[show abstract][hide abstract] ABSTRACT: The similarities between essential molecular mechanisms in Archaea and Eukarya make it possible to discover, using comparative genomics, new fundamental mechanisms conserved between these two domains. We are studying a complex of two proteins conserved in Archaea and Eukarya whose precise biological role and biochemical function remain unknown. One of them is a universal protein known as Kae1 (kinase-asociated endopeptidase 1). The second protein is a serine/threonine kinase corresponding to the proteins Bud32 in Saccharomyces cerevisiae and PRPK (p53-related protein kinase) in humans. The genes encoding the archaeal orthologues of Kae1 and PRPK are either contiguous or even fused in many archaeal genomes. In S. cerevisiae, Kae1 and Bud32 (PRPK) belong to a chromatin-associated complex [KEOPS (kinase, endopeptidase and other proteins of small size)/EKC (endopeptidase-like kinase chromatin-associated)] that is essential for telomere elongation and transcription of essential genes. Although Kae1 is annotated as O-sialoglycoprotein endopeptidase in most genomes, we found that the Kae1 protein from Pyrococcus abyssi has no protease activity, but is an atypical DNA-binding protein with an AP (apurinic) lyase activity. The structure of the fusion protein from Methanocaldococcus jannaschii revealed that Kae1 maintains the ATP-binding site of Bud32 [corrected] in an inactive configuration. We have in fact found that Kae1 inhibits the kinase activity of Bud32 (PRPK) in vitro. Understanding the precise biochemical function and biological role of these two proteins (which are probably essential for genome maintenance) remains a major challenge.
Biochemical Society Transactions 03/2009; 37(Pt 1):29-35. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: Topoisomerases are essential enzymes that solve topological problems arising from the double-helical structure of DNA. As a consequence, one should have naively expected to find homologous topoisomerases in all cellular organisms, dating back to their last common ancestor. However, as observed for other enzymes working with DNA, this is not the case. Phylogenomics analyses indicate that different sets of topoisomerases were present in the most recent common ancestors of each of the three cellular domains of life (some of them being common to two or three domains), whereas other topoisomerases families or subfamilies were acquired in a particular domain, or even a particular lineage, by horizontal gene transfers. Interestingly, two groups of viruses encode topoisomerases that are only distantly related to their cellular counterparts. To explain these observations, we suggest that topoisomerases originated in an ancestral virosphere, and that various subfamilies were later on transferred independently to different ancient cellular lineages. We also proposed that topoisomerases have played a critical role in the origin of modern genomes and in the emergence of the three cellular domains.
Nucleic Acids Research 03/2009; 37(3):679-92. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: DNA topoisomerases resolve DNA topological problems created during transcription, replication, and recombination. These ubiquitous enzymes are essential for cell viability and are highly potent targets for the development of antibacterial and antitumoral drugs. Type II enzymes catalyze the transfer of a DNA duplex through another one in an ATP-dependent mechanism. Because of its small size and sensitivity to antitumoral drugs, the archaeal DNA topoisomerase VI, a type II enzyme, is an excellent model for gaining further understanding of the organization and mechanism of these enzymes. We present the crystal structure of intact DNA topoisomerase VI bound to radicicol, an inhibitor of human topo II, and compare it to the conformation of the apo-protein as determined by small-angle X-ray scattering in solution. This structure, combined with a wealth of experimental data gathered on these enzymes, allows us to propose a structural model for the two-gate DNA transfer mechanism.
[show abstract][hide abstract] ABSTRACT: The DNA topoisomerases are essential for DNA replication, transcription, recombination, as well as for chromosome compaction and segregation. They may have appeared early during the formation of the modern DNA world. Several families and subfamilies of the two types of DNA topoisomerases (I and II) have been described in the three cellular domains of life (Archaea, Bacteria and Eukarya), as well as in viruses infecting eukaryotes or bacteria. The main families of DNA topoisomerases, Topo IA, Topo IB, Topo IC (Topo V), Topo IIA and Topo IIB (Topo VI) are not homologous, indicating that they originated independently. However, some of them share homologous modules or subunits that were probably recruited independently to produce different topoisomerase activities. The puzzling phylogenetic distribution of the various DNA topoisomerase families and subfamilies cannot be easily reconciled with the classical models of early evolution describing the relationships between the three cellular domains. A possible scenario is based on a Last Universal Common Ancestor (LUCA) with a RNA genome (i.e. without the need for DNA topoisomerases). Different families of DNA topoisomerases (some of them possibly of viral origin) would then have been independently introduced in the different cellular domains. We review here the main characteristics of the different families and subfamilies of DNA topoisomerases in a historical and evolutionary perspective, with the hope to stimulate further works and discussions on the origin and evolution of these fascinating enzymes.
[show abstract][hide abstract] ABSTRACT: The Kae1 (Kinase-associated endopeptidase 1) protein is a member of the recently identified transcription complex EKC and telomeres maintenance complex KEOPS in yeast. Kae1 homologues are encoded by all sequenced genomes in the three domains of life. Although annotated as putative endopeptidases, the actual functions of these universal proteins are unknown. Here we show that the purified Kae1 protein (Pa-Kae1) from Pyrococcus abyssi is an iron-protein with a novel type of ATP-binding site. Surprisingly, this protein did not exhibit endopeptidase activity in vitro but binds cooperatively to single and double-stranded DNA and induces unusual DNA conformational change. Furthermore, Pa-Kae1 exhibits a class I apurinic (AP)-endonuclease activity (AP-lyase). Both DNA binding and AP-endonuclease activity are inhibited by ATP. Kae1 is thus a novel and atypical universal DNA interacting protein whose importance could rival those of RecA (RadA/Rad51) in the maintenance of genome integrity in all living cells.
Nucleic Acids Research 02/2007; 35(18):6042-51. · 8.28 Impact Factor
[show abstract][hide abstract] ABSTRACT: Radicicol derivatives are currently investigated as promising antitumoral drugs because they inhibit the activity of the molecular chaperone heat shock protein (HSP90), causing the destabilization and eventual degradation of HSP90 client proteins that are often associated with tumor cells. These drugs interact with the ATP-binding site of HSP90 which is characterized by a structural element known as the Bergerat fold, also present in type II DNA topoisomerases (Topo II). We have previously shown that radicicol inhibits archaeal DNA topoisomerase VI, the prototype of Topo II of the B family (present in archaea, some bacteria and all the plants sequenced so far). We show here that radicicol also inhibits the human Topo II, a member of the A family (comprising the eukaryotic Topo II, bacterial gyrase, Topo IV and viral Topo II), which is a major target for antitumoral drugs. In addition, radicicol prevents in vitro induction of DNA cleavage by human Topo II in the presence of the antitumoral drug etoposide. The finding that radicicol can inhibit at least two different antitumoral drug targets in human, and interferes with drugs currently used in cancer treatment, could have implications in cancer therapy.
[show abstract][hide abstract] ABSTRACT: Type II DNA topoisomerases (Topo II) are essential enzymes implicated in key nuclear processes. The recent discovery of a novel kind of Topo II (DNA topoisomerase VI) in Archaea led to a division of these enzymes into two non-homologous families, (Topo IIA and Topo IIB) and to the identification of the eukaryotic protein that initiates meiotic recombination, Spo11. In the present report, we have updated the distribution of all Topo II in the three domains of life by a phylogenomic approach. Both families exhibit an atypical distribution by comparison with other informational proteins, with predominance of Topo IIA in Bacteria, Eukarya and viruses, and Topo IIB in Archaea. However, plants and some Archaea contain Topo II from both families. We confront this atypical distribution with current hypotheses on the evolution of the three domains of life and origin of DNA genomes.