Replication repair in real life: analysing how broken DNA replication machines are rebuilt inside cells.

DNA编码了为所有生命提供基础的基因蓝图。这种蓝图的任何破坏都可能对有机体造成非常有害的后果，因为它会导致突变，从而改变细胞的功能，甚至导致死亡。在人类中，这种突变可能会导致遗传病、癌症，可能还会导致老年的衰弱效应。细胞每次分裂时都必须复制其DNA，以便每个子细胞都能收到一套准确、完整的遗传指令，但不幸的是，这种复制过程也有可能破坏蓝图。这种腐败的发生是因为复制DNA的纳米机器在移动过程中不断遇到不可避免的障碍。这些障碍中的许多都可以清除或绕过，让最初的纳米机器继续前进。然而，其中一些障碍确实会导致纳米机器的故障。这些故障可能是灾难性的，因为它们可能导致部分遗传密码在细胞分裂之前没有被复制或被错误复制，导致代码损坏。这些故障及其相关风险是所有生物体都必须面对的问题。因此，进化出了将复制的纳米机器重新组装到DNA上的机制，允许复制重新开始，从而降低了错误引入遗传密码的风险。这些复制的纳米机器被重新加载到染色体上的方式在所有有机体中似乎都是相似的，这反映了遗传物质和复制这种材料的机器的保守性。然而，只有在细菌中，负责将这些纳米机器重新加载到DNA上的酶才被识别出来。细菌大肠杆菌提供了大量关于这种重新加载如何发生的信息，并提供了一个模型，用来了解这种修复是如何在所有生物体中发生的。然而，尽管经过了20年的研究，我们仍然对这种修复实际上是如何在细胞内发生的知之甚少。这种无知的部分原因是细胞内遇到的许多不同类型的障碍可能导致这些纳米机器的崩溃，以及这种纳米机器修复的复杂、重叠的性质。为了解决我们在细胞内如何实现DNA复制的理解中的这一差距，我们将使用最近开发的显微镜技术，使活细胞内的单分子得以可视化和跟踪。我们将利用细菌大肠杆菌中可用的信息来监控细胞内发生的纳米机器修复。这些信息将使我们能够生成一个模型，说明在这个简单的系统中，复制机器的重建如何支撑基因蓝图的复制，从而深入了解这种基本修复是如何在像我们这样的更复杂的生物体内发生的。

DNA encodes the genetic blueprint that provides the basis for all life. Any corruption of this blueprint can have very harmful consequences for the organism since it leads to mutations that can alter how cells function or even lead to death. In humans such mutations can lead to genetic diseases, cancer and possibly the debilitating effects of old age. Every time a cell divides it must copy its DNA so that each daughter cell receives an accurate, complete set of genetic instructions but unfortunately this copying process also has the potential to corrupt the blueprint. This corruption can occur because the nanomachines that duplicate DNA continually encounter unavoidable obstacles to their movement. Many of these obstacles can be cleared or bypassed, allowing the original nanomachine to continue on its way. However, some of these obstacles do lead to breakdown of the nanomachine. These breakdowns are potentially disastrous since they may lead to parts of the genetic code not being copied or miscopied prior to cell division, resulting in corruption of the code.These breakdowns, and the risks associated with them, are a problem that all organisms must face. Consequently mechanisms have evolved to reassemble the copying nanomachines back onto the DNA, allowing copying to resume and so reducing the risk of mistakes being introduced into the genetic code. The means by which these replicating nanomachines are reloaded back onto chromosomes appear to be similar in all organisms, a reflection of the conserved nature of the genetic material and the machines that copy this material. However, it is only in bacteria that the enzymes responsible for reloading these nanomachines back onto DNA have been identified. The bacterium Escherichia coli has provided a great deal of information about how this reloading occurs and has provided a model with which to understand how this repair occurs in all organisms. However, in spite of two decades of study, we still know very little about how this repair actually occurs inside cells. This ignorance is in part because of the many different types of obstacle encountered inside cells that may lead to breakdown of these nanomachines and also the complicated, overlapping nature of this nanomachine repair.To address this gap in our understanding of how the copying of DNA is achieved inside cells, we will use recently-developed microscopy techniques that allow single molecules to be visualised and tracked inside living cells. We will exploit the information available in the bacterium E. coli to monitor nanomachine repair as it happens inside cells. This information will allow us to generate a model of how the rebuilding of copying machines underpins duplication of the genetic blueprint in this simple system, providing insight into how this essential repair occurs inside more complicated organisms such as ourselves.