Replication fork repair at the single-molecule level

细胞每次分裂时复制数百万个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一复杂过程的准确性，每复制一百万个DNA碱基，只会出现不到一个错误。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。许多蛋白质都参与DNA复制，它们共同形成细胞的复制机制。在复制过程中产生的任何错误（突变）都可以通过检查DNA的酶来纠正，并在必要时进行改变（就像机器检查工厂生产线上的缺陷一样）。除了这些错误之外，机器还可能遇到DNA上的阻塞（类似于卡住的拉链）。这些损害可以通过代谢过程中内部产生的产物（即某些食物的副产品）或烟草烟雾或阳光中的紫外线成分等多种外部因素对DNA造成化学损伤。如果没有专门的酶，这种突变将无法被识别和修复，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。为了理解支撑DNA复制的机制，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。这种技术是基于荧光现象，即在不同颜色的光照射和吸收后发出的光。荧光标记（标记DNA的小分子）的使用，使DNA在光照在样品上时可见。传统的荧光测量，即所谓的集合法，同时测量许多分子，所得荧光信号是所有标记DNA分子信号的平均值。由于像DNA和蛋白质这样的复杂分子会在不同的时间做不同的事情，因此不可能研究复杂的动态行为，也不可能使用集成技术从不同的分子中分离信号。在这个项目中，我们将使用先进的荧光技术，允许分子在单分子水平上进行研究，提供关于细胞如何防止不可避免的破坏的前所未有的信息，这些破坏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.