Le rôle des protéines Universelles de Maintenance du Génome (Kae1, YgjD) et du complexe EKC/KEOPS dans les trois domaines du vivant – UGM Partners

Recently, we discovered that a universal protein annotated as 'O-syaloglycoprotein-endopeptidase' in all sequenced genomes, is in fact a DNA binding protein that exhibits an atypical apurinic endonuclease (AP-endo) activity (Hecker et al., NAR, 2007). We also obtained in vivo evidence suggesting that this protein, known under various names in the literature (YgjD/Ggc, Kae1, OSGEP or else Qri7/OSGEPL) is involved in genome maintenance. We propose here to call these proteins UGM for Universal Genome Maintenance. In S. cerevisiae, the UGM protein Kae1 interacts with a conserved protein-kinase, Bud32, and three other small proteins to form a complex that associated with chromatin. This complex, called EKC or KEOPS, is involved in the transcription and in telomere maintenance. Four of the five proteins of the EK/KEOPS complex have homologues in Eukarya and Archaea. This conservation indicates that this complex is an ancient molecular machine of fundamental importance. Although UGM proteins and the EKC/KEOPS complex are certainly as important as other universal protein such as RecA/Rad51, they are still studied by a very limited number of laboratories, and their biochemical function(s) and biological role(s) are still largely unknown. The objective of the present project is thus to determine the biochemical function(s) and biological role(s) of UGM proteins in the three domains of life and of the EKC/KEOPS complex in Archaea and Eukarya. For that purpose, we will combine informatics, structural, biochemical, genetics and genomic approaches in E. coli, P. abyssi, M. jannashii, S. cerevisiae, and human cells. Our consortium involves four partners with complementary expertises. Patrick Forterre (P1), who is an expert in archaeal molecular biology and hyperthermophilic proteins involved in DNA metabolism, has already collaborated with Herman van Tilbeurgh (P2), who is an expert in protein structure analyses. In the framework of a previous ANR project, they solved the structure of the archaeal Kae1 protein and of a KEOPS/EKC subcomplex (Hecker et al., NAR, 2007, EMBO J. 2008, Biochemical Transaction, 2009). Domenico Libri (P3) and Carl Mann (P4), who discovered the EKC complex (EMBO J, 2008), are expert in yeast genetics and in the analysis of chromatin associated proteins in human cells. P2 and P3 have already collaborated in the exploitation of structural data to demonstrate that the physical interaction between the UGM protein Kae1 and the kinase Bud32 is essential in vivo (Hecker et al., EMBO J. 2008). In the present project, we will complete the biochemical and structural analyses of UGM proteins and the EKC/KEOPS complex in the three domains of life. One of our main objectives will be to solve the structure of the complete yeast and archaeal KEOPS/EKC complex (P2) and to reconstitute these complexes from individual components (the archael complex probably includes a fifth subunit that is missing in yeast but is present in humans) (P1-4). For enzymatic analyses, we will focus on the role of an atypical ATP-iron binding site that P2 discovered in the UGM protein Kae1, and on the interaction of UGM proteins and the KEOPS/EKC complex with nucleic acids. Preliminary experiments by P3 show that the EKC/KEOPS complex binds specifically a telomeric-like sequence (P1-4). A major goal is now to determine how these proteins bind to DNA, if they also interact with RNA, and if they have other activities than the AP endonuclease activity previously detected. Phenotypic analyses of mutants of UGM proteins and subunits of the EKC/KEOPS complex will be performed in E. coli and S. cerevisiae. A major aspect of this project will be to focus on the identification of proteins that interact with UGM proteins in the three domains of life and/or the EKC/KEOPS complex in Archaea and Eukarya (P1, P3, P4). These protein partners will be sought using in parallel in silico (genome context analysis), biochemical (pull down assays, TAP-tag screening) and genetic/genomic approaches (transcriptome profiling, genome-wide interaction analysis) both in E. coli, Archaea, S. cerevisiae, and human cells. The rational is that identification of proteins partners will foster predictions on the biological role of the UGM proteins and/or the EKC/KEOPS complex. These predictions will then be tested by biochemical and genetic analyses. A major novelty of this project will be to initiate the study of the EKC/KEOPS complex in humans (P4). This will be a very important task since one protein of this complex is already known to phosphorylate p53 in humans and another (triplicated in humans) is known as a cancer testis antigen. In general, since the proteins studied in this project are either universal (UGM) or at least conserved in two of the three domains (the EKC/KEOPS complex), the deciphering of their biological roles will have extremely important consequences for biologists working in very different research fields.