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Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts.

Leach CA, Michael WM - J. Cell Biol. (2005)

Bottom Line: When sumoylation alone is prevented, replication occurs normally and neither monoubiquitylation nor sumoylation are required for the replication of simple single-strand DNA templates.Our findings expand the repertoire of functions for PCNA ubiquitylation and sumoylation by elucidating a role for these modifications during the replication of undamaged DNA.Furthermore, they suggest that PCNA monoubiquitylation serves as a molecular gas pedal that controls the speed of replisome movement during S phase.

View Article: PubMed Central - PubMed

Affiliation: The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

ABSTRACT
The homotrimeric DNA replication protein proliferating cell nuclear antigen (PCNA) is regulated by both ubiquitylation and sumoylation. We study the appearance and the impact of these modifications on chromosomal replication in frog egg extracts. Xenopus laevis PCNA is modified on lysine 164 by sumoylation, monoubiquitylation, and diubiquitylation. Sumoylation and monoubiquitylation occur during the replication of undamaged DNA, whereas diubiquitylation occurs specifically in response to DNA damage. When lysine 164 modification is prevented, replication fork movement through undamaged DNA slows down and DNA polymerase delta fails to associate with replicating chromatin. When sumoylation alone is prevented, replication occurs normally and neither monoubiquitylation nor sumoylation are required for the replication of simple single-strand DNA templates. Our findings expand the repertoire of functions for PCNA ubiquitylation and sumoylation by elucidating a role for these modifications during the replication of undamaged DNA. Furthermore, they suggest that PCNA monoubiquitylation serves as a molecular gas pedal that controls the speed of replisome movement during S phase.

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DNA damage induces polyubiquitylation of PCNA. (A) UV-damaged sperm chromatin was isolated from X. laevis egg extract after 30, 60, 90, or 120 min. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. The control portions of this experiment are reproduced from Fig. 1 A to facilitate comparison. (B) Duplicate samples from A contained 32P-dATP and DNA replication was measured as described in Materials and methods. Closed circles represent undamaged chromatin and open squares represent UV-damaged chromatin. (C) Undamaged chromatin, MMS-damaged chromatin, or UV-damaged chromatin was isolated from X. laevis egg extract after 45 min. Aphidicolin was added to 100 μg/ml at t = −10 min to the extract incubated with undamaged chromatin. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. (D) X. laevis egg extract, preincubated with 100 μg/ml aphidicolin, was incubated with sperm chromatin and either buffer, 0.2 μg/μL T7-PCNA (rWT), or 0.2 μg/μL T7-PCNA (rK164R). Chromatin was isolated and analyzed by immunoblotting with an antibody recognizing either PCNA or the T7 tag. (E) X. laevis egg extract was preincubated with 100 μg/ml aphidicolin. Undamaged sperm chromatin was incubated in this extract which also contained buffer, 0.5 μg/μL His-tagged ubiquitin, or 0.5 μg/μL His-tagged ubiquitin (K63R). Chromatin was isolated and the bound proteins were analyzed by Western blotting with an antibody recognizing PCNA.
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fig8: DNA damage induces polyubiquitylation of PCNA. (A) UV-damaged sperm chromatin was isolated from X. laevis egg extract after 30, 60, 90, or 120 min. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. The control portions of this experiment are reproduced from Fig. 1 A to facilitate comparison. (B) Duplicate samples from A contained 32P-dATP and DNA replication was measured as described in Materials and methods. Closed circles represent undamaged chromatin and open squares represent UV-damaged chromatin. (C) Undamaged chromatin, MMS-damaged chromatin, or UV-damaged chromatin was isolated from X. laevis egg extract after 45 min. Aphidicolin was added to 100 μg/ml at t = −10 min to the extract incubated with undamaged chromatin. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. (D) X. laevis egg extract, preincubated with 100 μg/ml aphidicolin, was incubated with sperm chromatin and either buffer, 0.2 μg/μL T7-PCNA (rWT), or 0.2 μg/μL T7-PCNA (rK164R). Chromatin was isolated and analyzed by immunoblotting with an antibody recognizing either PCNA or the T7 tag. (E) X. laevis egg extract was preincubated with 100 μg/ml aphidicolin. Undamaged sperm chromatin was incubated in this extract which also contained buffer, 0.5 μg/μL His-tagged ubiquitin, or 0.5 μg/μL His-tagged ubiquitin (K63R). Chromatin was isolated and the bound proteins were analyzed by Western blotting with an antibody recognizing PCNA.

Mentions: The results presented thus far indicate that PCNA is monoubiquitylated and sumoylated during a normal, uninterrupted S phase and that monoubiquitylation is required for efficient replication fork progression. Thus, these findings represent a departure from studies in yeast and human cells where monoubiquitylation is only readily observed after DNA damage (Introduction). Therefore, it was important to determine the status of PCNA in our system after DNA damage. For this we used sperm chromatin that had been damaged by exposure to UV light. This chromatin was incubated in egg extract and isolated, and PCNA was examined by immunoblotting. The inclusion of damaged chromatin resulted in a third slowly migrating band on the PCNA blot, suggesting that PCNA had undergone an additional modification in response to the damaged DNA, and all three slower migrating bands persisted throughout the entire time course of the experiment (Fig. 8 A). This persistence is consistent with the observation that damaged chromatin requires more time to replicate and that, even at 120 min, nucleotide was still being incorporated (Fig. 8 B). To determine if this banding pattern was unique to UV-damaged chromatin, we repeated the chromatin spin down using UV-damaged chromatin or MMS-damaged chromatin. We also tested undamaged chromatin in the presence of 100 μg/ml aphidicolin. As seen in Fig. 8 C, MMS-damaged chromatin exhibits the same banding pattern as UV-damaged chromatin. Aphidicolin treatment leads to an increase in the middle band, and there is no detectable PCNA sumoylation. To determine if the broad band induced by aphidicolin treatment merely masked sumoylated PCNA, we repeated the experiment in the presence of GST-SUMO1, which causes a large shift in sumoylated PCNA, but no band appeared, indicating that aphidicolin treatment prevents sumoylation of PCNA (not depicted). Importantly, neither UV, MMS, or aphidicolin treatment resulted in a significant increase in the appearance of the monoubiquitylated form of PCNA.


Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts.

Leach CA, Michael WM - J. Cell Biol. (2005)

DNA damage induces polyubiquitylation of PCNA. (A) UV-damaged sperm chromatin was isolated from X. laevis egg extract after 30, 60, 90, or 120 min. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. The control portions of this experiment are reproduced from Fig. 1 A to facilitate comparison. (B) Duplicate samples from A contained 32P-dATP and DNA replication was measured as described in Materials and methods. Closed circles represent undamaged chromatin and open squares represent UV-damaged chromatin. (C) Undamaged chromatin, MMS-damaged chromatin, or UV-damaged chromatin was isolated from X. laevis egg extract after 45 min. Aphidicolin was added to 100 μg/ml at t = −10 min to the extract incubated with undamaged chromatin. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. (D) X. laevis egg extract, preincubated with 100 μg/ml aphidicolin, was incubated with sperm chromatin and either buffer, 0.2 μg/μL T7-PCNA (rWT), or 0.2 μg/μL T7-PCNA (rK164R). Chromatin was isolated and analyzed by immunoblotting with an antibody recognizing either PCNA or the T7 tag. (E) X. laevis egg extract was preincubated with 100 μg/ml aphidicolin. Undamaged sperm chromatin was incubated in this extract which also contained buffer, 0.5 μg/μL His-tagged ubiquitin, or 0.5 μg/μL His-tagged ubiquitin (K63R). Chromatin was isolated and the bound proteins were analyzed by Western blotting with an antibody recognizing PCNA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171325&req=5

fig8: DNA damage induces polyubiquitylation of PCNA. (A) UV-damaged sperm chromatin was isolated from X. laevis egg extract after 30, 60, 90, or 120 min. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. The control portions of this experiment are reproduced from Fig. 1 A to facilitate comparison. (B) Duplicate samples from A contained 32P-dATP and DNA replication was measured as described in Materials and methods. Closed circles represent undamaged chromatin and open squares represent UV-damaged chromatin. (C) Undamaged chromatin, MMS-damaged chromatin, or UV-damaged chromatin was isolated from X. laevis egg extract after 45 min. Aphidicolin was added to 100 μg/ml at t = −10 min to the extract incubated with undamaged chromatin. The associated proteins were analyzed by Western blotting using an antibody recognizing PCNA. (D) X. laevis egg extract, preincubated with 100 μg/ml aphidicolin, was incubated with sperm chromatin and either buffer, 0.2 μg/μL T7-PCNA (rWT), or 0.2 μg/μL T7-PCNA (rK164R). Chromatin was isolated and analyzed by immunoblotting with an antibody recognizing either PCNA or the T7 tag. (E) X. laevis egg extract was preincubated with 100 μg/ml aphidicolin. Undamaged sperm chromatin was incubated in this extract which also contained buffer, 0.5 μg/μL His-tagged ubiquitin, or 0.5 μg/μL His-tagged ubiquitin (K63R). Chromatin was isolated and the bound proteins were analyzed by Western blotting with an antibody recognizing PCNA.
Mentions: The results presented thus far indicate that PCNA is monoubiquitylated and sumoylated during a normal, uninterrupted S phase and that monoubiquitylation is required for efficient replication fork progression. Thus, these findings represent a departure from studies in yeast and human cells where monoubiquitylation is only readily observed after DNA damage (Introduction). Therefore, it was important to determine the status of PCNA in our system after DNA damage. For this we used sperm chromatin that had been damaged by exposure to UV light. This chromatin was incubated in egg extract and isolated, and PCNA was examined by immunoblotting. The inclusion of damaged chromatin resulted in a third slowly migrating band on the PCNA blot, suggesting that PCNA had undergone an additional modification in response to the damaged DNA, and all three slower migrating bands persisted throughout the entire time course of the experiment (Fig. 8 A). This persistence is consistent with the observation that damaged chromatin requires more time to replicate and that, even at 120 min, nucleotide was still being incorporated (Fig. 8 B). To determine if this banding pattern was unique to UV-damaged chromatin, we repeated the chromatin spin down using UV-damaged chromatin or MMS-damaged chromatin. We also tested undamaged chromatin in the presence of 100 μg/ml aphidicolin. As seen in Fig. 8 C, MMS-damaged chromatin exhibits the same banding pattern as UV-damaged chromatin. Aphidicolin treatment leads to an increase in the middle band, and there is no detectable PCNA sumoylation. To determine if the broad band induced by aphidicolin treatment merely masked sumoylated PCNA, we repeated the experiment in the presence of GST-SUMO1, which causes a large shift in sumoylated PCNA, but no band appeared, indicating that aphidicolin treatment prevents sumoylation of PCNA (not depicted). Importantly, neither UV, MMS, or aphidicolin treatment resulted in a significant increase in the appearance of the monoubiquitylated form of PCNA.

Bottom Line: When sumoylation alone is prevented, replication occurs normally and neither monoubiquitylation nor sumoylation are required for the replication of simple single-strand DNA templates.Our findings expand the repertoire of functions for PCNA ubiquitylation and sumoylation by elucidating a role for these modifications during the replication of undamaged DNA.Furthermore, they suggest that PCNA monoubiquitylation serves as a molecular gas pedal that controls the speed of replisome movement during S phase.

View Article: PubMed Central - PubMed

Affiliation: The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.

ABSTRACT
The homotrimeric DNA replication protein proliferating cell nuclear antigen (PCNA) is regulated by both ubiquitylation and sumoylation. We study the appearance and the impact of these modifications on chromosomal replication in frog egg extracts. Xenopus laevis PCNA is modified on lysine 164 by sumoylation, monoubiquitylation, and diubiquitylation. Sumoylation and monoubiquitylation occur during the replication of undamaged DNA, whereas diubiquitylation occurs specifically in response to DNA damage. When lysine 164 modification is prevented, replication fork movement through undamaged DNA slows down and DNA polymerase delta fails to associate with replicating chromatin. When sumoylation alone is prevented, replication occurs normally and neither monoubiquitylation nor sumoylation are required for the replication of simple single-strand DNA templates. Our findings expand the repertoire of functions for PCNA ubiquitylation and sumoylation by elucidating a role for these modifications during the replication of undamaged DNA. Furthermore, they suggest that PCNA monoubiquitylation serves as a molecular gas pedal that controls the speed of replisome movement during S phase.

Show MeSH
Related in: MedlinePlus