaction of MutSalpha or MutLalpha with Rad17, Rad9, or replication protein A in the extract system. Use of purified proteins confirmed direct interaction of MutSalpha with ATR, TopBP1, and Chk1 and of MutLalpha with TopBP1. MutSalpha-Claspin and MutLalpha-Claspin interactions were not demonstrable with purified proteins, suggesting that extract interactions are indirect or depend on post-translational modification. Use of a modified chromatin immunoprecipitation assay showed that proliferating cell nuclear antigen, ATR, TopBP1, and Chk1 are recruited to chromatin in a MutLalpha- and MutSalpha-dependent fashion after N-methyl-N'-nitro-N-nitrosoguanidine treatment. However, chromatin enrichment of replication protein A, Claspin, Rad17-RFC, and Rad9-Rad1-Hus1 was not detected in these experiments. Although our failure to observe enrichment of the latter activities could be due to sensitivity limitations, these observations may indicate a novel mechanism for ATR activation.
在临床相关剂量下，化疗 S(N)1 DNA 甲基化试剂在人类细胞中诱导 ATR 介导的检查点反应，该反应依赖于功能性 MutSalpha 和 MutLalpha。这两种错配修复活性的缺乏使细胞对这类药物具有高度抗性，但将错配修复与检查点激活联系起来的机制仍然难以捉摸。在这项研究中，我们使用核提取物和纯化蛋白系统地研究了人类 MutSalpha 和 MutLalpha 与 ATR-Chk1 途径蛋白的相互作用。通过核共免疫沉淀，我们检测到 MutSalpha 与 ATR、TopBP1、Claspin 和 Chk1 的相互作用，以及 MutLalpha 与 TopBP1 和 Claspin 的相互作用。我们无法在提取物系统中检测到 MutSalpha 或 MutLalpha 与 Rad17、Rad9 或复制蛋白 A 的相互作用。使用纯化蛋白证实了 MutSalpha 与 ATR、TopBP1 和 Chk1 的直接相互作用，以及 MutLalpha 与 TopBP1 的直接相互作用。纯化蛋白未能证明 MutSalpha-Claspin 和 MutLalpha-Claspin 的相互作用，这表明提取物相互作用是间接的或依赖于翻译后修饰。使用改良的染色质免疫沉淀测定法显示，在 N-甲基-N'-硝基-N-亚硝基胍处理后，增殖细胞核抗原、ATR、TopBP1 和 Chk1 以 MutLalpha 和 MutSalpha 依赖的方式募集到染色质上。然而，在这些实验中未检测到复制蛋白 A、Claspin、Rad17-RFC 和 Rad9-Rad1-Hus1 的染色质富集。尽管我们未能观察到后一种活性的富集可能是由于灵敏度限制，但这些观察结果可能表明 ATR 激活的一种新机制。
