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

Rudra S, Skibbens RV - Cell Cycle (2012)

Bottom Line: 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.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.

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 2. Fen1 and Ctf7/Eco1 physically associate in vivo. Cells expressing Fen1:13Myc and Ctf7/Eco1:3HA were mechanically lysed and clarified by centrifugation, the clarified whole cell extract was co-immunoprecipitated using anti-Myc beads (2A) and anti HA Beads (2D) and analyzed by immunoblotting for Fen1:13Myc and Ctf7/Eco1:3HA. Whole cell extracts (WCE, lanes 1–4), Supernatants (SUP, lanes 5–8) and pull down fractions (PD, 9–12) are shown. (A) Co-immunoprecipitation of Fen1:13Myc and Ctf7:3HA with anti-MYC beads. Cells expressing only 3HA tag (Lane 11) and cells expressing Ctf7:3HA but untagged Fen1 (Lane 12) were used to determine the specificity of the co-immunoprecipitation. (B) Clarified whole cell extracts of cells co-expressing Fen1:13Myc and Ctf7:/Eco1:3HA were treated with and without DNaseI before 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 co-immunoprecipitation of cells expressing Fen1:13Myc and Ctf7:3HA using Anti-HA affinity beads. Cell expressing untagged Ctf7/Eco1 (Lane 9) and cells expressing 3HA tags alone (Lane 11) were used as a control to determine the specificity of the co-immunoprecipitation reaction.
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Figure 2: Figure 2. Fen1 and Ctf7/Eco1 physically associate in vivo. Cells expressing Fen1:13Myc and Ctf7/Eco1:3HA were mechanically lysed and clarified by centrifugation, the clarified whole cell extract was co-immunoprecipitated using anti-Myc beads (2A) and anti HA Beads (2D) and analyzed by immunoblotting for Fen1:13Myc and Ctf7/Eco1:3HA. Whole cell extracts (WCE, lanes 1–4), Supernatants (SUP, lanes 5–8) and pull down fractions (PD, 9–12) are shown. (A) Co-immunoprecipitation of Fen1:13Myc and Ctf7:3HA with anti-MYC beads. Cells expressing only 3HA tag (Lane 11) and cells expressing Ctf7:3HA but untagged Fen1 (Lane 12) were used to determine the specificity of the co-immunoprecipitation. (B) Clarified whole cell extracts of cells co-expressing Fen1:13Myc and Ctf7:/Eco1:3HA were treated with and without DNaseI before 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 co-immunoprecipitation of cells expressing Fen1:13Myc and Ctf7:3HA using Anti-HA affinity beads. Cell expressing untagged Ctf7/Eco1 (Lane 9) and cells expressing 3HA tags alone (Lane 11) were used as a control to determine the specificity of the co-immunoprecipitation reaction.

Mentions: If the above lagging strand-coupled establishment model is correct, then each of these proteins may physically interact. To test this prediction, we transformed cells expressing Fen1-13Myc with either a construct directing elevated expression of Ctf7/Eco1–3HA or 3HA alone as a control. Logarithmically growing cultures of the resulting transformants were lysed, and cell extracts were incubated with anti-MYC beads. After several washes, bound protein complexes were eluted and assayed by western blot. As expected, Fen1-13MYC was efficiently immunoprecipitated through this procedure (Fig. 2A). Probing duplicate membranes with HA-directed antibodies revealed that Ctf7/Eco1–3HA co-immunoprecipitated with Fen1-13MYC but was not pulled down from lysates obtained from cells expressing untagged Fen1 (Fig. 2A). The role of Fen1 in DNA modification raised the possibility that the Fen1-Ctf7/Eco1 association might be mediated through DNA. To address this possibility, we repeated the co-immunoprecipitation but included DNaseI in the cell lysate. The results show that Ctf7-3HA continues to efficiently co-immunoprecipitate with Fen1-13MYC in the absence of DNA (Fig. 2B). Complete degradation of lambda DNA that was spiked into the co-immunoprecipitation reaction confirmed the efficacy of the DNaseI treatment (Fig. 2C).


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

Rudra S, Skibbens RV - Cell Cycle (2012)

Figure 2. Fen1 and Ctf7/Eco1 physically associate in vivo. Cells expressing Fen1:13Myc and Ctf7/Eco1:3HA were mechanically lysed and clarified by centrifugation, the clarified whole cell extract was co-immunoprecipitated using anti-Myc beads (2A) and anti HA Beads (2D) and analyzed by immunoblotting for Fen1:13Myc and Ctf7/Eco1:3HA. Whole cell extracts (WCE, lanes 1–4), Supernatants (SUP, lanes 5–8) and pull down fractions (PD, 9–12) are shown. (A) Co-immunoprecipitation of Fen1:13Myc and Ctf7:3HA with anti-MYC beads. Cells expressing only 3HA tag (Lane 11) and cells expressing Ctf7:3HA but untagged Fen1 (Lane 12) were used to determine the specificity of the co-immunoprecipitation. (B) Clarified whole cell extracts of cells co-expressing Fen1:13Myc and Ctf7:/Eco1:3HA were treated with and without DNaseI before 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 co-immunoprecipitation of cells expressing Fen1:13Myc and Ctf7:3HA using Anti-HA affinity beads. Cell expressing untagged Ctf7/Eco1 (Lane 9) and cells expressing 3HA tags alone (Lane 11) were used as a control to determine the specificity of the co-immunoprecipitation reaction.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3368863&req=5

Figure 2: Figure 2. Fen1 and Ctf7/Eco1 physically associate in vivo. Cells expressing Fen1:13Myc and Ctf7/Eco1:3HA were mechanically lysed and clarified by centrifugation, the clarified whole cell extract was co-immunoprecipitated using anti-Myc beads (2A) and anti HA Beads (2D) and analyzed by immunoblotting for Fen1:13Myc and Ctf7/Eco1:3HA. Whole cell extracts (WCE, lanes 1–4), Supernatants (SUP, lanes 5–8) and pull down fractions (PD, 9–12) are shown. (A) Co-immunoprecipitation of Fen1:13Myc and Ctf7:3HA with anti-MYC beads. Cells expressing only 3HA tag (Lane 11) and cells expressing Ctf7:3HA but untagged Fen1 (Lane 12) were used to determine the specificity of the co-immunoprecipitation. (B) Clarified whole cell extracts of cells co-expressing Fen1:13Myc and Ctf7:/Eco1:3HA were treated with and without DNaseI before 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 co-immunoprecipitation of cells expressing Fen1:13Myc and Ctf7:3HA using Anti-HA affinity beads. Cell expressing untagged Ctf7/Eco1 (Lane 9) and cells expressing 3HA tags alone (Lane 11) were used as a control to determine the specificity of the co-immunoprecipitation reaction.
Mentions: If the above lagging strand-coupled establishment model is correct, then each of these proteins may physically interact. To test this prediction, we transformed cells expressing Fen1-13Myc with either a construct directing elevated expression of Ctf7/Eco1–3HA or 3HA alone as a control. Logarithmically growing cultures of the resulting transformants were lysed, and cell extracts were incubated with anti-MYC beads. After several washes, bound protein complexes were eluted and assayed by western blot. As expected, Fen1-13MYC was efficiently immunoprecipitated through this procedure (Fig. 2A). Probing duplicate membranes with HA-directed antibodies revealed that Ctf7/Eco1–3HA co-immunoprecipitated with Fen1-13MYC but was not pulled down from lysates obtained from cells expressing untagged Fen1 (Fig. 2A). The role of Fen1 in DNA modification raised the possibility that the Fen1-Ctf7/Eco1 association might be mediated through DNA. To address this possibility, we repeated the co-immunoprecipitation but included DNaseI in the cell lysate. The results show that Ctf7-3HA continues to efficiently co-immunoprecipitate with Fen1-13MYC in the absence of DNA (Fig. 2B). Complete degradation of lambda DNA that was spiked into the co-immunoprecipitation reaction confirmed the efficacy of the DNaseI treatment (Fig. 2C).

Bottom Line: 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.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.

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