Regulation of DNA Replication Timing by Origin Recognition Complex (ORC) Phosphorylation

项目摘要 基因组的成功复制需要数十亿个碱基的精确复制。不这样做 导致基因组不稳定并最终导致许多疾病。为了减轻这一点，DNA复制是一种 然而，这种调节也必须是灵活的，因为DNA复制是 通过发展和分化而发生变化。我们仍然不知道 DNA复制是受调控的，以及这种调控如何在发育过程中发生变化。一个这样的监管过程 还有很多有待发现的地方是复制定时程序，它控制某些段何时 在S期进行复制。复制时间（RT）是一个高度保守的现象，发现 从酵母菌到人类，控制复制时间的精确机制仍然存在， 未知RT还与某些肿瘤的突变率和耐药性的发展有关。 癌的Rif 1是RT的一个保守的活性调节因子，它提供了解开潜在分子机制的钥匙 Rif 1被认为延迟了复制解旋酶的激活，这些解旋酶位于逆转录酶的某些部分。 基因组，导致这些部分在S期后期复制。Rif 1如何靶向这些基因组区域， 延迟复制的原因尚不清楚。我们试图确定Rif 1是如何控制RT的， Rif 1的免疫沉淀，然后进行高分辨率质谱分析。我们发现Rif 1协同 免疫沉淀的起源识别复合物（ORC）。ORC是第一个与DNA结合的复合物， DNA复制的起始和控制基因组中DNA复制的起始位置。在这一提议中， 我们试图描述Rif 1和ORC之间的相互作用。为了实现这一目标，我们将确定分子 这种相互作用的相关性，表征Rif 1和ORC在细胞周期中与基因组结合的位置，以及 使用体外方法确定Rif 1/ORC相互作用是否直接。这项工作的发现可能 为Rif 1如何控制RT提供了一个新的机制范例，这可能适用于其他物种， Rif 1和ORC在功能上是保守的。这项工作还将提供对驱动复制的监管的深入了解 定时和DNA复制，这两者对于维持基因组的稳定性和完整性至关重要。此外，委员会认为， 这项建议将提供机会，学习生物信息学和生物化学技能，广泛适用于职业生涯 在科学方面，加强批判性思维和解决问题的能力，并通过以下方式提高科学沟通能力 正式介绍这项工作和全国会议和大学会议。我会和我的导师一起工作， ASPIRE计划通过职业发展办公室在范德比尔特办公室，以确定职业发展 机会最后，我将继续通过培训范德比尔特的本科生来发展导师技能， 寻求数据分析方面的培训机会，以便我成为健康数据科学领域的成功领导者。

PROJECT SUMMARY Successful duplication of the genome requires the accurate replication of billions of bases. Failure to do so results in genomic instability and ultimately a host of diseases. To mitigate against this, DNA replication is a coordinated and tightly regulated process; however, this regulation must also be flexible as DNA replication is subject to change through development and differentiation. Much still remains unknown regarding exactly how DNA replication is regulated and how this regulation changes through development. One such regulatory process where much remains to be discovered is the replication timing program, which controls when certain segments of the genome are replicated during S phase. Replication timing (RT) is a highly conserved phenomenon, found in species ranging from yeast to humans, yet the precise mechanisms that govern replication timing remains unknown. RT has also been implicated in mutation rates and the development of drug resistance in certain cancers. Rif1, a conserved active regulator of RT provides a key to unlock the underlying molecular mechanisms controlling RT. Rif1 is thought to delay the activation of replicative helicases that lie within certain sections of the genome, causing these sections to replicate later in S phase. How Rif1 is targeted to these genomic regions to cause delayed replication remains unclear. We sought to determine how Rif1 controls RT by carrying out immunoprecipitations of Rif1 followed by high-resolution mass spectrometry. We discovered that Rif1 co- immunoprecipated with the origin recognition complex (ORC). ORC is the first complex to bind to DNA during the initiation of DNA replication and controls where across the genome DNA replication will start. In this proposal, we seek to characterize the interaction between Rif1 and ORC. To achieve this, we will determine the molecular relevance of this interaction, characterize where Rif1 and ORC bind to the genome through the cell cycle, and use in vitro approaches to determine if the Rif1/ORC interaction is direct. The findings from this work could provide a new mechanism paradigm for how Rif1 controls RT, which will likely be applicable to other species as Rif1 and ORC are functionally conserved. This work will also provide insight into the regulation driving replication timing and DNA replication, both of which critical for maintaining genomic stability and integrity. Furthermore, this proposal will provide the opportunity to learn bioinformatic and biochemical skills widely applicable to careers in science, enhance critical thinking and problem-solving skills, and improve scientific communication skills by formally presenting this work and national conferences and university meetings. I will work with my mentors and the ASPIRE program through the office of career development office at Vanderbilt to identify career development opportunities. Lastly, I will continue to develop mentorship skills by training undergraduates at Vanderbilt and seek opportunities for training in data analytics so that I may become a successful leader in health data science.