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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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PCNA modification is not required for M13 replication. (A) p21-peptide beads were used to deplete PCNA from X. laevis egg extract as described in Materials and methods, and the resulting extracts were analyzed by SDS-PAGE and Western blotting using an antibody recognizing PCNA. (B) Either mock- or peptide-depleted extract was incubated with M13 DNA for 30 min. The peptide-depleted extract was supplemented with buffer, 0.2 μg/μL PCNA (wild type), or 0.2 μg/μL PCNA (K164R). The samples were then analyzed for DNA replication as described in Materials and methods.
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fig3: PCNA modification is not required for M13 replication. (A) p21-peptide beads were used to deplete PCNA from X. laevis egg extract as described in Materials and methods, and the resulting extracts were analyzed by SDS-PAGE and Western blotting using an antibody recognizing PCNA. (B) Either mock- or peptide-depleted extract was incubated with M13 DNA for 30 min. The peptide-depleted extract was supplemented with buffer, 0.2 μg/μL PCNA (wild type), or 0.2 μg/μL PCNA (K164R). The samples were then analyzed for DNA replication as described in Materials and methods.

Mentions: Previously, it had been shown that PCNA can be removed from X. laevis egg extracts using a peptide derived from the p21 protein (Mattock et al., 2001). We used this peptide to deplete PCNA from extracts, as shown in Fig. 3 A. Unfortunately, this extract was unable to replicate sperm chromatin after the addition of recombinant untagged PCNA (not depicted). This is consistent with published data and may be the result of co-depletion of some other factor (Mattock et al., 2001). The PCNA-depleted extract is unable to replicate single-stranded M13 DNA, but the addition of rPCNA restores the activity of the extract, as shown previously. Interestingly, the addition of mutant PCNA (rK164R) also restores the activity of the extract, demonstrating that modification of PCNA is not required for the replication of simple ssDNA templates (Fig. 3 B).


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

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

PCNA modification is not required for M13 replication. (A) p21-peptide beads were used to deplete PCNA from X. laevis egg extract as described in Materials and methods, and the resulting extracts were analyzed by SDS-PAGE and Western blotting using an antibody recognizing PCNA. (B) Either mock- or peptide-depleted extract was incubated with M13 DNA for 30 min. The peptide-depleted extract was supplemented with buffer, 0.2 μg/μL PCNA (wild type), or 0.2 μg/μL PCNA (K164R). The samples were then analyzed for DNA replication as described in Materials and methods.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: PCNA modification is not required for M13 replication. (A) p21-peptide beads were used to deplete PCNA from X. laevis egg extract as described in Materials and methods, and the resulting extracts were analyzed by SDS-PAGE and Western blotting using an antibody recognizing PCNA. (B) Either mock- or peptide-depleted extract was incubated with M13 DNA for 30 min. The peptide-depleted extract was supplemented with buffer, 0.2 μg/μL PCNA (wild type), or 0.2 μg/μL PCNA (K164R). The samples were then analyzed for DNA replication as described in Materials and methods.
Mentions: Previously, it had been shown that PCNA can be removed from X. laevis egg extracts using a peptide derived from the p21 protein (Mattock et al., 2001). We used this peptide to deplete PCNA from extracts, as shown in Fig. 3 A. Unfortunately, this extract was unable to replicate sperm chromatin after the addition of recombinant untagged PCNA (not depicted). This is consistent with published data and may be the result of co-depletion of some other factor (Mattock et al., 2001). The PCNA-depleted extract is unable to replicate single-stranded M13 DNA, but the addition of rPCNA restores the activity of the extract, as shown previously. Interestingly, the addition of mutant PCNA (rK164R) also restores the activity of the extract, demonstrating that modification of PCNA is not required for the replication of simple ssDNA templates (Fig. 3 B).

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