Recombination and Pol ζ Rescue Defective DNA Replication upon Impaired CMG Helicase—Pol ε Interaction

Abstract

The CMG complex (Cdc45, Mcm2–7, GINS (Psf1, 2, 3, and Sld5)) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.

Post-Translational Modifications of PCNA: Guiding for the Best DNA Damage Tolerance Choice

“…Defective-Replisome-Induced-Mutagenesis (DRIM) occurs when problems in replication factors, affecting replisome integrity or Polα, Polδ or Polε, promote the use of Polζ to continue DNA synthesis copying undamaged DNA. As a consequence, the low fidelity of Polζ causes an increase in the mutational rate [ 195 , 196 , 197 , 198 ]. PCNA monoubiquitination at K164 and Polζ recruitment were also observed during DRIM.…”

Two independent DNA repair pathways cause mutagenesis in template switching deficientSaccharomyces cerevisiae

“…While the error-prone TLS polymerases are implicated in bypassing DNA lesions, several lines of evidence indicate roles for TLS polymerases in repair events that do not involve DNA damage. DNA replisome mutants incur TLS polymerase-dependent mutations (Northam et al 2006;Aksenova et al 2010;Becker et al 2014;Denkiewicz-Kruk et al 2020), as do strains depleted of dNTPs (Northam et al 2010;Gallo et al 2019). Thus, replication stress in the form of DNA base lesions, and lesion-less replication stress induced genetically or chemically, are both overcome by the action of error-prone DNA polymerases, promoting cell survival at the cost of mutagenesis.Two independent DNA repair pathways cause mutagenesis in template switching deficient Saccharomyces cerevisiae…”