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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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Loss of PCNA modification interferes with chromatin binding of DNA polymerase δ. Sperm chromatin was incubated in X. laevis egg extract containing either buffer, 0.2 μg/μL PCNA (wild type), or 0.2 μg/μL PCNA (K164R). After 40 min the chromatin was isolated and resuspended in SDS sample buffer. The samples were then analyzed by SDS-PAGE and Western blotting with antibodies recognizing the indicated proteins.
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fig7: Loss of PCNA modification interferes with chromatin binding of DNA polymerase δ. Sperm chromatin was incubated in X. laevis egg extract containing either buffer, 0.2 μg/μL PCNA (wild type), or 0.2 μg/μL PCNA (K164R). After 40 min the chromatin was isolated and resuspended in SDS sample buffer. The samples were then analyzed by SDS-PAGE and Western blotting with antibodies recognizing the indicated proteins.

Mentions: In an attempt to determine the cause of the slow fork progression resulting from a knockdown of PCNA lysine 164 modification in the extracts, we examined the binding of polymerase δ to chromatin under these conditions. As can be seen in Fig. 7, the addition of mutant PCNA to the extract resulted in the elimination of lysine 164 modifications, as expected. In extracts containing this mutant, we observed a significant decrease in polymerase δ bound to chromatin when compared with the addition of wild-type PCNA. In contrast, we did not detect any effect of the mutant PCNA on the loading of the prereplication complex component Orc2, the ssDNA-binding protein replication protein A, or DNA polymerase α (p70 subunit) to chromatin. We conclude that lysine 164 modification of PCNA is required for both efficient chromosomal replication and for stable association of polymerase δ with replicating chromatin.


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

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

Loss of PCNA modification interferes with chromatin binding of DNA polymerase δ. Sperm chromatin was incubated in X. laevis egg extract containing either buffer, 0.2 μg/μL PCNA (wild type), or 0.2 μg/μL PCNA (K164R). After 40 min the chromatin was isolated and resuspended in SDS sample buffer. The samples were then analyzed by SDS-PAGE and Western blotting with antibodies recognizing the indicated proteins.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: Loss of PCNA modification interferes with chromatin binding of DNA polymerase δ. Sperm chromatin was incubated in X. laevis egg extract containing either buffer, 0.2 μg/μL PCNA (wild type), or 0.2 μg/μL PCNA (K164R). After 40 min the chromatin was isolated and resuspended in SDS sample buffer. The samples were then analyzed by SDS-PAGE and Western blotting with antibodies recognizing the indicated proteins.
Mentions: In an attempt to determine the cause of the slow fork progression resulting from a knockdown of PCNA lysine 164 modification in the extracts, we examined the binding of polymerase δ to chromatin under these conditions. As can be seen in Fig. 7, the addition of mutant PCNA to the extract resulted in the elimination of lysine 164 modifications, as expected. In extracts containing this mutant, we observed a significant decrease in polymerase δ bound to chromatin when compared with the addition of wild-type PCNA. In contrast, we did not detect any effect of the mutant PCNA on the loading of the prereplication complex component Orc2, the ssDNA-binding protein replication protein A, or DNA polymerase α (p70 subunit) to chromatin. We conclude that lysine 164 modification of PCNA is required for both efficient chromosomal replication and for stable association of polymerase δ with replicating chromatin.

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