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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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Figure 3. Chl1:13Myc physically associate with Fen1:3HA in vivo. Cells expressing Chl1:13Myc and Fen1:3HA were mechanically lysed and clarified by centrifugation. The clarified whole cell extract was co-immunoprecipitated using anti-Myc beads (3A) and anti HA beads (3B) and analyzed by immunoblotting for Chl1:13Myc and Fen1:3HA. Whole cell extracts (WCE, lanes 1–3), Supernatants (SUP, lanes 4–6) and pull down fractions (PD, 7–9) are shown. (A) Co-immunoprecipitation of Chl1:13Myc and Fen1:3HA using anti-MYC beads. Cells expressing untagged Fen1 cell extracts were used to determine the specificity of the co-immunoprecipitation reaction (Lane 8). (B) Clarified whole cell extracts of cells co-expressing Chl1:13Myc and Fen1:3HA were treated with or without DNaseI treatment before co-immunoprecipitation with anti-MYC beads. (C) 1 μg of λ DNA added in the clarified whole cell extract with and without DNaseI treatment, run on a 1% agarose gel. (D) Reciprocal immunoprecipitation of cells co-expressing Chl1:13Myc and Fen1:3HA with Anti-HA beads. Cells expressing untagged Fen1 were used to determine the specificity of the co-immunoprecipitation reaction (Lane 7).
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Figure 3: Figure 3. Chl1:13Myc physically associate with Fen1:3HA in vivo. Cells expressing Chl1:13Myc and Fen1:3HA were mechanically lysed and clarified by centrifugation. The clarified whole cell extract was co-immunoprecipitated using anti-Myc beads (3A) and anti HA beads (3B) and analyzed by immunoblotting for Chl1:13Myc and Fen1:3HA. Whole cell extracts (WCE, lanes 1–3), Supernatants (SUP, lanes 4–6) and pull down fractions (PD, 7–9) are shown. (A) Co-immunoprecipitation of Chl1:13Myc and Fen1:3HA using anti-MYC beads. Cells expressing untagged Fen1 cell extracts were used to determine the specificity of the co-immunoprecipitation reaction (Lane 8). (B) Clarified whole cell extracts of cells co-expressing Chl1:13Myc and Fen1:3HA were treated with or without DNaseI treatment before co-immunoprecipitation with anti-MYC beads. (C) 1 μg of λ DNA added in the clarified whole cell extract with and without DNaseI treatment, run on a 1% agarose gel. (D) Reciprocal immunoprecipitation of cells co-expressing Chl1:13Myc and Fen1:3HA with Anti-HA beads. Cells expressing untagged Fen1 were used to determine the specificity of the co-immunoprecipitation reaction (Lane 7).

Mentions: Fen1 flap endonuclease activity is stimulated by hChlR1,23 and both participate in cohesion.22,24-26 To test the possibility that Chl1 physically associates with Fen1, and thus provide positional information regarding Chl1 function relative to the DNA replication fork, cell lysates obtained from logarithmically growing cells co-expressing Chl1-13MYC and Fen1–3HA were incubated with anti-MYC beads. As before, bound proteins were eluted from washed beads and assayed by western blot. The results show efficient immunoprecipitation of Chl1-13MYC (Fig. 3A). Duplicate membranes probed for anti-HA antibodies reveals that Fen1–3HA co-immunoprecipitates with Chl1-13MYC but not with untagged Chl1 (Fig. 3A). We next tested whether Chl1 binding to Fen1 depended on DNA by including DNaseI in the cell lysate prior to pull down. The results show that Fen1–3HA efficiently co-immunoprecipitated with Chl1-13MYC even in the absence of DNA (Fig. 3B). Complete degradation of lambda DNA spiked into the co-immunoprecipitation reaction confirmed the efficacy of the DNaseI treatment (Fig. 3C).


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

Rudra S, Skibbens RV - Cell Cycle (2012)

Figure 3. Chl1:13Myc physically associate with Fen1:3HA in vivo. Cells expressing Chl1:13Myc and Fen1:3HA were mechanically lysed and clarified by centrifugation. The clarified whole cell extract was co-immunoprecipitated using anti-Myc beads (3A) and anti HA beads (3B) and analyzed by immunoblotting for Chl1:13Myc and Fen1:3HA. Whole cell extracts (WCE, lanes 1–3), Supernatants (SUP, lanes 4–6) and pull down fractions (PD, 7–9) are shown. (A) Co-immunoprecipitation of Chl1:13Myc and Fen1:3HA using anti-MYC beads. Cells expressing untagged Fen1 cell extracts were used to determine the specificity of the co-immunoprecipitation reaction (Lane 8). (B) Clarified whole cell extracts of cells co-expressing Chl1:13Myc and Fen1:3HA were treated with or without DNaseI treatment before co-immunoprecipitation with anti-MYC beads. (C) 1 μg of λ DNA added in the clarified whole cell extract with and without DNaseI treatment, run on a 1% agarose gel. (D) Reciprocal immunoprecipitation of cells co-expressing Chl1:13Myc and Fen1:3HA with Anti-HA beads. Cells expressing untagged Fen1 were used to determine the specificity of the co-immunoprecipitation reaction (Lane 7).
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Figure 3: Figure 3. Chl1:13Myc physically associate with Fen1:3HA in vivo. Cells expressing Chl1:13Myc and Fen1:3HA were mechanically lysed and clarified by centrifugation. The clarified whole cell extract was co-immunoprecipitated using anti-Myc beads (3A) and anti HA beads (3B) and analyzed by immunoblotting for Chl1:13Myc and Fen1:3HA. Whole cell extracts (WCE, lanes 1–3), Supernatants (SUP, lanes 4–6) and pull down fractions (PD, 7–9) are shown. (A) Co-immunoprecipitation of Chl1:13Myc and Fen1:3HA using anti-MYC beads. Cells expressing untagged Fen1 cell extracts were used to determine the specificity of the co-immunoprecipitation reaction (Lane 8). (B) Clarified whole cell extracts of cells co-expressing Chl1:13Myc and Fen1:3HA were treated with or without DNaseI treatment before co-immunoprecipitation with anti-MYC beads. (C) 1 μg of λ DNA added in the clarified whole cell extract with and without DNaseI treatment, run on a 1% agarose gel. (D) Reciprocal immunoprecipitation of cells co-expressing Chl1:13Myc and Fen1:3HA with Anti-HA beads. Cells expressing untagged Fen1 were used to determine the specificity of the co-immunoprecipitation reaction (Lane 7).
Mentions: Fen1 flap endonuclease activity is stimulated by hChlR1,23 and both participate in cohesion.22,24-26 To test the possibility that Chl1 physically associates with Fen1, and thus provide positional information regarding Chl1 function relative to the DNA replication fork, cell lysates obtained from logarithmically growing cells co-expressing Chl1-13MYC and Fen1–3HA were incubated with anti-MYC beads. As before, bound proteins were eluted from washed beads and assayed by western blot. The results show efficient immunoprecipitation of Chl1-13MYC (Fig. 3A). Duplicate membranes probed for anti-HA antibodies reveals that Fen1–3HA co-immunoprecipitates with Chl1-13MYC but not with untagged Chl1 (Fig. 3A). We next tested whether Chl1 binding to Fen1 depended on DNA by including DNaseI in the cell lysate prior to pull down. The results show that Fen1–3HA efficiently co-immunoprecipitated with Chl1-13MYC even in the absence of DNA (Fig. 3B). Complete degradation of lambda DNA spiked into the co-immunoprecipitation reaction confirmed the efficacy of the DNaseI treatment (Fig. 3C).

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