Investigating how replication fork rotation causes chromosomal instability during S phase

DNA是我们细胞的信息存储材料。它由两种很长的缠绕在一起的聚合物组成，每一种聚合物都由四个不同的核苷酸组成。这些核苷酸的序列一起编码了整个蓝图，或功能细胞的遗传密码。每次细胞分裂时，为了确保蓝图得以维持，DNA聚合物必须相互解开并精确地复制。这一非凡的壮举是由一组酶实现的，这些酶统称为DNA复制机制。据估计，DNA复制机制通常很少出错。然而，这种保真度在癌细胞和诱发过早衰老的人类疾病中降低了。在这些细胞中，核苷酸序列经常改变，染色体经常断裂并重新连接。然而，破损的部位并不是随机的。相反，破损通常发生在不同的区域，通常被称为“脆弱部位”。在脆弱的位点，染色体被认为特别难以分离和复制，从而导致错误和断裂。这些错误可以通过突变基因组而不可逆转地改变细胞的行为。这会导致细胞衰老，导致衰老或促进癌症的发展。偶尔发现DNA复制缺陷与罕见的人类发育障碍（如小头畸形）有关。因此，DNA复制过程中的错误可能具有广泛和特定的影响。为什么会这样是未知的，然而似乎有可能是复制过程中不同的问题对脆弱位点的影响不同，导致了不同的结果。我们理解为什么在脆弱的位点上复制似乎容易出错，这在很大程度上得益于对相对简单的真核细胞的研究，比如酵母，它以一种与人类细胞非常相似的方式复制DNA。这些研究表明，复制机制与DNA上的其他过程发生碰撞的基因组位点往往是“脆弱的”，它们周围似乎发生了更多的DNA损伤。这些研究促成了这样一种观点，即当复制机制遇到其他过程时，错误率会急剧增加。然而，这是如何发生的尚不清楚。在我们最近提交的工作中，我们发现了一种对脆弱地点发生的错误和损害的全新解释。我们发现，解开DNA的问题可能导致复制DNA的“编织”。这导致在复制未缠绕的DNA链时出现问题，在新复制的DNA中造成DNA损伤。该途径引起的损伤与通过候选酵母脆弱位点的复制密切相关。在本提案中，我们希望扩展这一分析，以确定酵母和人类的脆弱位点，其中这种新的途径DNA复制相关的DNA损伤是起作用的。然后，我们将评估可能由该途径引起的突变类型，并将这些突变与DNA复制可能诱导的不同细胞问题联系起来。为了做到这一点，我们将使用可以在整个基因组中量化DNA损伤的技术，并评估何时DNA损伤是在特定条件下引起的，这种条件下通过DNA编织放大了新途径造成的损伤。然后，我们将使用这些数据仔细描述通过新途径导致染色体脆弱的条件。在许多方面，我们一生中发生的多轮DNA复制是我们身体中衰老细胞和配子中“不老细胞”之间的关键区别。因此，了解DNA复制在哪里以及为什么改变了我们的遗传密码，改变了细胞功能，是理解衰老和潜在地抵消衰老的生物学方面的关键一步，这是现代社会最棘手的问题。

DNA is the information storage material of our cells. It is composed of two very long intertwined polymers, each made up of four distinct nucleotides. The sequence of these nucleotides together encodes the overall blueprint, or genetic code of the functioning cell. To ensure the blueprint is maintained every time a cell divides the DNA polymers have to untangled from one another and exactly duplicated. This remarkable feat is achieved by a collection of enzymes collectively known as the DNA replication machinery. It is estimated that the DNA replication machinery normally hardly ever makes a mistake. However, this fidelity is diminished in cancer cells and human diseases that induce premature aging. In these cells the nucleotide sequence often changes and the chromosomes are frequently broken and rejoined. However, the sites of breakage are not random. Instead, breakage often occurs in distinct areas commonly termed "fragile sites". At fragile sites it is thought that the chromosomes are especially difficult to separate and copy, leading to a errors and breakage. These errors can irreversibly change the behavior of cells by mutating the genome. This can cause cells to senesce, causing ageing or promote the development of cancer. Occasionally defects in DNA replication are found to be associated with rare human developmental disorders such as microcephaly. Therefore errors during DNA replication can have both widespread and specific effects. Why this is so is unknown, however it seems likely that distinct problems during replication affect fragile sites differently, leading to variable outcomes.Our understanding of why replication appears to be error prone at fragile sites has been greatly aided through studies of relatively simple eukaryotic cells, such as yeast that replicate DNA in a very similar fashion to human cells. These have shown that genomic sites where the replication machinery collides with other processes working on DNA are often "fragile" with increased DNA damage appearing to occur around them. These studies have contributed to the idea that when the replication machinery encounters other processes, the error rate dramatically increases. However, how this occurs is unknown.In our recently submitted work we have found a wholly novel explanation for errors and damage occurring at fragile sites. We have found that problems in untangling the DNA can lead to "braiding" of the replicating DNA. This leads to problems in duplicating the unwound strands, causing DNA damage in the newly replicated DNA. Damage caused by this pathway is closely linked to replication through candidate yeast fragile sites. In this proposal we wish to extend this analysis to define the yeast and human fragile sites where this novel pathway to DNA replication associated DNA damage is acting. We will then assess the types of mutations that are likely to be caused by this pathway and link these to the different cellular problems that DNA replication can induce. To do this we will use techniques where DNA damage can be quantified across an entire genome and assess when DNA damage is caused specifically under conditions that amplify the damage caused by the novel pathway through braiding of the DNA. We will then use this data to carefully describe the conditions that lead to chromosome fragility through the novel pathway.In many ways the multiple rounds of DNA replication that occur over our lifetime are the crucial difference between the ageing cells in our bodies and the "ageless cells" of our gametes. Therefore understanding where and why DNA replication changes our genetic code, changing cellular function, is a crucial step to understanding ageing and potentially counteracting the biological aspects of ageing most problematic for modern society.