From Comparative Genomics to Comparative Genetics - What is Required for Life Without DNA Replication Origins?

所有的细胞都含有生物体DNA的完整拷贝，被包装成染色体。在细胞分裂之前，它们的染色体必须复制。这被称为DNA复制，从染色体上被称为起源的特定位置开始。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，在一种情况下，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。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（如人类细胞）中的酶相似。火山盐黄貂鱼使用三个起源来复制它的染色体，但是当所有的起源都被删除时，细胞生长得更快。在人类身上做这些实验是不可能的。当真核生物或细菌的起源被删除时，DNA复制被阻止，细胞死亡。那么火山盐藻是如何存活下来的呢？没有起源的细胞通过一种叫做重组的过程来开始DNA复制。重组是DNA修复的一种形式，用于修复染色体上的断裂。我们发现重组在染色体上的随机位置开始DNA复制，而不是在特定的起点。但是，如果这种利用重组进行DNA复制的替代模式更有效，那么为什么会有起源呢？在古细菌的姊妹种地中海盐腐菌（Haloferax mediterranei）中，起源无法消除。当尝试这样做时，休眠的复制源将变为活动的。这意味着地中海盐黄貂鱼需要起源，而火山盐黄貂鱼可以通过重组来启动DNA复制。为什么这两种密切相关的微生物行为如此不同？我们认为火山盐黄貂具有地中海盐黄貂所缺少的关键基因，反之亦然。为了简化对这些关键基因的搜索，我们将研究这些和多达20个额外的盐铁杉物种的基因组。我们在罗马尼亚的同事已经发现，特兰西瓦尼亚的盐湖是盐柳属植物的丰富来源。我们将测试这些盐藻物种，看看它们的起源是否可以被消除（如火山盐藻），或者它们是否是必需的（如地中海盐藻）。然后我们将比较他们的基因组来定位负责的基因。同时，我们将检查使用重组开始DNA复制的后果。火山盐藻可以利用重组来启动DNA复制，但这可能是危险的。我们将测试它是否会导致突变或染色体重排，以及是否有避免重组的替代方案。与起源不同，重组可以发生在染色体上的任何地方，但发生的频率取决于DNA的长度。我们将测试这种DNA复制模式是否存在最小的染色体大小。我们的工作将增进我们对癌症的了解，从而为人类健康作出贡献。我们在火山盐藻中发现的东西与癌细胞有相似之处。黄貂鱼的染色体有很多拷贝，这被称为多倍体，这有助于它在复制和细胞分裂不协调时存活下来。癌细胞通常在控制DNA复制的基因中发生突变，多倍体是癌症的一个共同特征。不协调复制的另一个后果是癌细胞比普通细胞生长得快。这类似于我们观察到的无起源的火山盐藻的更快生长，它使用了另一种DNA复制模式。

All cells contain a complete copy of the organism's DNA, packaged into chromosomes. Before cells can divide, their chromosomes must be duplicated. This is called DNA replication and begins at specific sites on the chromosome called origins. Bacteria have a single replication origin but organisms with large chromosomes, such as humans, need many origins. We have found that in one case, 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. Haloferax volcanii comes from the Dead Sea, we chose it because the enzymes that carry out DNA replication in archaea are similar to those in eukaryotes, such as human cells.Haloferax volcanii uses three origins to replicate its chromosome but when all origins are deleted, the cells grow faster. Doing these experiments in humans would be impossible. When origins are deleted from eukaryotes or bacteria, DNA replication is prevented and cells die. So how is Haloferax volcanii able to survive?Cells without origins use a process called recombination to start DNA replication. Recombination is a form of DNA repair that is used to mend breaks in the chromosome. We found that recombination starts DNA replication at random locations on the chromosome, instead of at specific origins. But if this alternative mode of DNA replication using recombination is more efficient, why have origins at all?In a sister species of archaea called Haloferax mediterranei, origins cannot be eliminated. When this is attempted, a dormant replication origin becomes active. This means that Haloferax mediterranei needs origins, while Haloferax volcanii can instead use recombination to start DNA replication. Why do these two closely-related microbes behave so differently?We propose that Haloferax volcanii has critical genes that are missing from Haloferax mediterranei, or vice versa. To simplify the search for these critical genes, we will study the genomes of these and up to 20 additional Haloferax species. Our colleagues in Romania have already discovered that salt lakes in Transylvania are a rich source of Haloferax species. We will test these Haloferax species to see if their origins can be eliminated (as in Haloferax volcanii) or if they are essential (as in Haloferax mediterranei). Then we will compare their genomes to locate the genes responsible.At the same time, we will examine the consequences of using recombination to start DNA replication.Haloferax volcanii can use recombination to start DNA replication but this may be hazardous. We will test whether it leads to mutations or chromosome rearrangements, and whether there are alternatives that avoid recombination.Unlike origins, recombination can take place anywhere on the chromosome, but how often this happens depends on the length of DNA. We will test if there is a minimum size of chromosome for this alternative mode of DNA replication.Our work will contribute to human health by increasing our understanding of cancer. What we have discovered in Haloferax volcanii has parallels with cancer cells. Haloferax has many copies of its chromosome, this is called polyploidy and helps it to survive when replication and cell division are not coordinated. Cancer cells often have mutations in the genes that control DNA replication and polyploidy is a common feature of cancer. Another consequence of uncoordinated replication is that cancer cells grow faster than ordinary cells. This is similar to the faster growth we observe with origin-less Haloferax volcanii, which use an alternative mode of DNA replication.