An essential role for Orc6 in DNA replication through maintenance of pre-replicative complexes.
The heterohexameric origin recognition complex (ORC) acts as a scaffold for the G(1) phase assembly of pre-replicative complexes (pre-RC). Only the Orc1-5 subunits appear to be required for origin binding in budding yeast, yet Orc6 is an essential protein for cell proliferation. Imaging of Orc6-YFP in live cells revealed a punctate pattern consistent with the organization of replication origins into subnuclear foci. Orc6 was not detected at the site of division between mother and daughter cells, in contrast to observations for metazoans, and is not required for mitosis or cytokinesis. An essential role for Orc6 in DNA replication was identified by depleting it at specific cell cycle stages. Interestingly, Orc6 was required for entry into S phase after pre-RC formation, in contrast to previous models suggesting ORC is dispensable at this point in the cell cycle. When Orc6 was depleted in late G(1), Mcm2 and Mcm10 were displaced from chromatin, cells failed to progress through S phase, and DNA combing analysis following bromodeoxyuridine incorporation revealed that the efficiency of replication origin firing was severely compromised.
Semple
Jeffrey W
JW
Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
Da-Silva
Lance F
LF
Jervis
Eric J
EJ
Ah-Kee
Jennifer
J
Al-Attar
Hyder
H
Kummer
Lutz
L
Heikkila
John J
JJ
Pasero
Philippe
P
Duncker
Bernard P
BP
D018797:Cell Cycle Proteins / Q000378:metabolism; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D002868:Chromosomal Proteins, Non-Histone; D048749:Cytokinesis / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D016193:G1 Phase / Q000502:physiology; D026341:Minichromosome Maintenance 1 Protein; D064110:Minichromosome Maintenance Proteins; D008954:Models, Biological; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D018741:Replication Origin / Q000502:physiology*; D016196:S Phase / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism
D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; C498030:MCM1 protein, S cerevisiae; C076365:MCM10 protein, S cerevisiae; D026341:Minichromosome Maintenance 1 Protein; C497221:ORC6 protein, S cerevisiae; D051738:Origin Recognition Complex; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D064110:Minichromosome Maintenance Proteins