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Error-free DNA damage tolerance and sister chromatid proximity during DNA replication rely on the Polα/Primase/Ctf4 Complex.

Fumasoni M, Zwicky K, Vanoli F, Lopes M, Branzei D - Mol. Cell (2015)

Bottom Line: We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion.The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing.Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.

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Affiliation: IFOM, the FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy.

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Ctf4 Facilitates Error-Free DDT(A) sgs1 (HY1461) and sgs1 ctf4Δ (HY1472) cells were synchronized in G1 with alpha-factor (α) at 25°C prior to release at 30°C in media containing 0.033% MMS. Genomic DNA, extracted from samples collected at the indicated time points, was digested with NcoI and analyzed by 2D gel with a probe for ARS305. Schematic representation of major 2D gel signals, FACS, and X molecule quantification plots are displayed. The columns in the quantification graphs denote the data mean of two independent experiments and the bars indicate ranges.(B) Spontaneous mutations rates at CAN1 locus (×10−7) in WT (FY0001), rev3Δ (HY0008), ctf4Δ (HY3466), and ctf4Δ rev3Δ (HY3468). Mutation rates and 95% confidence intervals were estimated using the maximum-likelihood method. Non-overlapping confidence intervals indicate statistical significance.(C) WT (FY1000) and ctf4Δ (HY2193) cells were grown at 25°C and then shifted to 30°C for 2 hr in YPD or YPD containing 0.02% MMS. PCNA modifications were detected using a monoclonal antibody against PCNA. Ponceau staining serves as loading control. See also Figure S1.
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fig1: Ctf4 Facilitates Error-Free DDT(A) sgs1 (HY1461) and sgs1 ctf4Δ (HY1472) cells were synchronized in G1 with alpha-factor (α) at 25°C prior to release at 30°C in media containing 0.033% MMS. Genomic DNA, extracted from samples collected at the indicated time points, was digested with NcoI and analyzed by 2D gel with a probe for ARS305. Schematic representation of major 2D gel signals, FACS, and X molecule quantification plots are displayed. The columns in the quantification graphs denote the data mean of two independent experiments and the bars indicate ranges.(B) Spontaneous mutations rates at CAN1 locus (×10−7) in WT (FY0001), rev3Δ (HY0008), ctf4Δ (HY3466), and ctf4Δ rev3Δ (HY3468). Mutation rates and 95% confidence intervals were estimated using the maximum-likelihood method. Non-overlapping confidence intervals indicate statistical significance.(C) WT (FY1000) and ctf4Δ (HY2193) cells were grown at 25°C and then shifted to 30°C for 2 hr in YPD or YPD containing 0.02% MMS. PCNA modifications were detected using a monoclonal antibody against PCNA. Ponceau staining serves as loading control. See also Figure S1.

Mentions: Error-free DDT by TS can be molecularly monitored by examining the formation of X-shaped structures composed of sister chromatid junctions (SCJs) in the proximity of replication forks using 2D gel electrophoresis (Branzei et al., 2008). In this assay, yeast cells are released synchronously and allowed to replicate in media containing the alkylating reagent methyl methanesulfonate (MMS). The pattern of replication intermediates at genomic locations of interest is analyzed at different time points during DNA replication. Previous results showed that TS intermediates form during replication of damaged templates and accumulate when the Sgs1-Top3-Rmi1 complex is defective because of compromised resolution (Branzei et al., 2008; Liberi et al., 2005; Giannattasio et al., 2014). As a consequence, replication of damage templates leads to a progressive accumulation of SCJs in sgs1Δ mutant cells (Liberi et al., 2005), as well as in sgs1 hypomorphic mutants disrupted only in the helicase domain (Onoda et al., 2000; Vanoli et al., 2010) (Figure 1A).


Error-free DNA damage tolerance and sister chromatid proximity during DNA replication rely on the Polα/Primase/Ctf4 Complex.

Fumasoni M, Zwicky K, Vanoli F, Lopes M, Branzei D - Mol. Cell (2015)

Ctf4 Facilitates Error-Free DDT(A) sgs1 (HY1461) and sgs1 ctf4Δ (HY1472) cells were synchronized in G1 with alpha-factor (α) at 25°C prior to release at 30°C in media containing 0.033% MMS. Genomic DNA, extracted from samples collected at the indicated time points, was digested with NcoI and analyzed by 2D gel with a probe for ARS305. Schematic representation of major 2D gel signals, FACS, and X molecule quantification plots are displayed. The columns in the quantification graphs denote the data mean of two independent experiments and the bars indicate ranges.(B) Spontaneous mutations rates at CAN1 locus (×10−7) in WT (FY0001), rev3Δ (HY0008), ctf4Δ (HY3466), and ctf4Δ rev3Δ (HY3468). Mutation rates and 95% confidence intervals were estimated using the maximum-likelihood method. Non-overlapping confidence intervals indicate statistical significance.(C) WT (FY1000) and ctf4Δ (HY2193) cells were grown at 25°C and then shifted to 30°C for 2 hr in YPD or YPD containing 0.02% MMS. PCNA modifications were detected using a monoclonal antibody against PCNA. Ponceau staining serves as loading control. See also Figure S1.
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fig1: Ctf4 Facilitates Error-Free DDT(A) sgs1 (HY1461) and sgs1 ctf4Δ (HY1472) cells were synchronized in G1 with alpha-factor (α) at 25°C prior to release at 30°C in media containing 0.033% MMS. Genomic DNA, extracted from samples collected at the indicated time points, was digested with NcoI and analyzed by 2D gel with a probe for ARS305. Schematic representation of major 2D gel signals, FACS, and X molecule quantification plots are displayed. The columns in the quantification graphs denote the data mean of two independent experiments and the bars indicate ranges.(B) Spontaneous mutations rates at CAN1 locus (×10−7) in WT (FY0001), rev3Δ (HY0008), ctf4Δ (HY3466), and ctf4Δ rev3Δ (HY3468). Mutation rates and 95% confidence intervals were estimated using the maximum-likelihood method. Non-overlapping confidence intervals indicate statistical significance.(C) WT (FY1000) and ctf4Δ (HY2193) cells were grown at 25°C and then shifted to 30°C for 2 hr in YPD or YPD containing 0.02% MMS. PCNA modifications were detected using a monoclonal antibody against PCNA. Ponceau staining serves as loading control. See also Figure S1.
Mentions: Error-free DDT by TS can be molecularly monitored by examining the formation of X-shaped structures composed of sister chromatid junctions (SCJs) in the proximity of replication forks using 2D gel electrophoresis (Branzei et al., 2008). In this assay, yeast cells are released synchronously and allowed to replicate in media containing the alkylating reagent methyl methanesulfonate (MMS). The pattern of replication intermediates at genomic locations of interest is analyzed at different time points during DNA replication. Previous results showed that TS intermediates form during replication of damaged templates and accumulate when the Sgs1-Top3-Rmi1 complex is defective because of compromised resolution (Branzei et al., 2008; Liberi et al., 2005; Giannattasio et al., 2014). As a consequence, replication of damage templates leads to a progressive accumulation of SCJs in sgs1Δ mutant cells (Liberi et al., 2005), as well as in sgs1 hypomorphic mutants disrupted only in the helicase domain (Onoda et al., 2000; Vanoli et al., 2010) (Figure 1A).

Bottom Line: We show that Saccharomyces cerevisiae Polα/Primase/Ctf4 mutants, proficient in bulk DNA replication, are defective in recombination-mediated damage-bypass by template switching (TS) and have reduced sister chromatid cohesion.The decrease in error-free DDT is accompanied by increased usage of mutagenic DDT, fork reversal, and higher rates of genome rearrangements mediated by faulty strand annealing.Defects in this event impact on replication fork architecture and sister chromatid proximity, and represent a frequent source of chromosome lesions upon replication dysfunctions.

View Article: PubMed Central - PubMed

Affiliation: IFOM, the FIRC Institute of Molecular Oncology, Via Adamello 16, 20139 Milan, Italy.

Show MeSH
Related in: MedlinePlus