Conservation of chromatin recruitment mechanisms in metazoan DNA replication licensing factors

生命需要通过复制基因组DNA将遗传信息从父母忠实地传递给后代。所有动物都必须复制它们的DNA，然而复制所需的蛋白质在不同动物之间有很大的不同。这个项目将通过研究进化上古老的和更现代的动物的DNA复制过程，来研究整个动物界DNA复制的异同。这些研究将从海绵延伸到人类，并应该澄清DNA复制是如何启动的，以及进化如何在保持复制蛋白质功能的同时导致复制蛋白质的差异。该项目还将为社区大学生提供暑期研究机会，否则他们将没有机会在R1机构的学术实验室接受培训。这一更广泛的影响计划专门旨在增加少数族裔对科学的参与，并鼓励学生过渡到四年制STEM学位和博士课程。DNA复制的第一步是将复制的解旋酶Mcm2-7装载到染色质上。这个许可过程需要三种蛋白质：orc1-6、cdc6和cdt1。Fly ORC1具有一个内在无序区域(IDR)，该区域是体内染色质募集所必需的和充分的，并在体外介导DNA依赖的相分离。这些活性受磷酸化的负调控。有趣的是，虽然所有的后生动物ORC1直系物都有IDR，但这些区域的长度高度可变，缺乏线性序列相似性，这引发了功能保守的问题。ORC1IDR同源基因确实有相似的氨基酸组成，从而导致假设序列组成可能定义这个IDR类的功能。这一假设将通过研究不同ORC1IDR同源物的体外DNA结合和体内染色质募集活性的研究来验证。这一结果预计将促进对复制许可机制的保守的理解，并将通过发展组成同源性的概念来广泛地为内在无序蛋白质的研究提供信息，这可能适用于许多IDR类别。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Life requires the faithful transmission of genetic information from parents to offspring through replication of genomic DNA. All animals must replicate their DNA, yet the proteins required for replication differ substantially between animals. This project will investigate similarities and differences in DNA replication across the animal kingdom by studying the process in evolutionarily ancient and more modern animals. The studies will extend from sea sponge to humans and should clarify how DNA replication is initiated and how evolution might result in differences in replication proteins while still maintaining their function. The project will also provide summer research opportunities to community college students who would not otherwise have the opportunity to train in an academic laboratory at an R1 institution. This broader impacts program is specifically aimed at increasing minority participation in science and encouraging student transition to four-year STEM degree and Ph.D. programs. The first step in DNA replication is loading of the replicative helicase, Mcm2-7 onto chromatin. This licensing process requires three proteins: Orc1-6, Cdc6, and Cdt1. Fly Orc1 possesses an intrinsically disordered region (IDR) that is necessary and sufficient for chromatin recruitment in vivo and mediates DNA-dependent phase separation in vitro. These activities are negatively regulated by phosphorylation. Interestingly, while all metazoan Orc1 orthologs possess IDRs, these regions are highly variable in length and lack linear sequence similarity, raising questions about functional conservation. Orc1 IDR orthologs do share similar amino acid composition, leading to the hypothesis that sequence composition may define the functions of this IDR class. This hypothesis will be tested by studies that investigate in vitro DNA binding and in vivo chromatin recruitment activity of different Orc1 IDR orthologs. The outcomes are expected to advance understanding of the conservation of replication licensing mechanisms and will also broadly inform the study of intrinsically disordered proteins by developing the concept of compositional homology, which may be applicable to many IDR classes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.