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首页> 外文期刊>Epigenetics & Chromatin >DNA replication and the GINS complex: localization on extended chromatin fibers
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DNA replication and the GINS complex: localization on extended chromatin fibers

机译：DNA复制和GINS复合物：定位在延伸的染色质纤维上

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Background The GINS complex is thought to be essential for the processes of initiation and elongation of DNA replication. This complex contains four subunits, one of which (Psf1) is proposed to bind to both chromatin and DNA replication-associated proteins. To date there have been no microscopic analyses to evaluate the chromatin distribution of this complex. Here, we show the organization of GINS complexes on extended chromatin fibers in relation to sites of DNA replication and replication-associated proteins. Results Using immunofluorescence microscopy we were able to visualize ORC1, ORC2, PCNA, and GINS complex proteins Psf1 and Psf2 bound to extended chromatin fibers. We were also able to detect these proteins concurrently with the visualization of tracks of recently replicated DNA where EdU, a thymidine analog, was incorporated. This allowed us to assess the chromatin association of proteins of interest in relation to the process of DNA replication. ORC and GINS proteins were found on chromatin fibers before replication could be detected. These proteins were also associated with newly replicated DNA in bead-like structures. Additionally, GINS proteins co-localized with PCNA at sites of active replication. Conclusion In agreement with its proposed role in the initiation of DNA replication, GINS proteins associated with chromatin near sites of ORC binding that were devoid of EdU (absence of DNA replication). The association of GINS proteins with PCNA was consistent with a role in the process of elongation. Additionally, the large size of our chromatin fibers (up to approximately 7 Mb) allowed for a more expansive analysis of the distance between active replicons than previously reported.
机译：背景技术GINS复合物被认为对DNA复制的起始和延伸过程至关重要。该复合物包含四个亚基，其中一个（Psf1）被提议与染色质和DNA复制相关蛋白结合。迄今为止，还没有显微镜分析来评估该复合物的染色质分布。在这里，我们显示了与DNA复制和复制相关蛋白的位点有关的扩展染色质纤维上的GINS复合物的组织。结果使用免疫荧光显微镜，我们能够观察到与延伸的染色质纤维结合的ORC1，ORC2，PCNA和GINS复合蛋白Psf1和Psf2。我们还能够在可视化最近复制的DNA的痕迹的同时检测这些蛋白质，其中结合了胸腺嘧啶核苷类似物EdU。这使我们能够评估与DNA复制过程有关的目的蛋白的染色质缔合。在可以检测到复制之前，在染色质纤维上发现了ORC和GINS蛋白。这些蛋白质也与新复制的DNA呈珠状结构相关。另外，GINS蛋白与PCNA共定位在活性复制位点。结论与提议的在DNA复制中的作用一致，与INC结合位点附近的染色质相关的GINS蛋白不含EdU（DNA复制缺失）。 GINS蛋白与PCNA的缔合与延伸过程中的作用一致。此外，我们染色质纤维的大尺寸（最高约7 Mb）使得对活性复制子之间距离的分析比以前报道的更为广泛。

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