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Sister chromatid cohesion establishment occurs in concert with lagging strand synthesis.

Rudra S, Skibbens RV - Cell Cycle (2012)

Bottom Line: Our genetic and biochemical studies link Ctf7/Eco1 to the Okazaki fragment flap endonuclease, Fen1.We further report genetic and biochemical interactions between Fen1 and the cohesion-associated DNA helicase, Chl1.These results raise a new model wherein cohesin deposition and establishment occur in concert with lagging strand-processing events and in the presence of both sister chromatids.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA.

ABSTRACT
Cohesion establishment is central to sister chromatid tethering reactions and requires Ctf7/Eco1-dependent acetylation of the cohesin subunit Smc3. Ctf7/Eco1 is essential during S phase, and a number of replication proteins (RFC complexes, PCNA and the DNA helicase Chl1) all play individual roles in sister chromatid cohesion. While the mechanism of cohesion establishment is largely unknown, a popular model is that Ctf7/Eco1 acetylates cohesins encountered by and located in front of the fork. In turn, acetylation is posited both to allow fork passage past cohesin barriers and convert cohesins to a state competent to capture subsequent production of sister chromatids. Here, we report evidence that challenges this pre-replicative cohesion establishment model. Our genetic and biochemical studies link Ctf7/Eco1 to the Okazaki fragment flap endonuclease, Fen1. We further report genetic and biochemical interactions between Fen1 and the cohesion-associated DNA helicase, Chl1. These results raise a new model wherein cohesin deposition and establishment occur in concert with lagging strand-processing events and in the presence of both sister chromatids.

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Related in: MedlinePlus

Figure 1.ctf7eco1–1 is synthetically lethal in combination with fen1. Yeast cells harboring ctf7eco1–1: ADE were crossed with fen1::KANr mutant cells and the resulting diploids were transformed with a CEN: URA3:CTF7ECO1 plasmid and sporulated. The resulting fen1 and ctf7eco1–1: ADE single mutants and fen1, ctf7eco1–1: ADE double mutants were plated on media with or without FOA (See also Table 1). Two independent isolates are shown for each strain. (A) Growth of fen1, ctf7eco1–1 single mutants and fen1::KAN ctf7eco1–1 CTF7: URA double mutants strains at 23°C on YPD. (B) Growth of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutants on FOA plates at 23°C. (C) Growth of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutants on FOA plates at 30°C (See also Fig. S1). (D) Schematic representation of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutant strains.
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Figure 1: Figure 1.ctf7eco1–1 is synthetically lethal in combination with fen1. Yeast cells harboring ctf7eco1–1: ADE were crossed with fen1::KANr mutant cells and the resulting diploids were transformed with a CEN: URA3:CTF7ECO1 plasmid and sporulated. The resulting fen1 and ctf7eco1–1: ADE single mutants and fen1, ctf7eco1–1: ADE double mutants were plated on media with or without FOA (See also Table 1). Two independent isolates are shown for each strain. (A) Growth of fen1, ctf7eco1–1 single mutants and fen1::KAN ctf7eco1–1 CTF7: URA double mutants strains at 23°C on YPD. (B) Growth of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutants on FOA plates at 23°C. (C) Growth of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutants on FOA plates at 30°C (See also Fig. S1). (D) Schematic representation of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutant strains.

Mentions: In contrast to the popular notion that Ctf7/Eco1 acts on cohesins positioned in front of the DNA replication fork, Ctf7/Eco1 positioning relative to the fork remains unknown. Several studies point to factors that function behind the fork, such as the flap endonuclease Fen1, as critical for sister chromatid pairing and formally raise a model that establishment occurs as sister chromatids emerge from behind the fork.23,27 To test this alternate model, yeast cells harboring ctf7eco1-1:ADE were crossed with fen1::KANr mutant cells, and the resulting diploids were placed in sporulation medium. Notably, heterozygous ctf7eco1-1:ADE/CTF7 FEN1/fen1 cells exhibited extremely poor sporulation efficiency (< 2%). When diploid cells were first transformed with CEN-URA3-CTF7ECO1 plasmid, however, the resulting transformants sporulated with high efficiency (approximately 85%). Similar haplo-insuffiency in sporulation was previously observed in crosses involving ctf7/eco1 (Brands and Skibbens, unpublished observation), revealing that Ctf7/Eco1 performs an essential but dosage-dependent role during meiosis. Of the 112 spores obtained from sporulated transformed diploids, we recovered the expected number of wild-type cells and both fen1 and ctf7/eco1 single mutant cells (Table 1). In contrast, only seven double mutant fen1 ctf7/eco1 cells were recovered at 23°C, six of which harbored CTF7/ECO1 plasmid. Upon plating onto media supplemented with 5' FOA,28 all six plasmid-bearing double mutant strains were inviable, revealing a synthetic lethal interaction between ctf7/eco1 and fen1 mutations (Fig. 1). The single double mutant spore exhibited robust growth at all temperatures and, thus, likely results from meiotic gene conversion or incorporation of an extragenic suppressor (Fig. S1).


Sister chromatid cohesion establishment occurs in concert with lagging strand synthesis.

Rudra S, Skibbens RV - Cell Cycle (2012)

Figure 1.ctf7eco1–1 is synthetically lethal in combination with fen1. Yeast cells harboring ctf7eco1–1: ADE were crossed with fen1::KANr mutant cells and the resulting diploids were transformed with a CEN: URA3:CTF7ECO1 plasmid and sporulated. The resulting fen1 and ctf7eco1–1: ADE single mutants and fen1, ctf7eco1–1: ADE double mutants were plated on media with or without FOA (See also Table 1). Two independent isolates are shown for each strain. (A) Growth of fen1, ctf7eco1–1 single mutants and fen1::KAN ctf7eco1–1 CTF7: URA double mutants strains at 23°C on YPD. (B) Growth of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutants on FOA plates at 23°C. (C) Growth of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutants on FOA plates at 30°C (See also Fig. S1). (D) Schematic representation of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutant strains.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Figure 1.ctf7eco1–1 is synthetically lethal in combination with fen1. Yeast cells harboring ctf7eco1–1: ADE were crossed with fen1::KANr mutant cells and the resulting diploids were transformed with a CEN: URA3:CTF7ECO1 plasmid and sporulated. The resulting fen1 and ctf7eco1–1: ADE single mutants and fen1, ctf7eco1–1: ADE double mutants were plated on media with or without FOA (See also Table 1). Two independent isolates are shown for each strain. (A) Growth of fen1, ctf7eco1–1 single mutants and fen1::KAN ctf7eco1–1 CTF7: URA double mutants strains at 23°C on YPD. (B) Growth of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutants on FOA plates at 23°C. (C) Growth of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutants on FOA plates at 30°C (See also Fig. S1). (D) Schematic representation of fen1::KAN, ctf7eco1–1 single mutants and fen1 ctf7eco1–1: ADE CTF7: URA double mutant strains.
Mentions: In contrast to the popular notion that Ctf7/Eco1 acts on cohesins positioned in front of the DNA replication fork, Ctf7/Eco1 positioning relative to the fork remains unknown. Several studies point to factors that function behind the fork, such as the flap endonuclease Fen1, as critical for sister chromatid pairing and formally raise a model that establishment occurs as sister chromatids emerge from behind the fork.23,27 To test this alternate model, yeast cells harboring ctf7eco1-1:ADE were crossed with fen1::KANr mutant cells, and the resulting diploids were placed in sporulation medium. Notably, heterozygous ctf7eco1-1:ADE/CTF7 FEN1/fen1 cells exhibited extremely poor sporulation efficiency (< 2%). When diploid cells were first transformed with CEN-URA3-CTF7ECO1 plasmid, however, the resulting transformants sporulated with high efficiency (approximately 85%). Similar haplo-insuffiency in sporulation was previously observed in crosses involving ctf7/eco1 (Brands and Skibbens, unpublished observation), revealing that Ctf7/Eco1 performs an essential but dosage-dependent role during meiosis. Of the 112 spores obtained from sporulated transformed diploids, we recovered the expected number of wild-type cells and both fen1 and ctf7/eco1 single mutant cells (Table 1). In contrast, only seven double mutant fen1 ctf7/eco1 cells were recovered at 23°C, six of which harbored CTF7/ECO1 plasmid. Upon plating onto media supplemented with 5' FOA,28 all six plasmid-bearing double mutant strains were inviable, revealing a synthetic lethal interaction between ctf7/eco1 and fen1 mutations (Fig. 1). The single double mutant spore exhibited robust growth at all temperatures and, thus, likely results from meiotic gene conversion or incorporation of an extragenic suppressor (Fig. S1).

Bottom Line: Our genetic and biochemical studies link Ctf7/Eco1 to the Okazaki fragment flap endonuclease, Fen1.We further report genetic and biochemical interactions between Fen1 and the cohesion-associated DNA helicase, Chl1.These results raise a new model wherein cohesin deposition and establishment occur in concert with lagging strand-processing events and in the presence of both sister chromatids.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Lehigh University, Bethlehem, PA, USA.

ABSTRACT
Cohesion establishment is central to sister chromatid tethering reactions and requires Ctf7/Eco1-dependent acetylation of the cohesin subunit Smc3. Ctf7/Eco1 is essential during S phase, and a number of replication proteins (RFC complexes, PCNA and the DNA helicase Chl1) all play individual roles in sister chromatid cohesion. While the mechanism of cohesion establishment is largely unknown, a popular model is that Ctf7/Eco1 acetylates cohesins encountered by and located in front of the fork. In turn, acetylation is posited both to allow fork passage past cohesin barriers and convert cohesins to a state competent to capture subsequent production of sister chromatids. Here, we report evidence that challenges this pre-replicative cohesion establishment model. Our genetic and biochemical studies link Ctf7/Eco1 to the Okazaki fragment flap endonuclease, Fen1. We further report genetic and biochemical interactions between Fen1 and the cohesion-associated DNA helicase, Chl1. These results raise a new model wherein cohesin deposition and establishment occur in concert with lagging strand-processing events and in the presence of both sister chromatids.

Show MeSH
Related in: MedlinePlus