A novel DNA segregation model system from Archaea revealing bacterial and eukaryotic linkages

点击翻译按钮获取中文摘要

Archaea evolved as a domain of life billions of years ago, but they are a relatively recent addition to the map of the universal tree of living organisms. Their discovery 40 years ago represented a major milestone. Archaea are unicellular organisms that populate our planet together with bacteria and eukaryotes. Both bacteria and archaea are prokaryotes, i.e. their genetic material is not wrapped by a membrane into a separate compartment, called nucleus, which is instead a hallmark of eukaryotes (baker yeast, fungi, plants, animals and humans to mention some). Archaea are known to be ubiquitous, constituting a considerable fraction of the biosphere. Their ubiquity and abundance make them key players in regulating biogeochemical cycles on Earth. From a functional and mechanistic standpoint, archaea are a mosaic of features from bacteria and eukaryotes, but they are also characterized by unique features like methane production.Heat-loving archaea are super microbes thriving at 80 degrees C and higher temperatures in hot springs, volcanoes, deep sea vents and exhibiting unusual properties, which make these organisms valuable for the development of novel biotechnological applications, but also extremely interesting for studies on life pushed to extremes. Their ability to grow in extreme environments where no other terrestrial organism can survive has also rejuvenated hopes of discovering extraterrestrial life. Despite the significant progress made in decoding molecular mechanisms in these organisms in the last four decades, to date little information is available on the fundamental process of DNA segregation in archaea and the subject remains a black box awaiting investigation. Genome segregation is a crucial stage of the life cycle of every cell: the DNA is first duplicated, then separated and equally distributed into the two daughter cells. We intend to study this process in a strain of the heat-loving archaeon Sulfolobus isolated from an acidic hot spring in Japan. This microbe contains a large and a small ring of DNA. The large is called chromosome and the small is designated as plasmid. We have recently investigated three proteins that are encoded by the plasmid, pNOB8, and solved their three-dimensional structures. These proteins assemble into a nanomachine that drives duplicated sister plasmids apart, so that each daughter cell receives the same copy number. We intend to introduce changes in the DNA of the host Sulfolobus strain, so that mutations can be introduced in the genes encoding the proteins to show that these factors are essential for the inheritance of the plasmid. One of the proteins responsible for the inheritance of pNOB8 is AspA. It binds strongly to a special site on the plasmid that acts as a docking site and then associates to adjacent regions spreading on the DNA and forming a helix. We want to investigate whether the plamid contains multiple docking sites for AspA and the process through which this protein stretches on the DNA. The other two proteins of the plasmid segregation nanomachine are ParB and ParA. ParB is an adaptor sitting between AspA and ParA in the complex. Adaptors need to be pliable and ParB is indeed flexible: the protein consists of two domains connected by a flexible linker. One of the ParB domains has a bacterial flavour, whereas the other looks like a eukaryotic protein. We suspect that the ParB region that binds ParA is the flexible linker and we want to test this hypothesis. We intend to investigate if other proteins in Sulfolobus associate with the eukaryotic domain of ParB, modifying and regulating it. Finally, we are eager to gain a snapshot of these proteins in Sulfolobus cells. We are going to use microscopes that will provide high-resolution images of the protein complex and will tell us where it localizes relative to the DNA mass of the chromosome. This analysis will allow to identify patterns that will shed light on the life of the pNOB8 DNA segregation nanomachine.