Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, North Carolina, USA.
Epigenetics Chromatin. 2009 May 14;2(1):6. doi: 10.1186/1756-8935-2-6.
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.
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.
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.
GINS 复合物被认为对于 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）被提议与染色质和与 DNA 复制相关的蛋白质结合。迄今为止，还没有进行微观分析来评估该复合物在染色质上的分布。在这里，我们展示了 GINS 复合物在延伸的染色质纤维上的组织方式，与 DNA 复制和与复制相关的蛋白质的位置有关。
我们使用免疫荧光显微镜能够可视化 ORC1、ORC2、PCNA 和 GINS 复合物蛋白 Psf1 和 Psf2 结合到延伸的染色质纤维上。我们还能够同时检测到这些蛋白质，以及最近复制的 DNA 的轨迹，其中 EdU（胸腺嘧啶类似物）被掺入。这使我们能够评估感兴趣的蛋白质与 DNA 复制过程相关的染色质关联。在能够检测到复制之前，ORC 和 GINS 蛋白就已经存在于染色质纤维上。这些蛋白质也与珠状结构中的新复制 DNA 相关联。此外，GINS 蛋白与 PCNA 在活跃复制的位点共定位。
与它在 DNA 复制起始中的预期作用一致，GINS 蛋白与 ORC 结合位点附近的染色质相关联，而这些位点没有 EdU（没有 DNA 复制）。GINS 蛋白与 PCNA 的关联与延伸过程中的作用一致。此外，我们的染色质纤维的大尺寸（长达约 7 Mb）允许比以前报道的更广泛地分析活跃复制子之间的距离。
