Replication Termination in the Archaea

DNA是生物体的蓝图。任何物种要繁殖，无论是单细胞微生物还是人类，都必须能够复制自己的DNA，从而确保将遗传指令传递给后代。这项研究计划概述了一系列实验，这些实验将研究DNA复制的最后一步。在细胞中，DNA形成双螺旋结构。为了让分子复制，双螺旋解压，两条链都被复制，产生两个子分子。当DNA解压并被复制时形成的结构被称为复制叉。在复制接近尾声时，两个分叉将彼此靠近并发生碰撞。这两个分叉的碰撞必须小心地解决，这样才能产生两个完整的子分子。此过程称为复制终止。我们建议研究一类被称为古生菌的微生物的复制终止的分子基础。我们研究的古生物生长在温泉环境中，比如在黄石国家公园发现的。由于生物体在80摄氏度的温度下生长，它们的细胞机械异常坚固，这种稳定性极大地有助于我们对有机体的生化分析。此外，在过去的几年里，越来越明显的是，古生物用来复制DNA的机器是我们自己细胞使用的机器的简化版本。因此，除了揭示古生菌中复制终止的过程外，我们的研究也可能对人类细胞中DNA复制终止的进化和机制产生宝贵的见解。我们提出的实验将确定复制分叉是否只是随机碰撞，或者DNA中是否存在定义的路障，为复制分叉在定义的终止点创建特定的停止点。如果发现后一种可能性，我们将调查路障的作用机理。我们的研究将扩展到调查我们先前确定的复制分叉暂停位置。此暂停站点不会导致终止，而只是在短时间内停止分叉。虽然我们之前的工作证明了这个短暂停顿站点的存在，但我们还不知道它为什么或如何导致叉子在这个站点短暂停顿。我们建议进行一系列实验，以确定叉子运动中的这种停顿是如何影响的。

DNA is the blueprint for living organisms. For any species to propagate, be it a single celled microbe or a human being, it must be able to duplicate its DNA; thereby ensuring that it passes its genetic instructions onto its progeny. This research proposal outlines a series of experiments that will investigate the final step in the replication of DNA. In cells, DNA forms a double helix. For the molecule to replicate, the double helix unzips and both strands are copied to generate two daughter molecules. The structure formed as DNA unzips and is copied is termed the replication fork. Towards the end of replication, two forks will approach each other and collide. This collision of the two forks must resolved carefully so that two complete daughter molecules are generated. This process is called replication termination. We propose to study the molecular basis of replication termination in a class of microbe, termed the Archaea. The Archaea we work on grow in hot spring environments, such as found in Yellowstone National Park. Because the organisms grow at 80 degrees centigrade, their cellular machinery is unusually robust and this stability greatly aids our analyses of the organism's biochemistry. Furthermore, over the last few years it has become increasingly apparent that the machinery that Archaea use to duplicate their DNA is a simplified version of the machinery that our own cells use. Therefore, as well as shedding light on the process of replication termination in the archaea, our studies may also yield invaluable insights into the evolution and mechanism of DNA replication termination in human cells too. The experiments that we propose will determine if replication forks simply collide randomly or whether there are defined road blocks in the DNA that create specific stop sites for the replication forks at defined termination sites. If the latter possibility is found to be the case, we will investigate the mechanism by which the road block functions. Our studies will be extended to investigate a replication fork pausing site that we have previously identified. This pausing site does not lead to termination, but rather simply stalls the fork for a short period of time. Although our earlier work demonstrated the existence of this transient pause site, we do not yet know why or how it causes forks to briefly arrest at this site. We propose to perform a series of experiments to establish how this pause in fork movement is effected.