Bilateral NSF/BIO-BBSRC- Remodelling Replication Roadblocks: Regulatory Systems that Integrate DNA Replication, Recombination and Protein Modification

在细胞分裂之前，它们的染色体必须复制。这个过程被称为DNA复制，它开始于染色体上被称为复制起点的特定位置。细菌只有一个复制起点，但是像人类这样的具有大染色体的生物体需要多个复制起点。我们发现起源是不必要的，没有它们的细胞可以比正常细胞生长得更快。我们的DNA复制研究是在古细菌的成员火山盐藻中进行的。生命之树分为三大类：真核生物、细菌和古细菌。古生菌是一种以生活在极端条件下而闻名的微生物，比如酸性水池和盐湖。火山盐藻来自死海，我们选择它是因为古细菌中进行DNA复制的酶与真核生物中使用的酶相似。没有起源的火山盐藻细胞使用一种叫做重组的替代方法来开始DNA复制。重组是DNA修复的一种形式，它用于修复染色体上的断裂。当DNA复制停滞时，就会出现这些断裂，如果DNA受损而无法复制，就会发生这种情况。事实上，在所有生物体中，重组被用来重新启动停滞的DNA复制，这可能是它的主要功能。在重组可以用来重新启动停滞的DNA复制之前，必须首先去除用于复制DNA的酶，以便重组酶可以取代它们的位置。这些酶复合物的分解是由一个系统来完成的，这个系统用一种叫做泛素的分子来标记蛋白质——这种标记可以识别那些注定要被重塑或破坏的蛋白质。我们已经在火山盐铁ax中发现了一个在DNA复制、重组和蛋白质破坏中起作用的酶网络。我们将对属于该网络的其他酶进行系统的搜索。我们的目标是揭示细胞对DNA复制停滞的反应背后的调节机制。为了做到这一点，我们将使用我们的实验数据来创建一个监管系统的计算机模型。反过来，这个计算机模型将告诉我们哪些基因和蛋白质是这个过程的关键，我们应该详细研究。这种实验方法被称为系统生物学。通过使用无起源的火山盐藻细胞，我们可以确保所有的DNA复制都是通过重组开始的。在像人类这样的复杂生物体中，起源已经与细胞过程结合在一起，不可能在不产生有害影响的情况下删除它们。因此，简化后的制度可让我们详细研究规管机制。

Before cells can divide, their chromosomes must be duplicated. This process is called DNA replication and it begins at specific locations on the chromosome called replication origins. Bacteria have a single replication origin but organisms with large chromosomes, such as humans, need many origins. We have found that origins are unnecessary, and that cells without them can grow faster than normal.Our research on DNA replication was carried out in Haloferax volcanii, a member of the archaea. The tree of life is split into three groups: eukaryotes, bacteria and archaea. Archaea are microbes renowned for living in extreme conditions such as acid pools and salt lakes. Haloferax volcanii comes from the Dead Sea, we chose it because the enzymes that carry out DNA replication in archaea are similar to those used in eukaryotes.Haloferax volcanii cells without origins use an alternative method called recombination to start DNA replication. Recombination is a form of DNA repair, it is used to mend breaks in the chromosome. These breaks can arise when DNA replication is stalled, which happens if the DNA is damaged and cannot be duplicated. In fact, recombination is used to restart stalled DNA replication in all organisms, and this may be its primary function.Before recombination can be used to restart stalled DNA replication, the enzymes that are being used to duplicate the DNA must first be removed, so that recombination enzymes can take their place. The disassembly of these enzyme complexes is carried out by a system that tags the proteins with a molecule called ubiquitin - this tag identifies the proteins that are destined for remodelling or destruction.We have identified in Haloferax volcanii a network of enzymes that act in DNA replication, recombination and protein destruction. We will carry out a systematic search for other enzymes that belong to this network. Our goal is to uncover the regulatory mechanism behind the cell's response to stalled DNA replication. To do this, we will use our experimental data to create a computer model of the regulatory system. In turn, this computer model will tell us which genes and proteins are key to the process, and that we should examine in detail. This experimental approach is called systems biology.By using Haloferax volcanii cells without origins, we can ensure that all DNA replication is started by recombination. In complex organisms such as humans, origins have become integrated with cellular processes and it is impossible to delete them without detrimental effects. Therefore, our simplified system will allow us to examine the regulatory mechanisms in detail.