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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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Ubiquitylation of PCNA facilitates sperm chromatin replication. (A) Sperm chromatin was incubated in X. laevis egg extract supplemented with buffer, 0.2 μg PCNA (wild type), or 0.2 μg PCNA (K164R) for 45 min. The chromatin was then isolated and analyzed by SDS-PAGE followed by Western blotting with an antibody recognizing PCNA. (B) Duplicate samples containing 32P-dATP were used to measure DNA replication 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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fig5: Ubiquitylation of PCNA facilitates sperm chromatin replication. (A) Sperm chromatin was incubated in X. laevis egg extract supplemented with buffer, 0.2 μg PCNA (wild type), or 0.2 μg PCNA (K164R) for 45 min. The chromatin was then isolated and analyzed by SDS-PAGE followed by Western blotting with an antibody recognizing PCNA. (B) Duplicate samples containing 32P-dATP were used to measure DNA replication 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: Despite repeated attempts using a variety of strategies, we could not selectively eliminate PCNA monoubiquitylation while leaving sumoylation intact. However, we have shown that sumoylation is not important for DNA replication (Fig. 4). To get at a possible function for monoubiquitylation in replication, we determined the effect of eliminating lysine 164 modification on chromosomal replication. To do this we added either untagged wild-type or mutant (K164R) rPCNA protein to extracts and analyzed the effect on both replication and PCNA modification in the extract. As shown in Fig. 5 A, addition of the mutant PCNA results in a decrease in the amount of modified PCNA bound to chromatin. This is presumably attributable to the mutant recombinant protein outcompeting the endogenous PCNA for binding sites on chromatin. In Fig. 5 B, it is clear that the addition of this mutant PCNA inhibits replication at both the 30 and 60 min time points. We conclude that modification of PCNA lysine 164 is important for efficient replication. This is likely the result of the loss of monoubiquitylation of PCNA because the Ubc9-DN protein had no inhibitory effect on replication, but did eliminate sumoylation of lysine 164. It is also possible that PCNA monoubiquitylation and sumoylation act redundantly during replication, as our experiment does not rule this out.


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

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

Ubiquitylation of PCNA facilitates sperm chromatin replication. (A) Sperm chromatin was incubated in X. laevis egg extract supplemented with buffer, 0.2 μg PCNA (wild type), or 0.2 μg PCNA (K164R) for 45 min. The chromatin was then isolated and analyzed by SDS-PAGE followed by Western blotting with an antibody recognizing PCNA. (B) Duplicate samples containing 32P-dATP were used to measure DNA replication 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.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Ubiquitylation of PCNA facilitates sperm chromatin replication. (A) Sperm chromatin was incubated in X. laevis egg extract supplemented with buffer, 0.2 μg PCNA (wild type), or 0.2 μg PCNA (K164R) for 45 min. The chromatin was then isolated and analyzed by SDS-PAGE followed by Western blotting with an antibody recognizing PCNA. (B) Duplicate samples containing 32P-dATP were used to measure DNA replication 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: Despite repeated attempts using a variety of strategies, we could not selectively eliminate PCNA monoubiquitylation while leaving sumoylation intact. However, we have shown that sumoylation is not important for DNA replication (Fig. 4). To get at a possible function for monoubiquitylation in replication, we determined the effect of eliminating lysine 164 modification on chromosomal replication. To do this we added either untagged wild-type or mutant (K164R) rPCNA protein to extracts and analyzed the effect on both replication and PCNA modification in the extract. As shown in Fig. 5 A, addition of the mutant PCNA results in a decrease in the amount of modified PCNA bound to chromatin. This is presumably attributable to the mutant recombinant protein outcompeting the endogenous PCNA for binding sites on chromatin. In Fig. 5 B, it is clear that the addition of this mutant PCNA inhibits replication at both the 30 and 60 min time points. We conclude that modification of PCNA lysine 164 is important for efficient replication. This is likely the result of the loss of monoubiquitylation of PCNA because the Ubc9-DN protein had no inhibitory effect on replication, but did eliminate sumoylation of lysine 164. It is also possible that PCNA monoubiquitylation and sumoylation act redundantly during replication, as our experiment does not rule this out.

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