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J Cell Biol
2005 Dec 19;1716:947-54. doi: 10.1083/jcb.200508100.
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Ubiquitin/SUMO modification of PCNA promotes replication fork progression in Xenopus laevis egg extracts.
Leach CA
,
Michael WM
.
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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.
Figure 1. PCNA undergoes secondary modifications during DNA replication. (A) Undamaged 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. (B) Duplicate samples from A contained 32P-dATP and DNA replication was measured as described in Materials and methods. (C) Sperm chromatin was incubated in X. laevis egg extract for 30 min with or without 250 μM of recombinant GST-geminin (rGeminin). The chromatin was isolated and resuspended in SDS sample buffer. A sample was also collected from the supernatant fraction to analyze the unbound fraction.
Figure 2. PCNA is monoubiquitylated and sumoylated during DNA replication. (A) X. laevis egg extract was incubated with sperm chromatin and either buffer, 0.5 μg/μL His-tagged ubiquitin, 0.5 μg/μL GST-SUMO1, or 0.5 μg/μL GST-SUMO2. Chromatin was isolated and analyzed by Western blotting with an antibody recognizing PCNA. The bottom blot shows a Western blot, using an antibody recognizing GST, of the sample before chromatin isolation. (B) X. laevis egg extract was incubated with sperm chromatin and either buffer, 200 ng/μL T7-PCNA (rWT), or 200 ng/μL T7-PCNA (rK164R). Chromatin was isolated and analyzed by immunoblotting with an antibody recognizing either PCNA or the T7 tag.
Figure 3. 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.
Figure 4. 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.
Figure 5. 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.
Figure 6. Loss of PCNA ubiquitylation slows replication fork progression. (A) Sperm chromatin was incubated in X. laevis egg extract containing either 0.2 μg/μL PCNA (wild type) or 0.2 μg/μL PCNA (K164R). Aliquots were removed after 25, 30, 35, 40, 45, and 50 min and prepared for alkaline agarose gel analysis as described in Materials and methods. (B) Fragment lengths were calculated by comparison to a DNA ladder and plotted against pixel intensity obtained with National Institutes of Health Image software. (C) Sperm chromatin was incubated in X. laevis egg extract containing either 0.2 μg/μL PCNA (wild type) or 0.2 μg/μL PCNA (K164R). Cold dATP was added after 25 min. Aliquots were removed after 25, 30, 35, 40, 45, and 50 min and prepared for alkaline agarose gel analysis as described in Materials and methods.
Figure 7. 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.
Figure 8. 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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