Investigating how replication fork rotation causes chromosomal instability during S phase

DNA是我们细胞的信息存储材料。它由两个很长的缠绕在一起的聚合物组成，每个聚合物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。为了确保每次细胞分裂时都能保持蓝图，DNA聚合物必须彼此解开并精确复制。这一非凡的壮举是通过一系列统称为DNA复制机制的酶来实现的。据估计，DNA复制机制通常几乎不会出错。然而，在癌细胞和导致过早衰老的人类疾病中，这种保真度会降低。在这些细胞中，核苷酸序列经常改变，染色体经常断裂和重新连接。然而，破损的位置并不是随机的。相反，破损通常发生在通常被称为“脆弱部位”的不同区域。在脆弱的部位，人们认为染色体特别难分离和复制，导致错误和断裂。这些错误可以通过突变基因组来不可逆转地改变细胞的行为。这可能会导致细胞衰老，导致衰老或促进癌症的发展。偶尔，DNA复制缺陷被发现与罕见的人类发育障碍有关，如小头畸形。因此，DNA复制过程中的错误可能会产生广泛和特定的影响。原因尚不清楚，然而，复制过程中的不同问题似乎可能以不同的方式影响脆弱部位，导致不同的结果。通过对相对简单的真核细胞的研究，我们对为什么脆弱部位的复制似乎容易出错的理解得到了很大的帮助，例如以与人类细胞非常相似的方式复制DNA的酵母。这些研究表明，复制机制与处理DNA的其他过程发生碰撞的基因组位置往往是“脆弱的”，DNA损伤似乎发生在它们周围。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，这是如何发生的还不清楚。在我们最近提交的工作中，我们发现了一种全新的解释，可以解释发生在脆弱部位的错误和损坏。我们已经发现，解开DNA的问题可能会导致复制的DNA“辫子”。这导致在复制解开的链时出现问题，导致新复制的DNA中的DNA损伤。这一途径造成的损伤与候选酵母脆性部位的复制密切相关。在这项提案中，我们希望将这一分析扩展到定义酵母和人类的脆弱部位，在这些部位，这种与DNA复制相关的DNA损伤的新途径正在发挥作用。然后，我们将评估这一途径可能引起的突变类型，并将这些突变与DNA复制可能引发的不同细胞问题联系起来。为了做到这一点，我们将使用可以量化整个基因组的DNA损伤的技术，并评估DNA损伤是在通过DNA编织放大新途径造成的损伤的条件下具体造成的。然后，我们将使用这些数据来仔细描述通过新的途径导致染色体脆弱性的条件。在许多方面，在我们一生中发生的多轮DNA复制是我们身体中老化的细胞和我们配子中不衰老的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我们的遗传密码，改变细胞功能，是理解衰老以及潜在地对抗衰老最具现代社会问题的生物学方面的关键一步。

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.