| 1. |
- Dmowski, Michal, et al.
(författare)
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Impairment of the non-catalytic subunit Dpb2 of DNA Pol ɛ results in increased involvement of Pol δ on the leading strand
- 2023
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Ingår i: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 129
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Tidskriftsartikel (refereegranskat)abstract
- The generally accepted model assumes that leading strand synthesis is performed by Pol ε, while lagging-strand synthesis is catalyzed by Pol δ. Pol ε has been shown to target the leading strand by interacting with the CMG helicase [Cdc45 Mcm2–7 GINS(Psf1–3, Sld5)]. Proper functioning of the CMG-Pol ɛ, the helicase-polymerase complex is essential for its progression and the fidelity of DNA replication. Dpb2p, the essential non-catalytic subunit of Pol ε plays a key role in maintaining the correct architecture of the replisome by acting as a link between Pol ε and the CMG complex. Using a temperature-sensitive dpb2–100 mutant previously isolated in our laboratory, and a genetic system which takes advantage of a distinct mutational signature of the Pol δ-L612M variant which allows detection of the involvement of Pol δ in the replication of particular DNA strands we show that in yeast cells with an impaired Dpb2 subunit, the contribution of Pol δ to the replication of the leading strand is significantly increased.
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| 2. |
- Dmowski, Michal, et al.
(författare)
-
Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex
- 2022
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Ingår i: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 110
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Tidskriftsartikel (refereegranskat)abstract
- DNA replication is performed by replisome proteins, which are highly conserved from yeast to humans. The CMG [Cdc45-Mcm2–7-GINS(Psf1–3, Sld5)] helicase unwinds the double helix to separate the leading and lagging DNA strands, which are replicated by the specialized DNA polymerases epsilon (Pol ε) and delta (Pol δ), respectively. This division of labor was confirmed by both genetic analyses and in vitro studies. Exceptions from this rule were described mainly in cells with impaired catalytic polymerase ε subunit. The central role in the recruitment and establishment of Pol ε on the leading strand is played by the CMG complex assembled on DNA during replication initiation. In this work we analyzed the consequences of impaired functioning of the CMG complex for the division labor between DNA polymerases on the two replicating strands. We showed in vitro that the GINSPsf1–1 complex poorly bound the Psf3 subunit. In vivo, we observed increased rates of L612M Pol δ-specific mutations during replication of the leading DNA strand in psf1–1 cells. These findings indicated that defective functioning of GINS impaired leading strand replication by Pol ε and necessitated involvement of Pol δ in the synthesis on this strand with a possible impact on the distribution of mutations and genomic stability. These are the first results to imply that the division of labor between the two main replicases can be severely influenced by a defective nonpolymerase subunit of the replisome.
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