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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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Sumoylation of PCNA is not required for sperm chromatin replication. (A) X. laevis egg extract was supplemented with either buffer or 5 μg/μL GST-Ubc9 dominant-negative protein (Ubc9-DN). Sperm chromatin was incubated in these extracts for 30 min and isolated. The resulting samples were subjected to SDS-PAGE and Western blotting using an antibody recognizing PCNA. (B) Duplicate samples containing 32P-dATP were used to calculate the amount of DNA replication occurring in these samples after 30 and 60 min as described in Materials and methods. The average of three independent experiments is shown with error bars representing the SEM.
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fig4: Sumoylation of PCNA is not required for sperm chromatin replication. (A) X. laevis egg extract was supplemented with either buffer or 5 μg/μL GST-Ubc9 dominant-negative protein (Ubc9-DN). Sperm chromatin was incubated in these extracts for 30 min and isolated. The resulting samples were subjected to SDS-PAGE and Western blotting using an antibody recognizing PCNA. (B) Duplicate samples containing 32P-dATP were used to calculate the amount of DNA replication occurring in these samples after 30 and 60 min as described in Materials and methods. The average of three independent experiments is shown with error bars representing the SEM.

Mentions: We hypothesized that the sumoylation of PCNA during S phase might play a role in chromatin replication. To test this we used a dominant-negative version of Ubc9 (Ubc9-DN), the only known E2 enzyme involved in the conjugation of SUMO to substrate proteins. As shown in Fig. 4 A, the addition of recombinant Ubc9-DN results in the disappearance of SUMO-modified PCNA. We then tested the effect of this dominant-negative protein on DNA replication. We combined X. laevis crude extract with 0.8 μg/μL of either buffer or Ubc9-DN. After a short incubation on ice, an ATP regeneration system, 32P-dATP, and sperm chromatin were added. Samples were collected after 30 and 60 min, and DNA replication was measured as described in Materials and methods. Fig. 4 B clearly shows that, despite the ability of Ubc9-DN to prevent sumoylation of PCNA, it has no effect on DNA replication. The Ubc9-DN will inhibit the sumoylation of all proteins in the extract, but because there is no impact on replication we can conclude that sumoylation is not required for DNA replication and, more specifically, that PCNA sumoylation is not required. These data are consistent with a previous paper showing that Ubc9-DN does not affect replication in X. laevis egg extracts (Azuma et al., 2003).


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

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

Sumoylation of PCNA is not required for sperm chromatin replication. (A) X. laevis egg extract was supplemented with either buffer or 5 μg/μL GST-Ubc9 dominant-negative protein (Ubc9-DN). Sperm chromatin was incubated in these extracts for 30 min and isolated. The resulting samples were subjected to SDS-PAGE and Western blotting using an antibody recognizing PCNA. (B) Duplicate samples containing 32P-dATP were used to calculate the amount of DNA replication occurring in these samples after 30 and 60 min as described in Materials and methods. The average of three independent experiments is shown with error bars representing the SEM.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171325&req=5

fig4: Sumoylation of PCNA is not required for sperm chromatin replication. (A) X. laevis egg extract was supplemented with either buffer or 5 μg/μL GST-Ubc9 dominant-negative protein (Ubc9-DN). Sperm chromatin was incubated in these extracts for 30 min and isolated. The resulting samples were subjected to SDS-PAGE and Western blotting using an antibody recognizing PCNA. (B) Duplicate samples containing 32P-dATP were used to calculate the amount of DNA replication occurring in these samples after 30 and 60 min as described in Materials and methods. The average of three independent experiments is shown with error bars representing the SEM.
Mentions: We hypothesized that the sumoylation of PCNA during S phase might play a role in chromatin replication. To test this we used a dominant-negative version of Ubc9 (Ubc9-DN), the only known E2 enzyme involved in the conjugation of SUMO to substrate proteins. As shown in Fig. 4 A, the addition of recombinant Ubc9-DN results in the disappearance of SUMO-modified PCNA. We then tested the effect of this dominant-negative protein on DNA replication. We combined X. laevis crude extract with 0.8 μg/μL of either buffer or Ubc9-DN. After a short incubation on ice, an ATP regeneration system, 32P-dATP, and sperm chromatin were added. Samples were collected after 30 and 60 min, and DNA replication was measured as described in Materials and methods. Fig. 4 B clearly shows that, despite the ability of Ubc9-DN to prevent sumoylation of PCNA, it has no effect on DNA replication. The Ubc9-DN will inhibit the sumoylation of all proteins in the extract, but because there is no impact on replication we can conclude that sumoylation is not required for DNA replication and, more specifically, that PCNA sumoylation is not required. These data are consistent with a previous paper showing that Ubc9-DN does not affect replication in X. laevis egg extracts (Azuma et al., 2003).

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