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.
Highlights
Proper functioning of the eukaryotic genome depends on the accu rate replication of genetic material, which relies on the coordinated synthesis of the leading and lagging DNA strands by replicative DNA polymerases in concert with other proteins
Pol ζ belongs to translesion synthesis polymerases (TLS); it participates in the process of DNA synthesis when replicative polymerases or accessory subunits of the replisome are impaired, causing defective replisome-induced mutagenesis (DRIM) [11,19,20,21,22,23,24,25]
The genetic system for analysis of leading/lagging strand replication Given that the CMG complex is involved in the specific recruitment of Pol ε through its interaction with GINS, our goal was to investigate whether GINS impairment due to defective complex formation could result in an increased contribution of Pol δ to leading strand replication
Summary
Proper functioning of the eukaryotic genome depends on the accu rate replication of genetic material, which relies on the coordinated synthesis of the leading and lagging DNA strands by replicative DNA polymerases in concert with other proteins. Yeast Pol δ consists of three subunits: catalytic Pol and two noncatalytic Pol and Pol32 [9] These two polymerases possess 3′→5′ proofreading exonuclease activity, which contributes to high-fidelity DNA synthesis. A polymerase that has a significant impact on the level of spontaneous mutagenesis is Pol ζ (zeta) [10,11,12,13] This polymerase has no 3′→5′ proofreading exonuclease activity and, compared with the main replicative polymerases, generates more errors during DNA synthesis [14,15]. Pol ζ belongs to translesion synthesis polymerases (TLS); it participates in the process of DNA synthesis when replicative polymerases or accessory subunits of the replisome are impaired, causing defective replisome-induced mutagenesis (DRIM) [11,19,20,21,22,23,24,25]
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