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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 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.
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fig2: 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.

Mentions: To determine if these bands corresponded to either ubiquitylated or sumoylated PCNA, we combined freshly prepared crude extract with 0.5 μg/μL of recombinant histidine (His)-tagged ubiquitin, GST-tagged small ubiquitin-related modifier (SUMO) 1, or GST-tagged SUMO2. Sperm chromatin and an ATP-regeneration mix was added to the extract, and the reactions were incubated at room temperature for 40 min. The chromatin was then purified and the resulting samples analyzed, as in Fig. 1 A. If one of the slower migrating bands was the result of ubiquitin conjugation we would expect that band to undergo an additional shift of ∼1 kD as a result of the His-tag on the recombinant ubiquitin. Likewise, if one of the bands was the result of SUMO conjugation we would expect that band to undergo an additional shift of 25 kD, corresponding to the GST tag on the recombinant SUMO. As seen in Fig. 2 A, when His-tagged ubiquitin (His-Ub) is added, the lower of the two bands undergoes an additional shift. When either GST-tagged SUMO1 (GST-SUMO1) or GST-tagged SUMO2 (GST-SUMO2) is added, the upper band undergoes an additional shift. Interestingly, despite adding equal amounts of GST-SUMO1 or GST-SUMO2 we observe that the conjugation of GST-SUMO1 is much more efficient. We conclude that PCNA undergoes both monoubiquitylation and sumoylation during normal DNA replication in X. laevis egg extracts.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171325&req=5

fig2: 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.
Mentions: To determine if these bands corresponded to either ubiquitylated or sumoylated PCNA, we combined freshly prepared crude extract with 0.5 μg/μL of recombinant histidine (His)-tagged ubiquitin, GST-tagged small ubiquitin-related modifier (SUMO) 1, or GST-tagged SUMO2. Sperm chromatin and an ATP-regeneration mix was added to the extract, and the reactions were incubated at room temperature for 40 min. The chromatin was then purified and the resulting samples analyzed, as in Fig. 1 A. If one of the slower migrating bands was the result of ubiquitin conjugation we would expect that band to undergo an additional shift of ∼1 kD as a result of the His-tag on the recombinant ubiquitin. Likewise, if one of the bands was the result of SUMO conjugation we would expect that band to undergo an additional shift of 25 kD, corresponding to the GST tag on the recombinant SUMO. As seen in Fig. 2 A, when His-tagged ubiquitin (His-Ub) is added, the lower of the two bands undergoes an additional shift. When either GST-tagged SUMO1 (GST-SUMO1) or GST-tagged SUMO2 (GST-SUMO2) is added, the upper band undergoes an additional shift. Interestingly, despite adding equal amounts of GST-SUMO1 or GST-SUMO2 we observe that the conjugation of GST-SUMO1 is much more efficient. We conclude that PCNA undergoes both monoubiquitylation and sumoylation during normal DNA replication in X. laevis egg extracts.

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