Defining a fundamental role for histone methylation in preventing DNA damage-induced replication catastrophe

细胞基因组的复制对生命的延续至关重要，这必须以有效和适时的方式进行，以确保每个子细胞获得正确数量的遗传物质。细胞内DNA损伤的存在可能会减缓或阻碍DNA复制机制，从而阻止基因组复制的及时完成。如果未修复的损伤太大，这可能会导致细胞死亡，或者当细胞分裂时，损伤会转移到子细胞，这可能会导致突变。随着时间的推移，DNA损伤和突变的积累是正常健康细胞转化为肿瘤细胞过程的基本部分。因此，很明显，DNA复制过程中出现的修复损伤问题是人类疾病和癌症的重要原因。为了防止遗传损伤持续存在，细胞进化出了一个高度复杂的细胞路径网络，可以识别和修复DNA损伤。Fanconi贫血(FA)途径是DNA复制过程中最重要的DNA修复途径之一。FA途径是以一种罕见的人类疾病Fanconi贫血命名的，这种疾病与严重的临床症状有关，如生长迟缓、肢体畸形、脑异常、进行性骨髓衰竭导致贫血和癌症易感性增加。到目前为止，人们已经发现，16个不同基因中的任何一个的突变都可以导致FA，这些基因都在同一条途径上工作。对罕见的人类综合征，如FA的研究，极大地增加了我们对DNA修复途径如何保护细胞免受遗传损伤以及这一过程失败时的后果的了解。然而，尽管我们取得了进展，但我们仍然不完全了解这些途径如何发挥作用的一些基本方面，或者实际上是否存在尚待发现的其他因素。事实上，患有FA样疾病的患者尚未被发现致病突变。在这方面，我们已经确定了一种新的蛋白质(PFAP；假定的Fanconi贫血蛋白)，它的功能是帮助修复DNA复制过程中出现的DNA损伤。有趣的是，PFAP缺失导致的缺陷与FA患者细胞中观察到的缺陷惊人地相似，这表明PFAP在FA途径中发挥作用，甚至可能在表现出FA样症状的患者中发生突变。此外，我们还发现PFAP与一种名为HMT-1(组蛋白甲基转移酶1)的酶结合，该酶的主要作用是通过甲基化过程改变DNA周围蛋白质(称为染色质)的结构。值得注意的是，HMT-1的缺失会引起与我们在缺乏PFAP的细胞中观察到的相同的问题，这表明了染色质甲基化和FA修复途径之间的生化联系。虽然我们知道染色质甲基化可以改变细胞对某些类型DNA损伤的反应方式，但它如何影响FA途径尚不清楚。因此，这个应用程序旨在使用三种不同的方法来研究这一点。首先，我们的目标是确定PFAP在FA途径中的作用，并确定PFAP是否在表现出FA样疾病的患者的细胞中发生突变。其次，我们的目标是了解在存在DNA损伤的情况下，PFAP如何控制DNA复制。最后，我们的目标是确定由PFAP/HMT-1进行的染色质甲基化是否有助于复制相关的DNA损伤的修复。拟议中的研究对细胞对DNA损伤的这一基本和高度保守的方面进行研究，有可能对改善人类健康和福祉产生重大的长期影响。除了加深我们对正常细胞如何转化为肿瘤细胞的理解外，它还可能有助于FA样疾病的诊断和/或促进治疗癌症的新化疗药物的开发。

Duplication of the cell's genome is essential for the continuation of life, and this must occur in an efficient and well-timed manner to ensure that each daughter cell gets the correct amount of genetic material. The presence of damage within the cell's DNA can slow or stall the DNA replication machinery, preventing the timely completion of genome duplication. This can result in either cell death, if the unrepaired damage is too great, or transfer of the damage into the daughter cells when the cell divides, which can cause mutations. The accumulation of DNA damage and mutations over time is a fundamental part of the process in which normal healthy cells undergo transformation into tumour cells. It is therefore clear that problems with repairing damage that occurs during DNA replication are a significant cause of human disease and cancer.To prevent genetic damage from persisting, cells have evolved a highly complex network of cellular pathways that recognise and repair DNA damage. One of the most important DNA repair pathways that functions during DNA replication is the Fanconi Anaemia (FA) pathway. The FA pathway is named after the rare human disease, Fanconi Anaemia, which is associated with severe clinical symptoms such as growth retardation, limb malformation, brain abnormalities, progressive bone marrow failure leading to anaemia and an increased predisposition for developing cancer. To date, it has been found that mutations in any one of 16 different genes, all working within the same pathway, can give rise to FA. The study of rare human syndromes, such as FA, has dramatically increased our knowledge about how DNA repair pathways protect the cell from genetic damage, and the consequences when this process fails. However, despite our progress, we still do not fully understand some fundamental aspects of how these pathways function or indeed whether additional factors exist that are yet to be discovered. Indeed, patients suffering from an FA-like disorder have been identified in whom the disease-causing mutation has yet to be discovered.In this respect, we have identified a new protein (PFAP; Putative Fanconi Anaemia Protein) that functions to help repair DNA damage that arises during DNA replication. Interestingly, the defects caused by loss of PFAP are strikingly similar to those observed in cells from FA patients, suggesting that PFAP functions within the FA pathway, and may even be mutated in patients exhibiting FA-like symptoms. In addition, we have discovered that PFAP binds to an enzyme called HMT-1 (Histone Methyltransferase 1), whose primary role is to modify the structure of proteins surrounding DNA (called chromatin) through a process called methylation. Strikingly, loss of HMT-1 gives rise to the same problems as we observed in cells lacking PFAP, indicative of a biochemical link between 'chromatin methylation' and the FA repair pathway.Whilst we know that chromatin methylation can alter the way cells respond to certain types of DNA damage, it is unknown how it affects the FA pathway. Therefore, this application aims to investigate this, using three different approaches. Firstly, we aim to establish where PFAP functions within the FA pathway and ascertain whether PFAP is mutated in cells derived from patients exhibiting an FA-like disorder. Secondly, we aim to understand how PFAP controls DNA replication in the presence of DNA damage. Lastly, we aim to determine if chromatin methylation carried out by PFAP/HMT-1 contributes to how replication-associated DNA damage is repaired.The proposed research on this fundamental and highly conserved aspect of the cellular response to DNA damage has the potential to make a significant long-term impact on improving human health and wellbeing. In addition to furthering our understanding of how normal cells transform into tumour cells, it could also aid the diagnosis of FA-like disorders and/or facilitate the development of new chemotherapeutic drugs to treat cancer.