Janoušková Eliška, Nečasová Ivona, Pavloušková Jana, Zimmermann Michal, Hluchý Milan, Marini Victoria, Nováková Monika, Hofr Ctirad
Chromatin Molecular Complexes, CEITEC and Laboratory of Functional Genomics and Proteomics, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Brno CZ-62500, Czech Republic.
Department of Biology, Faculty of Medicine, Masaryk University, Brno CZ-62500, Czech Republic.
Nucleic Acids Res. 2015 Mar 11;43(5):2691-700. doi: 10.1093/nar/gkv097. Epub 2015 Feb 11.
More than two decades of genetic research have identified and assigned main biological functions of shelterin proteins that safeguard telomeres. However, a molecular mechanism of how each protein subunit contributes to the protecting function of the whole shelterin complex remains elusive. Human Repressor activator protein 1 (Rap1) forms a multifunctional complex with Telomeric Repeat binding Factor 2 (TRF2). Rap1-TRF2 complex is a critical part of shelterin as it suppresses homology-directed repair in Ku 70/80 heterodimer absence. To understand how Rap1 affects key functions of TRF2, we investigated full-length Rap1 binding to TRF2 and Rap1-TRF2 complex interactions with double-stranded DNA by quantitative biochemical approaches. We observed that Rap1 reduces the overall DNA duplex binding affinity of TRF2 but increases the selectivity of TRF2 to telomeric DNA. Additionally, we observed that Rap1 induces a partial release of TRF2 from DNA duplex. The improved TRF2 selectivity to telomeric DNA is caused by less pronounced electrostatic attractions between TRF2 and DNA in Rap1 presence. Thus, Rap1 prompts more accurate and selective TRF2 recognition of telomeric DNA and TRF2 localization on single/double-strand DNA junctions. These quantitative functional studies contribute to the understanding of the selective recognition of telomeric DNA by the whole shelterin complex.
二十多年的基因研究已经确定并赋予了保护端粒的端粒保护蛋白的主要生物学功能。然而，每个蛋白质亚基如何对整个端粒保护蛋白复合体的保护功能做出贡献的分子机制仍然难以捉摸。人类阻遏激活蛋白1（Rap1）与端粒重复序列结合因子2（TRF2）形成多功能复合体。Rap1-TRF2复合体是端粒保护蛋白的关键部分，因为在缺乏Ku 70/80异二聚体的情况下，它能抑制同源定向修复。为了了解Rap1如何影响TRF2的关键功能，我们通过定量生化方法研究了全长Rap1与TRF2的结合以及Rap1-TRF2复合体与双链DNA的相互作用。我们观察到，Rap1降低了TRF2对DNA双链的整体结合亲和力，但增加了TRF2对端粒DNA的选择性。此外，我们还观察到Rap1诱导TRF2从DNA双链中部分释放。在有Rap1存在的情况下，TRF2与DNA之间不太明显的静电吸引力导致了TRF2对端粒DNA的选择性提高。因此，Rap1促使TRF2更准确、更有选择性地识别端粒DNA，并使TRF2定位在单/双链DNA交界处。这些定量功能研究有助于理解整个端粒保护蛋白复合体对端粒DNA的选择性识别。
