Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Pittsburgh, PA 15219, USA.
Nucleic Acids Res. 2010 Jul;38(12):3984-98. doi: 10.1093/nar/gkq144. Epub 2010 Mar 9.
WRN protein loss causes Werner syndrome (WS), which is characterized by premature aging as well as genomic and telomeric instability. WRN prevents telomere loss, but the telomeric protein complex must regulate WRN activities to prevent aberrant telomere processing. Telomere-binding TRF2 protein inhibits telomere t-loop deletion by blocking Holliday junction (HJ) resolvase cleavage activity, but whether TRF2 also modulates HJ displacement at t-loops is unknown. In this study, we used multiplex fluorophore imaging to track the fate of individual strands of HJ substrates. We report the novel finding that TRF2 inhibits WRN helicase strand displacement of HJs with telomeric repeats in duplex arms, but unwinding of HJs with a telomeric center or lacking telomeric sequence is unaffected. These data, together with results using TRF2 fragments and TRF2 HJ binding assays, indicate that both the TRF2 B- and Myb domains are required to inhibit WRN HJ activity. We propose a novel model whereby simultaneous binding of the TRF2 B-domain to the HJ core and the Myb domain to telomeric arms promote and stabilize HJs in a stacked arm conformation that is unfavorable for unwinding. Our biochemical study provides a mechanistic basis for the cellular findings that TRF2 regulates WRN activity at telomeres.
WRN 蛋白缺失会导致 Werner 综合征（WS），其特征是早衰以及基因组和端粒不稳定。WRN 可防止端粒丢失，但端粒蛋白复合物必须调节 WRN 的活性，以防止异常的端粒加工。端粒结合蛋白 TRF2 通过阻断 Holliday 结（HJ）解旋酶切割活性来抑制端粒 t 环缺失，但 TRF2 是否也调节 t 环上的 HJ 位移尚不清楚。在这项研究中，我们使用多重荧光成像来跟踪 HJ 底物的单链命运。我们报告了一个新的发现，即 TRF2 抑制带有双链臂端粒重复的 HJ 中 WRN 解旋酶的链移位，但带有端粒中心或缺乏端粒序列的 HJ 解旋不受影响。这些数据，以及使用 TRF2 片段和 TRF2 HJ 结合测定的结果表明，TRF2 的 B 结构域和 Myb 结构域都需要抑制 WRN HJ 活性。我们提出了一个新的模型，即 TRF2 B 结构域与 HJ 核心的同时结合以及 Myb 结构域与端粒臂的同时结合促进和稳定了 HJ 处于不利解旋的堆叠臂构象。我们的生化研究为 TRF2 在端粒上调节 WRN 活性的细胞发现提供了机制基础。
