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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.

View Article: PubMed Central - PubMed

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

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Recombination Pathways Supporting the Viability of CTF4 Mutants(A–C) Tetrad dissection of ctf4Δ X sgs1 rad52Δ and ctf4Δ X sgs1 rad51Δ (A), ctf4Δ X rad59Δ (B), and ctf4Δ X rfa1-T11 (C) crossings. The expected genotypes are indicated. In (B), the line indicates elimination of superfluous lanes from the tetrad dissection plate image.(D) Deletion rate assay: single colonies of WT, ctf4Δ, pri1-M4, rad51Δ, ctf4Δ rad51Δ, and pri1-M4 rad51Δ cells (obtained from crossing FY1162 carrying the direct repeat construct, with ctf4Δ rad51Δ and pri1-M4 rad51Δ, respectively) were suspended in water and diluted before being plated on YPD, 5-FOA and low-adenine plates. Red sectors/colonies characteristic of deletion events are shown. Intra-chromosomal deletion rates and 95% confidence intervals were estimated using the maximum-likelihood method. See also Figure S5.
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fig5: Recombination Pathways Supporting the Viability of CTF4 Mutants(A–C) Tetrad dissection of ctf4Δ X sgs1 rad52Δ and ctf4Δ X sgs1 rad51Δ (A), ctf4Δ X rad59Δ (B), and ctf4Δ X rfa1-T11 (C) crossings. The expected genotypes are indicated. In (B), the line indicates elimination of superfluous lanes from the tetrad dissection plate image.(D) Deletion rate assay: single colonies of WT, ctf4Δ, pri1-M4, rad51Δ, ctf4Δ rad51Δ, and pri1-M4 rad51Δ cells (obtained from crossing FY1162 carrying the direct repeat construct, with ctf4Δ rad51Δ and pri1-M4 rad51Δ, respectively) were suspended in water and diluted before being plated on YPD, 5-FOA and low-adenine plates. Red sectors/colonies characteristic of deletion events are shown. Intra-chromosomal deletion rates and 95% confidence intervals were estimated using the maximum-likelihood method. See also Figure S5.

Mentions: To identify the TS-sensitive step that is defective in Polα/Primase/Ctf4 complex mutants, when re-priming is either affected or uncoupled from the MCM helicase, we combined ctf4Δ and pri1-M4 with a series of mutations affecting distinct HR-associated processes. We found that ctf4Δ shows synthetic sickness/lethality with rad52Δ (Kouprina et al., 1992), but not with rad51Δ (Figure 5A) or other Rad51 mediators, such as Rad55, that assist Rad51-mediated strand invasion (data not shown). We also observed a similar pattern of genetic interactions for pri1-M4 (Figure S5A).


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)

Recombination Pathways Supporting the Viability of CTF4 Mutants(A–C) Tetrad dissection of ctf4Δ X sgs1 rad52Δ and ctf4Δ X sgs1 rad51Δ (A), ctf4Δ X rad59Δ (B), and ctf4Δ X rfa1-T11 (C) crossings. The expected genotypes are indicated. In (B), the line indicates elimination of superfluous lanes from the tetrad dissection plate image.(D) Deletion rate assay: single colonies of WT, ctf4Δ, pri1-M4, rad51Δ, ctf4Δ rad51Δ, and pri1-M4 rad51Δ cells (obtained from crossing FY1162 carrying the direct repeat construct, with ctf4Δ rad51Δ and pri1-M4 rad51Δ, respectively) were suspended in water and diluted before being plated on YPD, 5-FOA and low-adenine plates. Red sectors/colonies characteristic of deletion events are shown. Intra-chromosomal deletion rates and 95% confidence intervals were estimated using the maximum-likelihood method. See also Figure S5.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
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fig5: Recombination Pathways Supporting the Viability of CTF4 Mutants(A–C) Tetrad dissection of ctf4Δ X sgs1 rad52Δ and ctf4Δ X sgs1 rad51Δ (A), ctf4Δ X rad59Δ (B), and ctf4Δ X rfa1-T11 (C) crossings. The expected genotypes are indicated. In (B), the line indicates elimination of superfluous lanes from the tetrad dissection plate image.(D) Deletion rate assay: single colonies of WT, ctf4Δ, pri1-M4, rad51Δ, ctf4Δ rad51Δ, and pri1-M4 rad51Δ cells (obtained from crossing FY1162 carrying the direct repeat construct, with ctf4Δ rad51Δ and pri1-M4 rad51Δ, respectively) were suspended in water and diluted before being plated on YPD, 5-FOA and low-adenine plates. Red sectors/colonies characteristic of deletion events are shown. Intra-chromosomal deletion rates and 95% confidence intervals were estimated using the maximum-likelihood method. See also Figure S5.
Mentions: To identify the TS-sensitive step that is defective in Polα/Primase/Ctf4 complex mutants, when re-priming is either affected or uncoupled from the MCM helicase, we combined ctf4Δ and pri1-M4 with a series of mutations affecting distinct HR-associated processes. We found that ctf4Δ shows synthetic sickness/lethality with rad52Δ (Kouprina et al., 1992), but not with rad51Δ (Figure 5A) or other Rad51 mediators, such as Rad55, that assist Rad51-mediated strand invasion (data not shown). We also observed a similar pattern of genetic interactions for pri1-M4 (Figure S5A).

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