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Fidelity consequences of the impaired interaction between DNA polymerase epsilon and the GINS complex.

DNA Repair (Amst.). 2015 May;29:23-35. Epub 2015 Feb 16
Marta Garbacz 1 , Hiroyuki Araki 2 , Krzysztof Flis 1 , Anna Bebenek 3 , Anna E Zawada 1 , Piotr Jonczyk 1 , Karolina Makiela-Dzbenska 4 , Iwona J Fijalkowska 5
Marta Garbacz 1 , Hiroyuki Araki 2 , Krzysztof Flis 1 , Anna Bebenek 3 , Anna E Zawada 1 , Piotr Jonczyk 1 , Karolina Makiela-Dzbenska 4 , Iwona J Fijalkowska 5
+ et al

[No authors listed]

Author information
  • 1 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Laboratory of Mutagenesis and DNA Repair, Pawinskiego 5A, Warsaw 02-106, Poland.
  • 2 National Institute of Genetics, Division of Microbial Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
  • 3 Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Department of Molecular Biology, Pawinskiego 5A, Warsaw 02-106, Poland.
  • 4 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.
  • 5 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.

摘要


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.

KEYWORDS: DNA leading strand, DNA polymerase delta, DNA polymerase epsilon, DNA polymerase zeta, Fidelity of replication, GINS complex

基因