title: Dpb2 integrates the leading-strand DNA polymerase into the eukaryotic replisome.
abstract: BACKGROUND
  The eukaryotic replisome is a critical determinant of genome integrity with a complex structure that remains poorly characterized. A central unresolved issue is how the Cdc45-MCM-GINS helicase is linked to DNA polymerase epsilon, which synthesizes the leading strand at replication forks and is an important focus of regulation.
  
  
  RESULTS
  Here, we use budding yeast to show that a conserved amino-terminal domain of the Dpb2 subunit of Pol ε (Dpb2NT) interacts with the Psf1 component of GINS, via the unique "B domain" of the latter that is dispensable for assembly of the GINS complex but is essential for replication initiation. We show that Dpb2NT is required during initiation for assembly of the Cdc45-MCM-GINS helicase. Moreover, overexpressed Dpb2NT is sufficient to support assembly of the Cdc45-MCM-GINS helicase during initiation, upon depletion of endogenous Dpb2. This produces a replisome that lacks DNA polymerase epsilon, and although cells are viable, they grow extremely poorly. Finally, we use a novel in vitro assay to show that Dpb2NT is essential for Pol ε to interact with the replisome after initiation.
  
  
  CONCLUSIONS
  These findings indicate that the association of Dpb2 with the B domain of Psf1 plays two critical roles during chromosome replication in budding yeast. First, it is required for initiation, because it facilitates the incorporation of GINS into the Cdc45-MCM-GINS helicase at nascent forks. Second, it plays an equally important role after initiation, because it links the leading strand DNA polymerase to the Cdc45-MCM-GINS helicase within the replisome.
authors.affiliation: Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK.
authors.forename: Sugopa
authors.initials: S
authors.lastname: Sengupta
authors.forename: Frederick
authors.initials: F
authors.lastname: van Deursen
authors.forename: Giacomo
authors.initials: G
authors.lastname: de Piccoli
authors.forename: Karim
authors.initials: K
authors.lastname: Labib
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism; D002850:Chromatography, Gel; D004265:DNA Helicases / Q000378:metabolism*; D004257:DNA Polymerase II / Q000737:chemistry / Q000378:metabolism*; D004261:DNA Replication / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D012394:Rosaniline Dyes; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D004718:Saccharomycetales; D013056:Spectrophotometry, Ultraviolet; D020798:Two-Hybrid System Techniques
chemical_list: C102209:CDC45 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C441579:MRC1 protein, S cerevisiae; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; C077018:PRP8 protein, S cerevisiae; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D012394:Rosaniline Dyes; D029701:Saccharomyces cerevisiae Proteins; C048139:Coomassie blue; C529202:Dpb2 protein, S cerevisiae; D004257:DNA Polymerase II; D004265:DNA Helicases