What regulates replication origin activation?

所有细胞都含有生物体DNA的完整拷贝，这是生命的遗传蓝图，被包装成称为染色体的离散单元。由于新细胞需要遗传物质的拷贝，因此在细胞分裂之前，染色体必须被完整而准确地复制。真核生物，如酵母和人类，拥有巨大的基因组，包含数百万个碱基编码遗传信息。为了确保这些基因组在允许的时间内完成复制，DNA复制过程从每条染色体的多个位点开始，称为复制起点。这些复制起点是专门的DNA序列，它们组装细胞机器，然后沿着DNA读取和复制遗传物质移动。细胞激活足够的复制起始点以确保染色体的完整复制是至关重要的。染色体复制失控可能导致基因组不稳定，这凸显了控制复制起始激活的重要性。尽管DNA复制起源很重要，但我们对指定和控制它们的DNA序列知之甚少。DNA复制过程的失败导致遗传不稳定和癌症和先天性疾病等疾病。我希望对确保遗传完整性的基本生物学的更好理解将提供新的见解，从而改进这些疾病的诊断和治疗。除了DNA复制，遗传物质也被读取，然后被翻译成蛋白质。这个过程的第一步叫做转录。我最近发现，转录对复制起始功能是有害的，因此可能在决定哪些DNA序列可以作为起始功能方面发挥关键作用。该项目旨在了解细胞如何协调读取遗传信息的两个关键过程，DNA复制和DNA转录，以确保基因组的稳定性。我将研究出芽酵母和裂变酵母，因为它们的基因组被很好地理解，并且很容易修改以提出实验问题，重要的是对DNA复制的控制与人类细胞相似。此外，我已经精确地确定了超过一半的出芽酵母复制起点的位置，提供了一个大型数据集来帮助我了解复制起点的属性。通过与领先的裂变酵母实验室合作，我将确定该物种复制起源的位置。这将首次允许对两种生物体之间的复制起源特征进行全基因组比较，以确定哪些特性是共享的，因此可能具有重要的功能。我将直接讨论转录是如何影响复制的以及细胞使用什么分子机制来保护复制，特别是复制起点，不受转录的影响。这些实验不仅能让我了解细胞如何协调复制和转录，还能让我了解是什么决定了分子水平上的复制起源行为。利用这些结果，我将建立一个基于计算机的染色体复制过程模型，并通过将计算机预测与实验结果进行比较来测试该模型。预测和观察之间的差异将突出我们对DNA复制的理解的局限性，为进一步的实验指明重要的方向。这项工作将揭示DNA复制起源是如何被指定的，以及它们的行为是如何被调节的。通过在分子水平上理解控制整个基因组复制起源的过程，我将能够模拟整个染色体是如何复制的。这个模型将使我能够预测染色体复制过程中的弱点，这可能是癌症等遗传疾病的基础。

All cells contain a complete copy of the organism's DNA, the genetic blue print of life, packaged into discrete units called chromosomes. Since new cells need a copy of the genetic material, the chromosomes must be completely and accurately replicated before the cell can divide. Eukaryotes, such as yeast and humans, have large genomes with millions of bases encoding the genetic information. To ensure complete replication of these genomes within the allowed time, the process of DNA replication starts at multiple sites along each chromosome, called replication origins. These replication origins are specialised DNA sequences that assemble the cellular machinery that then moves along the DNA reading and copying the genetic material. It is essential that the cell activates sufficient replication origins to ensure complete replication of the chromosomes. The importance of controlling replication origin activation is highlighted by the genome instability that may result from uncontrolled chromosome replication. Despite the importance of DNA replication origins we understand little about the DNA sequences that specify and control them. Failures in the processes of DNA replication lead to genetic instability and diseases such as cancer and congenital disorders. I hope that a better understanding of the basic biology that ensures genetic integrity will give new insights that will allow improved diagnosis and treatment of these diseases. In addition to DNA replication, the genetic material is also read and then translated to make proteins. The initial step in this process is called transcription. I have recently found that transcription is detrimental to replication origin function and may therefore play a key role in determining which DNA sequences can function as origins. This project aims to understand how the cell coordinates the two key processes that read the genetic information, DNA replication and DNA transcription, to ensure genomic stability. I will work with budding and fission yeasts, because their genomes are well understood and easily modified to ask experimental questions, and importantly the controls over DNA replication are similar to those in human cells. Furthermore, I have already precisely identified the location of more than half of the budding yeast replication origins providing a large dataset to help me understand the properties of replication origins. By collaborating with leading fission yeast laboratories I will identify the location of replication origins in this species. This will allow, for the first time, genome-wide comparisons of replication origin characteristics between two organisms to determine which properties are shared and therefore likely to be of functional importance. I will go on to look directly at how replication is affected by transcription and what molecular mechanisms are used by the cell to protect replication, and specifically replication origins, from transcription. These experiments will not only allow me to understand how the cell coordinates replication and transcription, but will also give an understanding of what determines replication origin behaviour at the molecular level. Using these results, I will build a computer-based model of the processes of chromosome replication and test the model by comparing the computer predictions with experimental results. Differences between prediction and observation will highlight the limitations in our understanding of DNA replication, indicating important directions for further experiments. This work will uncover how DNA replication origins are specified and how their behaviour is regulated. By understanding, at the molecular level, the processes that control replication origins throughout the genome I will be able to model how whole chromosomes are replicated. This model will allow me to predict weaknesses in the chromosome replication process that may underlie genetic diseases such as cancer.