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RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks.

Hashimoto Y, Puddu F, Costanzo V - Nat. Struct. Mol. Biol. (2011)

Bottom Line: We found that, upon fork collapse, the active CDC45-MCM-GINS (CMG) helicase complex loses its GINS subunit.PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity.These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process.

View Article: PubMed Central - PubMed

Affiliation: Genome Stability Unit, Clare Hall Laboratories, London Research Institute, South Mimms, Hertfordshire, UK.

ABSTRACT
In higher eukaryotes, the dynamics of replisome components during fork collapse and restart are poorly understood. Here we have reconstituted replication fork collapse and restart by inducing single-strand DNA lesions that create a double-strand break in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45-MCM-GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and polymerase ɛ onto DNA in a fashion that is dependent on RAD51 and MRE11 but independent of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process.

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The role of PCNA in DNA replication and chromatin association of replication proteins upon fork collapse. In (a) replication of sperm nuclei incubated in extracts for 80 min in the presence of 1 μg ml−1 aphidicolin and 0, 0.73, 0.37, 0.18 U μl−1 S1 nuclease and PCNA wild type (WT), PCNA K164R (KR), PCNA Y249A Y250A (YA) or PCNA K164R Y249A Y250A (KR YA) recombinant proteins. Replication products were resolved by neutral agarose gel and subjected to autoradiography (left). Signal intensities were quantified and reported in the graph (right). (b) Binding to chromatin of the indicated proteins was monitored by immunoblotting of chromatin treated with 200 J m−2 UV or incubated in extracts treated with 1 μg ml−1 aphidicolin, 0.97 U μl−1 S1 nuclease or 0.1 U μl−1 EcoR1 and recombinant PCNA wild type (WT), PCNA K164R (KR) or PCNA Y249A Y250A (YA) as indicated. 0.5 μl egg extract was loaded as a control (Ext). (c) The interaction of PCNA and replication proteins in egg extract was monitored by incubation of His-tagged wild type and mutant PCNA proteins followed by pull down with Ni-NTA sepharose. The interacting proteins were detected by immunoblotting as indicated.
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Figure 5: The role of PCNA in DNA replication and chromatin association of replication proteins upon fork collapse. In (a) replication of sperm nuclei incubated in extracts for 80 min in the presence of 1 μg ml−1 aphidicolin and 0, 0.73, 0.37, 0.18 U μl−1 S1 nuclease and PCNA wild type (WT), PCNA K164R (KR), PCNA Y249A Y250A (YA) or PCNA K164R Y249A Y250A (KR YA) recombinant proteins. Replication products were resolved by neutral agarose gel and subjected to autoradiography (left). Signal intensities were quantified and reported in the graph (right). (b) Binding to chromatin of the indicated proteins was monitored by immunoblotting of chromatin treated with 200 J m−2 UV or incubated in extracts treated with 1 μg ml−1 aphidicolin, 0.97 U μl−1 S1 nuclease or 0.1 U μl−1 EcoR1 and recombinant PCNA wild type (WT), PCNA K164R (KR) or PCNA Y249A Y250A (YA) as indicated. 0.5 μl egg extract was loaded as a control (Ext). (c) The interaction of PCNA and replication proteins in egg extract was monitored by incubation of His-tagged wild type and mutant PCNA proteins followed by pull down with Ni-NTA sepharose. The interacting proteins were detected by immunoblotting as indicated.

Mentions: RAD51-dependent and RAD51-independent BIR has been hypothesized to be responsible for restoration of collapsed replication forks38-41. The fork structure produced by S1 or Mung bean nucleases (Fig.1B) might trigger BIR, which plays an important role in fork restart after DSB formation in S. cerevisiae42. Recently, PCNA alleles specifically defective in BIR pol30-89 (F248A F249A) and pol30-92 (R80A), which act as dominant negative inhibitors of BIR, have been described14. To verify whether BIR operates in higher eukaryotes and is responsible for fork restart we made the equivalent mutant (Y249A Y250A) of the PCNA allele that shows the most severe phenotype in yeast and tested its effect on DNA replication and chromatin association of replication proteins in the presence of S1 nuclease (Fig. 5). PCNA mutant proteins added in excess to egg extract equilibrate with endogenous PCNA forming mutant complexes that can be loaded onto chromatin20. Under these conditions similar replication activities were obtained in the presence of wild type PCNA or PCNA K164R and S1 nuclease (Fig 5A). However, DNA replication efficiency was substantially decreased in the presence of PCNA Y249A Y250A or PCNA K164R Y249A Y250A mutant proteins (Fig.5A), suggesting that S1 nuclease treatments require BIR to promote efficient DNA replication. Consistently, PCNA Y249A Y250A decreased the chromatin binding of PSF2 in the presence of S1 (Fig.5B). In addition, we found that Pol eta and RAD51 were also decreased by PCNA Y249A Y250A (Fig.5B), suggesting that the inability of this PCNA allele to support BIR is due to defective chromatin binding of Pol eta and RAD51. We then performed a pull-down assay to examine the physical interaction between PCNA and replication proteins in egg extracts (Fig.5C). Pol delta and Pol eta were efficiently pulled down by wild type PCNA and PCNA K164R, but not by PCNA Y249A Y250A and PCNA K164R Y249A Y250A, suggesting that physical interaction with PCNA is necessary to recruit polymerase eta (Pol eta). RAD51 was instead not pulled down by PCNA, suggesting that the effects of PCNA mutant alleles on RAD51 loading onto chromatin are not due to a direct interaction. Overall, these results indicate that the BIR-defective allele of PCNA is unable to support the proper loading of RAD51 and Pol eta onto chromatin to ensure efficient replication restart.


RAD51- and MRE11-dependent reassembly of uncoupled CMG helicase complex at collapsed replication forks.

Hashimoto Y, Puddu F, Costanzo V - Nat. Struct. Mol. Biol. (2011)

The role of PCNA in DNA replication and chromatin association of replication proteins upon fork collapse. In (a) replication of sperm nuclei incubated in extracts for 80 min in the presence of 1 μg ml−1 aphidicolin and 0, 0.73, 0.37, 0.18 U μl−1 S1 nuclease and PCNA wild type (WT), PCNA K164R (KR), PCNA Y249A Y250A (YA) or PCNA K164R Y249A Y250A (KR YA) recombinant proteins. Replication products were resolved by neutral agarose gel and subjected to autoradiography (left). Signal intensities were quantified and reported in the graph (right). (b) Binding to chromatin of the indicated proteins was monitored by immunoblotting of chromatin treated with 200 J m−2 UV or incubated in extracts treated with 1 μg ml−1 aphidicolin, 0.97 U μl−1 S1 nuclease or 0.1 U μl−1 EcoR1 and recombinant PCNA wild type (WT), PCNA K164R (KR) or PCNA Y249A Y250A (YA) as indicated. 0.5 μl egg extract was loaded as a control (Ext). (c) The interaction of PCNA and replication proteins in egg extract was monitored by incubation of His-tagged wild type and mutant PCNA proteins followed by pull down with Ni-NTA sepharose. The interacting proteins were detected by immunoblotting as indicated.
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Related In: Results  -  Collection

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Figure 5: The role of PCNA in DNA replication and chromatin association of replication proteins upon fork collapse. In (a) replication of sperm nuclei incubated in extracts for 80 min in the presence of 1 μg ml−1 aphidicolin and 0, 0.73, 0.37, 0.18 U μl−1 S1 nuclease and PCNA wild type (WT), PCNA K164R (KR), PCNA Y249A Y250A (YA) or PCNA K164R Y249A Y250A (KR YA) recombinant proteins. Replication products were resolved by neutral agarose gel and subjected to autoradiography (left). Signal intensities were quantified and reported in the graph (right). (b) Binding to chromatin of the indicated proteins was monitored by immunoblotting of chromatin treated with 200 J m−2 UV or incubated in extracts treated with 1 μg ml−1 aphidicolin, 0.97 U μl−1 S1 nuclease or 0.1 U μl−1 EcoR1 and recombinant PCNA wild type (WT), PCNA K164R (KR) or PCNA Y249A Y250A (YA) as indicated. 0.5 μl egg extract was loaded as a control (Ext). (c) The interaction of PCNA and replication proteins in egg extract was monitored by incubation of His-tagged wild type and mutant PCNA proteins followed by pull down with Ni-NTA sepharose. The interacting proteins were detected by immunoblotting as indicated.
Mentions: RAD51-dependent and RAD51-independent BIR has been hypothesized to be responsible for restoration of collapsed replication forks38-41. The fork structure produced by S1 or Mung bean nucleases (Fig.1B) might trigger BIR, which plays an important role in fork restart after DSB formation in S. cerevisiae42. Recently, PCNA alleles specifically defective in BIR pol30-89 (F248A F249A) and pol30-92 (R80A), which act as dominant negative inhibitors of BIR, have been described14. To verify whether BIR operates in higher eukaryotes and is responsible for fork restart we made the equivalent mutant (Y249A Y250A) of the PCNA allele that shows the most severe phenotype in yeast and tested its effect on DNA replication and chromatin association of replication proteins in the presence of S1 nuclease (Fig. 5). PCNA mutant proteins added in excess to egg extract equilibrate with endogenous PCNA forming mutant complexes that can be loaded onto chromatin20. Under these conditions similar replication activities were obtained in the presence of wild type PCNA or PCNA K164R and S1 nuclease (Fig 5A). However, DNA replication efficiency was substantially decreased in the presence of PCNA Y249A Y250A or PCNA K164R Y249A Y250A mutant proteins (Fig.5A), suggesting that S1 nuclease treatments require BIR to promote efficient DNA replication. Consistently, PCNA Y249A Y250A decreased the chromatin binding of PSF2 in the presence of S1 (Fig.5B). In addition, we found that Pol eta and RAD51 were also decreased by PCNA Y249A Y250A (Fig.5B), suggesting that the inability of this PCNA allele to support BIR is due to defective chromatin binding of Pol eta and RAD51. We then performed a pull-down assay to examine the physical interaction between PCNA and replication proteins in egg extracts (Fig.5C). Pol delta and Pol eta were efficiently pulled down by wild type PCNA and PCNA K164R, but not by PCNA Y249A Y250A and PCNA K164R Y249A Y250A, suggesting that physical interaction with PCNA is necessary to recruit polymerase eta (Pol eta). RAD51 was instead not pulled down by PCNA, suggesting that the effects of PCNA mutant alleles on RAD51 loading onto chromatin are not due to a direct interaction. Overall, these results indicate that the BIR-defective allele of PCNA is unable to support the proper loading of RAD51 and Pol eta onto chromatin to ensure efficient replication restart.

Bottom Line: We found that, upon fork collapse, the active CDC45-MCM-GINS (CMG) helicase complex loses its GINS subunit.PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity.These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process.

View Article: PubMed Central - PubMed

Affiliation: Genome Stability Unit, Clare Hall Laboratories, London Research Institute, South Mimms, Hertfordshire, UK.

ABSTRACT
In higher eukaryotes, the dynamics of replisome components during fork collapse and restart are poorly understood. Here we have reconstituted replication fork collapse and restart by inducing single-strand DNA lesions that create a double-strand break in one of the replicated sister chromatids after fork passage. We found that, upon fork collapse, the active CDC45-MCM-GINS (CMG) helicase complex loses its GINS subunit. A functional replisome is restored by the reloading of GINS and polymerase ɛ onto DNA in a fashion that is dependent on RAD51 and MRE11 but independent of replication origin assembly and firing. PCNA mutant alleles defective in break-induced replication (BIR) are unable to support restoration of replisome integrity. These results show that, in higher eukaryotes, replisomes are partially dismantled after fork collapse and fully re-established by a recombination-mediated process.

Show MeSH