Functional proteomic identification of DNA replication proteins by induced proteolysis in vivo.
Evolutionarily diverse eukaryotic cells share many conserved proteins of unknown function. Some are essential for cell viability, emphasising their importance for fundamental processes of cell biology but complicating their analysis. We have developed an approach to the large-scale characterization of such proteins, based on conditional and rapid degradation of the target protein in vivo, so that the immediate consequences of bulk protein depletion can be examined. Budding yeast strains have been constructed in which essential proteins of unknown function have been fused to a 'heat-inducible-degron' cassette that targets the protein for proteolysis at 37 degrees C (ref. 4). By screening the collection for defects in cell-cycle progression, here we identify three DNA replication factors that interact with each other and that have uncharacterized homologues in human cells. We have used the degron strains to show that these proteins are required for the establishment and normal progression of DNA replication forks. The degron collection could also be used to identify other, essential, proteins with roles in many other processes of eukaryotic cell biology.
Kanemaki
Masato
M
Cancer Research UK, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester, M20 4BX, UK.
Sanchez-Diaz
Alberto
A
Gambus
Agnieszka
A
Labib
Karim
K
D018797:Cell Cycle Proteins / Q000378:metabolism; D002470:Cell Survival; D004261:DNA Replication / Q000187:drug effects; D004318:Doxycycline / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D020043:Genes, Essential; D008025:Ligases / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D018741:Replication Origin / Q000187:drug effects; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D044767:Ubiquitin-Protein Ligases
D018797:Cell Cycle Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; C066066:UBR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; D008025:Ligases; D004318:Doxycycline