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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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RAD51 is required for DNA replication in the presence of forks collapsed by a single strand break in the template. (a) The requirement of RAD51 and PCNA modification at Lys 164 for replication of undamaged sperm DNA (control) or MMS or UV treated sperm DNA was tested using GST-BRC4 (BRC4), which sequesters RAD51, and PCNA K164R mutant. Replication products (labelled with 32P-dATP) were resolved by neutral agarose gel electrophoresis and subjected to autoradiography (left). The quantification of the signal is shown in the graph as photon emission intensity (Intensity) expressed in arbitrary units (AU) (right). The data shown here and hereafter represent typical findings of 3 or more experiments. (b) A model for ssDNA specific endonuclease-dependent fork collapse and RAD51-dependent restart. (c) The requirement of RAD51 for DNA replication in the presence of S1 nuclease was tested using sperm nuclei (4000 nuclei per μl) incubated for 80 min in the presence or absence of 1 μg ml−1 aphidicolin (Low aph) and S1 nuclease (0, 2.92, 1.46, 0.73, 0.37 U μl−1). Replication products were detected by autoradiography with quantification shown on the right, as in 1a. Sybr Green staining shows total DNA (Sybr Green).
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Figure 1: RAD51 is required for DNA replication in the presence of forks collapsed by a single strand break in the template. (a) The requirement of RAD51 and PCNA modification at Lys 164 for replication of undamaged sperm DNA (control) or MMS or UV treated sperm DNA was tested using GST-BRC4 (BRC4), which sequesters RAD51, and PCNA K164R mutant. Replication products (labelled with 32P-dATP) were resolved by neutral agarose gel electrophoresis and subjected to autoradiography (left). The quantification of the signal is shown in the graph as photon emission intensity (Intensity) expressed in arbitrary units (AU) (right). The data shown here and hereafter represent typical findings of 3 or more experiments. (b) A model for ssDNA specific endonuclease-dependent fork collapse and RAD51-dependent restart. (c) The requirement of RAD51 for DNA replication in the presence of S1 nuclease was tested using sperm nuclei (4000 nuclei per μl) incubated for 80 min in the presence or absence of 1 μg ml−1 aphidicolin (Low aph) and S1 nuclease (0, 2.92, 1.46, 0.73, 0.37 U μl−1). Replication products were detected by autoradiography with quantification shown on the right, as in 1a. Sybr Green staining shows total DNA (Sybr Green).

Mentions: RAD51 is required for replication restart in yeast and mammals9-11,19. However, the mechanism of replication fork reassembly after collapse remains obscure. Using Xenopus laevis (X. laevis) egg extract as model system we set out to uncover the mechanism underlying RAD51 mediated replication fork restart. We first tested which DNA lesions produce replication fork collapse that requires RAD51 to be restarted. To this end we analysed the effects of DNA damaging agents such as methyl methanesulfonate (MMS) and ultraviolet radiation (UV) on DNA replication in the absence of RAD51 bound to chromatin. RAD51 chromatin binding was inhibited by the BRC4 domain of BRCA2 protein fused to GST, as previously shown20. Replication products were resolved on neutral agarose gel21, where the major signals could be observed as two bands; the upper one contains branched DNA, whereas the lower one corresponds to branch-free DNA (Fig.1A). The signal present in the entire lane was quantified to measure DNA replication and reported in the accompanying graph. Consistent with previous results, although DNA damage decreased the number of active replicons due to physical blockage and activation of the S-phase checkpoint20 the absence of RAD51 bound to chromatin did not cause any further impairment of DNA replication (Fig.1A). As RAD51 is involved in HR dependent post-replication repair, which can be redundantly carried out by translesion polymerases20 we tested the contribution of translesion DNA synthesis to the DNA replication efficiency in the presence of UV and MMS- treated templates by using PCNA-K164R, which suppresses the chromatin loading of translesion polymerases20. The suppression of this pathway did not affect the efficiency of DNA replication of damaged templates under the conditions used in these experiments (Fig.1A). These observations suggest RAD51 and translesion synthesis play a minor role in replication restart during MMS- or UV-challenged replication in egg extract.


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)

RAD51 is required for DNA replication in the presence of forks collapsed by a single strand break in the template. (a) The requirement of RAD51 and PCNA modification at Lys 164 for replication of undamaged sperm DNA (control) or MMS or UV treated sperm DNA was tested using GST-BRC4 (BRC4), which sequesters RAD51, and PCNA K164R mutant. Replication products (labelled with 32P-dATP) were resolved by neutral agarose gel electrophoresis and subjected to autoradiography (left). The quantification of the signal is shown in the graph as photon emission intensity (Intensity) expressed in arbitrary units (AU) (right). The data shown here and hereafter represent typical findings of 3 or more experiments. (b) A model for ssDNA specific endonuclease-dependent fork collapse and RAD51-dependent restart. (c) The requirement of RAD51 for DNA replication in the presence of S1 nuclease was tested using sperm nuclei (4000 nuclei per μl) incubated for 80 min in the presence or absence of 1 μg ml−1 aphidicolin (Low aph) and S1 nuclease (0, 2.92, 1.46, 0.73, 0.37 U μl−1). Replication products were detected by autoradiography with quantification shown on the right, as in 1a. Sybr Green staining shows total DNA (Sybr Green).
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Related In: Results  -  Collection

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Figure 1: RAD51 is required for DNA replication in the presence of forks collapsed by a single strand break in the template. (a) The requirement of RAD51 and PCNA modification at Lys 164 for replication of undamaged sperm DNA (control) or MMS or UV treated sperm DNA was tested using GST-BRC4 (BRC4), which sequesters RAD51, and PCNA K164R mutant. Replication products (labelled with 32P-dATP) were resolved by neutral agarose gel electrophoresis and subjected to autoradiography (left). The quantification of the signal is shown in the graph as photon emission intensity (Intensity) expressed in arbitrary units (AU) (right). The data shown here and hereafter represent typical findings of 3 or more experiments. (b) A model for ssDNA specific endonuclease-dependent fork collapse and RAD51-dependent restart. (c) The requirement of RAD51 for DNA replication in the presence of S1 nuclease was tested using sperm nuclei (4000 nuclei per μl) incubated for 80 min in the presence or absence of 1 μg ml−1 aphidicolin (Low aph) and S1 nuclease (0, 2.92, 1.46, 0.73, 0.37 U μl−1). Replication products were detected by autoradiography with quantification shown on the right, as in 1a. Sybr Green staining shows total DNA (Sybr Green).
Mentions: RAD51 is required for replication restart in yeast and mammals9-11,19. However, the mechanism of replication fork reassembly after collapse remains obscure. Using Xenopus laevis (X. laevis) egg extract as model system we set out to uncover the mechanism underlying RAD51 mediated replication fork restart. We first tested which DNA lesions produce replication fork collapse that requires RAD51 to be restarted. To this end we analysed the effects of DNA damaging agents such as methyl methanesulfonate (MMS) and ultraviolet radiation (UV) on DNA replication in the absence of RAD51 bound to chromatin. RAD51 chromatin binding was inhibited by the BRC4 domain of BRCA2 protein fused to GST, as previously shown20. Replication products were resolved on neutral agarose gel21, where the major signals could be observed as two bands; the upper one contains branched DNA, whereas the lower one corresponds to branch-free DNA (Fig.1A). The signal present in the entire lane was quantified to measure DNA replication and reported in the accompanying graph. Consistent with previous results, although DNA damage decreased the number of active replicons due to physical blockage and activation of the S-phase checkpoint20 the absence of RAD51 bound to chromatin did not cause any further impairment of DNA replication (Fig.1A). As RAD51 is involved in HR dependent post-replication repair, which can be redundantly carried out by translesion polymerases20 we tested the contribution of translesion DNA synthesis to the DNA replication efficiency in the presence of UV and MMS- treated templates by using PCNA-K164R, which suppresses the chromatin loading of translesion polymerases20. The suppression of this pathway did not affect the efficiency of DNA replication of damaged templates under the conditions used in these experiments (Fig.1A). These observations suggest RAD51 and translesion synthesis play a minor role in replication restart during MMS- or UV-challenged replication in egg extract.

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
Related in: MedlinePlus