Replication fork repair at the single-molecule level

细胞每次分裂时都能复制数百万个DNA碱基对的能力，DNA复制，是生物学的奇迹之一。同样值得注意的是，这个复杂过程的准确性，每复制100万个DNA碱基，错误不到一个。这一壮举是通过DNA与酶的微妙相互作用实现的，酶是蛋白质分子，充当细胞的主力，催化所有细胞过程。许多蛋白质参与DNA复制，它们共同作用形成细胞的复制机制。复制过程中的任何错误（突变）都可以通过检测DNA的酶来纠正，并在必要时进行修改（就像机器在工厂生产线上检查缺陷一样）。除了这些错误，机器还可能遇到DNA上的块（类似于卡住的拉链）。这些可以采取对DNA的化学损伤的形式，由代谢过程中内部产生的产物（即某些食物的副产品）或广泛的外部因素（如烟草烟雾或阳光的紫外线成分）引起。如果没有专门的酶，这些突变就无法被识别和修复，这可能导致细胞死亡，或者在人类等高等生物中，导致癌症的发生。为了了解DNA复制的基础机制，我们正在利用先进的技术来确定细胞如何尝试和克服这些障碍。这些技术是基于荧光现象，即在照射后发光和吸收不同颜色的光。使用荧光标记，标记DNA的小分子，使DNA在光照射在样品上时可见。传统的荧光测量，即所谓的整体方法，同时测量大量的分子，所得的荧光信号是所有标记的DNA分子的信号的平均值。由于像DNA和蛋白质这样的复杂分子在不同的时间会做不同的事情，因此不可能研究复杂的动态行为或使用集成技术将信号从不同的分子中分离出来。在这个项目中，我们将使用先进的荧光技术，允许在单分子水平上研究分子，提供有关细胞如何确保不可避免的故障的前所未有的信息，这些故障被认为发生在所有生物体中，包括我们自己。

The ability of cells to duplicate millions of base pairs of DNA every time they divide, DNA replication, is one of the wonders of biology. Equally remarkable is the accuracy with which this complex process is achieved, with less than one mistake made for every million bases of DNA copied. This feat is achieved through the subtle interplay of the DNA with enzymes, which are protein molecules that act as the workhorses of the cell, catalyzing all cellular processes. Many proteins are involved in DNA replication and they work together forming the replication machinery of the cell. Any mistakes (mutations) made during replication can be corrected by enzymes that inspect the DNA and make alterations if necessary (just as machines check for defects on a factory production line). In addition to these kinds of mistakes, the machinery can also encounter blocks on the DNA (analogous to a jammed zip fastener). These can take the form of chemical damage to the DNA caused by products generated internally during metabolism (i.e. byproducts from certain foods) or by a wide range of outside agents such as tobacco smoke or the ultraviolet component of sunlight. Without specialised enzymes such mutations would not be recognized and repaired, which could lead to the death of the cell or, in higher organisms such as man, the onset of cancer. To understand the mechanisms that underpin DNA replication, we are utilising advanced techniques to determine how cells try and overcome such blocks. The techniques are based on the phenomenon of fluorescence, which is the emission of light following irradiation and absorption of light of a different colour. The use of fluorescent labels, small molecules that tag DNA, allow the DNA to be visible when light is shone on a sample. Conventional fluorescence measurements, so called ensemble methods, measure a great many molecules simultaneously, and the resultant fluorescence signal is the average of the signal from all labelled DNA molecules. Since complex molecules like DNA and proteins will be doing different things at different times, it is impossible to study complex dynamic behaviour or to separate the signals from different molecules using ensemble techniques. In this project, we will use advanced fluorescence technology that allows molecules to be studied at the single-molecule level, providing unprecedented information about how cells insure against the inevitable breakdowns that are thought to occur in all organisms, including ourselves.