title: Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex.
abstract: DNA polymerase epsilon interacts with the CMG (Cdc45-MCM-GINS) complex by Dpb2p, the non-catalytic subunit of DNA polymerase epsilon. It is postulated that CMG is responsible for targeting of Pol ɛ to the leading strand. We isolated a mutator dpb2-100 allele which encodes the mutant form of Dpb2p. We showed previously that Dpb2-100p has impaired interactions with Pol2p, the catalytic subunit of Pol ɛ. Here, we present that Dpb2-100p has strongly impaired interaction with the Psf1 and Psf3 subunits of the GINS complex. Our in vitro results suggest that while dpb2-100 does not alter Pol ɛ's biochemical properties including catalytic efficiency, processivity or proofreading activity - it moderately decreases the fidelity of DNA synthesis. As the in vitro results did not explain the strong in vivo mutator effect of the dpb2-100 allele we analyzed the mutation spectrum in vivo. The analysis of the mutation rates in the dpb2-100 mutant indicated an increased participation of the error-prone DNA polymerase zeta in replication. However, even in the absence of Pol ζ activity the presence of the dpb2-100 allele was mutagenic, indicating that a significant part of mutagenesis is Pol ζ-independent. A strong synergistic mutator effect observed for transversions in the triple mutant dpb2-100 pol2-4 rev3Δ as compared to pol2-4 rev3Δ and dpb2-100 rev3Δ suggests that in the presence of the dpb2-100 allele the number of replication errors is enhanced. We hypothesize that in the dpb2-100 strain, where the interaction between Pol ɛ and GINS is weakened, the access of Pol δ to the leading strand may be increased. The increased participation of Pol δ on the leading strand in the dpb2-100 mutant may explain the synergistic mutator effect observed in the dpb2-100 pol3-5DV double mutant.
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Pawinskiego 5A, Warsaw 02-106, Poland.
authors.forename: Marta
authors.initials: M
authors.lastname: Garbacz
authors.affiliation: National Institute of Genetics, Division of Microbial Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Araki
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Pawinskiego 5A, Warsaw 02-106, Poland.
authors.forename: Krzysztof
authors.initials: K
authors.lastname: Flis
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Department of Molecular Biology, Pawinskiego 5A, Warsaw 02-106, Poland.
authors.forename: Anna
authors.initials: A
authors.lastname: Bebenek
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Pawinskiego 5A, Warsaw 02-106, Poland.
authors.forename: Anna E
authors.initials: AE
authors.lastname: Zawada
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Pawinskiego 5A, Warsaw 02-106, Poland.
authors.forename: Piotr
authors.initials: P
authors.lastname: Jonczyk
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Pawinskiego 5A, Warsaw 02-106, Poland. Electronic address: kmakiela@ibb.waw.pl.
authors.forename: Karolina
authors.initials: K
authors.lastname: Makiela-Dzbenska
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Pawinskiego 5A, Warsaw 02-106, Poland. Electronic address: fijalkowska.iwona@gmail.com.
authors.forename: Iwona J
authors.initials: IJ
authors.lastname: Fijalkowska
mesh_terms: D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004257:DNA Polymerase II / Q000235:genetics* / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D009154:Mutation; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000378:metabolism*; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; C077018:PRP8 protein, S cerevisiae; C513448:PSF3 protein, S cerevisiae; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D017415:Ribonucleoprotein, U5 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; C425929:DNA polymerase zeta; C529202:Dpb2 protein, S cerevisiae; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase
keywords: DNA leading strand; DNA polymerase delta; DNA polymerase epsilon; DNA polymerase zeta; Fidelity of replication; GINS complex