Probing DNA segregation in archaea: molecular dissection of an atypical tricistronic partition system from Sulfolobus

基因组分离的过程是每个细胞生命周期的一个基本阶段：基因内容首先被复制，然后被分离并平均分配到两个子细胞中。遗传物质（被组织成线性DNA染色体）在高等生物（如植物、动物、人类）细胞中分离的机制已经被广泛研究和充分理解。在这些细胞中，负责染色体分离的分子机器被称为“有丝分裂纺锤体”：它由称为微管的电缆组成，这些电缆固定在染色体的特定位置，称为“着丝粒”。在细胞分裂之前，微管将姐妹染色体拉到纺锤体的相反两极。在细菌中，情况更加难以捉摸。细菌的遗传遗传由单个圆形（很少是线性的）染色体和称为质粒的小圆圈DNA组成。历史上，人们认为细菌染色体和质粒的分离是一个被动的过程，不需要专门的仪器，可能涉及到新复制的遗传元件附着在生长的细胞壁上。然而，近年来已有证据表明存在一种负责染色体和质粒分离的主动机制，需要专用因子的参与。在细菌中，最具特征的DNA分离系统是那些由质粒指定的，它们在细胞中数量很少。这些质粒拥有自己的生存工具箱，一个由两个基因组成的分离盒，通常被称为parA和parB，以及一个着丝粒样的位点。这种卡带确保在细胞分裂时质粒从一代到下一代的准确分离。这一过程背后的分子机制尚未完全阐明；然而，最近的发现暗示有丝分裂纺锤体一样的机器的存在。古生菌是生命的第三个领域：1977年它们的发现代表了一个重要的生物学里程碑。根据它们的RNA或核糖核酸所含的序列，它们最初被确定为不同的生物群。对其生理学、生物化学和遗传学的进一步表征提供了明确的证据，证明它们是一组独特的生物（不同于细菌和高等多细胞生物）。第一批被研究的古细菌都来自极端环境，但现在已知它们在地球上大多数生态位中茁壮成长，它们占生物圈的20%左右。超嗜热古细菌生长在80℃及以上，并表现出不同寻常的特性，这使这些生物成为开发新型生物技术过程的宝贵资源。工业应用包括生产在高温下稳定的古细菌衍生酶，纤维素降解酶，使用它们的膜作为药物和基因的传递系统。尽管对古细菌的基本生物学过程进行了大量研究，但迄今为止还没有关于这些生物基因组分离机制的信息。我们打算在一种名为Sulfolobus NOB8H2的古菌中分析这一过程，该古菌是从日本北海道岛的温泉中分离出来的。这个古菌含有一个质粒pNOB8，其中包含一个假定的DNA分离盒，包括三个基因（指定为orf44， parB, parA）。本项目拟重点研究上述基因编码的因子的特征、它们在细胞分裂时pNOB8分裂中的功能和作用以及它们之间的动态相互作用。我们还打算鉴定pNOB8的着丝粒，并剖析该位点与ParB、ParA和Orf44之间的相互作用。此外，研究还将探讨ParA蛋白是否会像在一些细菌ParA对应物中观察到的那样组装成电缆状结构。

The process of genome segregation is a fundamental stage of the life cycle of each cell: the genetic content is first duplicated, then separated and equally distributed into the two daughter cells. The mechanism whereby the genetic material (that is organized into linear DNA chromosomes) is separated in cells of higher organisms (like plants, animals, humans) has been extensively studied and is well understood. In these cells the molecular machine responsible for chromosome segregation is known as 'mitotic spindle': it consists of cables, called microtubules, which are anchored to chromosomes at a specific site, known as 'centromere'. The microtubules pull sister chromosomes to opposite poles of the spindle, before the cell divides. In bacteria the picture is more elusive. The genetic patrimony of bacteria consists of a single circular (more rarely linear) chromosome and sometimes smaller circles of DNA called plasmids. Historically, it was presumed that segregation of bacterial chromosomes and plasmids was a passive process, not requiring a dedicated apparatus and perhaps involving attachment of the newly-replicated genetic elements to the growing cell wall. However, in recent years evidence has been provided pointing to the existence of an active mechanism responsible for the segregation of chromosomes and plasmids, requiring the participation of dedicated factors. In bacteria, the most well-characterized DNA segregation systems are those specified by plasmids, which are present in the cell in low numbers. These plasmids harbour their own survival kit, a segregation cassette consisting of two genes, often termed parA and parB, and a centromere-like site. This cassette ensures an accurate segregation of the plasmids from one generation to the next at cell division. The molecular mechanisms underlying this process have not been thoroughly elucidated as yet; however, recent discoveries hint at the existence of mitotic spindle-like machineries. Archaea are the third domain of life: their discovery in 1977 represented a major biological milestone. They were initially identified as a different group of organisms on the basis of sequences contained in their RNA or ribonucleic acid. Further characterization of their physiology, biochemistry and genetics has provided unequivocal evidence that they are a distinct group of organisms (different from bacteria and from higher multicellular organisms). The first archaea to be studied were all from extreme environments, but they are now known to thrive in most of the earth's ecological niches and they constitute ~20% of the biosphere. Hyperthermophilic archaea grow at 80 C and above and exhibit unusual properties, which make these organisms a valuable resource for the development of novel biotechnological processes. Industrial applications include the production of archaea-derived enzymes, which are stable at high temperature, cellulose degrading enzymes, the use of their membranes as delivery systems for drugs and genes. Despite numerous studies on fundamental biological processes in archaea, to date no information is available on the mechanism of genome segregation in these organisms. We intend to analyze this process in an archaeon called Sulfolobus NOB8H2, which has been isolated from hot springs in the island of Hokkaido, Japan. This archaeon contains a plasmid, pNOB8, which harbours a putative DNA segregation cassette comprising three genes (designated as orf44, parB, parA). The project here proposed will focus on the characterization of the factors encoded by the genes above, their function and respective role in pNOB8 partitioning at cell division and their dynamic interactions. We also intend to identify the centromere of pNOB8 and dissect the interactions between this site and ParB, ParA and perhaps Orf44. Furthermore, investigations will be conducted to probe whether the ParA protein assembles into cable-like structures as observed for some bacterial ParA counterparts.