Recombination and the clearance of replicative blocks - to bypass or not to bypass?

一个细胞形成两个新的子细胞的能力使生物体得以生长和繁殖。这个过程需要复制大量的DNA，以便每个子代都能获得生存所需的所有遗传信息的精确副本。由于精确复制DNA的重要性，生物体进化出了非常复杂的DNA复制机器，以减少出错的机会。不幸的是，我们现在知道，这些复制机器遇到了许多障碍，如果不克服这些障碍，就会在复制过程中造成错误。这样的错误会导致基因信息的错误，从而导致致命的后果。包裹在DNA上的蛋白质经常对DNA复制机制形成障碍。这些蛋白质在所有生物体中都是维持、读取和包装遗传信息所必需的，因此无法避免。尽管DNA复制机制可以成功地将这些蛋白质从DNA中分离出来进行复制，但与DNA结合的蛋白质的绝对数量意味着复制过程偶尔会停止。然而，一类特殊的酶可以修复和重新启动损坏的DNA复制机器。很明显，这些重组酶可以帮助复制与蛋白质结合的DNA，但它们是如何做到的还不清楚。一种说法是，如果DNA复制机器被蛋白质阻断，那么重组酶可以在蛋白质阻断的另一边重新开始DNA复制。这条途径会跳过这个片段，允许复制继续进行，但也会导致靠近蛋白质片段的基因不被复制。因此，重组酶的参与可能被视为有害的，导致无法复制两个子细胞生存所需的所有遗传信息。或者，我们提出重组酶可能只是在DNA最初停止的地方重新开始复制。这可能会给DNA复制机制第二次机会，将阻断蛋白从DNA上推开，继续复制所有的遗传信息。因此，这一过程可能提供了一种机制，以确保精确复制被蛋白质包裹的DNA。我们将使用模型细菌大肠杆菌来确定重组酶在复制被蛋白质包裹的DNA中的作用。我们对大肠杆菌中DNA复制和重组的基本机制了解很多，这使我们能够分析这两个非常复杂的过程是如何相互作用的。我们将确定重组酶帮助DNA复制机器通过蛋白质块移动的机制。我们还将建立准确和不准确重组机制之间的平衡，以了解这些过程何时可能对遗传物质产生潜在的非常有害的变化。复制DNA的需要和将蛋白质与DNA结合的需要之间的冲突是所有生物体都必须以某种方式解决的问题。因此，这项工作将准确地解决是什么驱动了基因内突变的积累，以及什么机制有助于减少这种积累。遗传物质中突变的获得是进化的动力，但这种突变往往是有害的，而不是有益的，因此必须加以控制。突变在人类遗传疾病的获得和癌症发展中的重要性说明了这一点。了解大肠杆菌中DNA复制和重组的基本机制大大提高了我们对更复杂生物（如我们自己）遗传稳定性的认识。我们现在可以利用大肠杆菌来解决这些关键过程之间的相互作用，这种相互作用对于理解基因如何以尽可能准确的方式复制至关重要。

The ability of a cell to form two new daughter cells allows organisms to grow and reproduce. This process requires the copying of vast amounts of DNA so that each daughter receives an accurate copy of all the genetic information required for survival. Because of the importance of generating accurate copies of the DNA, organisms have evolved very complex DNA replication machines that reduce the chances of mistakes being made. Unfortunately, we now know that many obstacles are encountered by these replication machines that, if not overcome, can cause mistakes in the copying process. Such mistakes can lead to errors in the genetic information that can have fatal consequences.Proteins that coat the DNA form frequent obstacles to the DNA copying machinery. These proteins are essential in all organisms for maintaining, reading and packing the genetic information, and so cannot be avoided. Although the DNA replication machinery can successfully push off most of these proteins from the DNA to be copied, the sheer number of proteins bound to the DNA mean that occasionally the copying process is stopped in its tracks. However, a specific class of enzymes can repair and restart broken down DNA replication machines. It is clear that these recombination enzymes can help to copy DNA that is bound by proteins but how they do so remains unclear. One proposal is that if a DNA replication machine becomes blocked by a protein then recombination enzymes can restart DNA replication on the other side of the protein block. This pathway would skip over the block, allowing copying to continue, but would also result in genes near the protein block not being copied. Consequently the involvement of recombination enzymes could be seen as harmful, resulting in failure to copy all the genetic information needed by the two daughter cells to survive. Alternatively, we have proposed that recombination enzymes might simply restart DNA replication near to where it initially came to a halt. This might give the DNA replication machinery a second chance to push the blocking protein off the DNA and continue to copy all of the genetic information. Such a process might therefore provide a mechanism to ensure accurate copying of DNA coated with proteins.We will use the model bacterium E. coli to determine the roles of recombination enzymes in copying DNA coated with proteins. We know a great deal about the basic mechanisms of both DNA replication and recombination in E. coli, allowing us to analyse how these two very complicated processes interact. We will determine the mechanisms by which recombination enzymes can help DNA replication machines to move through protein blocks. We will also establish what dictates the balance between accurate and inaccurate recombination mechanisms to understand when such processes might generate potentially very harmful changes to the genetic material.The conflict between the need to copy DNA and the need to have proteins bound to the DNA is one that all organisms must somehow resolve. This work will address therefore exactly what drives the accumulation of mutations within genes and what mechanisms help to minimise this accumulation. Acquisition of mutations in the genetic material is the driving force of evolution but such mutations are frequently harmful rather than beneficial and so must be kept in check. This is illustrated by the importance of mutations in the acquisition of human genetic disorders and the development of cancer. Understanding fundamental mechanisms of DNA replication and recombination in E. coli has greatly advanced our knowledge of genetic stability in more complex organisms such as ourselves. We are now in a position to use E. coli to address the interplay between these critical processes, an interplay that is central to understanding how genes are copied in as accurate a manner as possible.