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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 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.
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fig1: 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.

Mentions: X. laevis egg extracts can be used to synchronously replicate sperm chromatin and have proven essential to increasing our understanding of the events that occur during DNA replication. We first wanted to determine if PCNA underwent secondary protein modifications during DNA synthesis in this cell-free system. We prepared a crude extract from X. laevis eggs, added sperm chromatin in the presence of an ATP-regeneration system, and incubated it at room temperature. After the indicated amounts of time, the chromatin was isolated from the extracts by sequential centrifugation through two sucrose cushions and resuspended in SDS sample buffer. The samples were separated by PAGE and subjected to Western blotting analysis with an antibody recognizing PCNA. Duplicate samples also contained 32P-dATP. Samples were collected at the indicated times, and DNA replication was measured as described in Materials and methods. As seen in Fig. 1 A, two slower migrating bands are detected at 30 and 60 min, coincident with the majority of nucleotide incorporation (Fig. 1, A and B). To further investigate the dependence of these bands on DNA replication we repeated the chromatin spin down, but in the presence or absence of recombinant GST-tagged geminin. Geminin inhibits the function of the essential replication factor Cdt1 and thereby blocks replication fork assembly and subsequent DNA replication. We also examined the unbound fraction of these chromatin isolations to assess whether or not the modification of PCNA was limited to the chromatin-bound PCNA. Fig. 1 C clearly demonstrates that only chromatin-bound PCNA exhibits slower migrating versions of PCNA. Also, it is clear that inhibition of replication by the addition of GST-tagged geminin prevents the appearance of the slower migrating forms of PCNA.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: 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.
Mentions: X. laevis egg extracts can be used to synchronously replicate sperm chromatin and have proven essential to increasing our understanding of the events that occur during DNA replication. We first wanted to determine if PCNA underwent secondary protein modifications during DNA synthesis in this cell-free system. We prepared a crude extract from X. laevis eggs, added sperm chromatin in the presence of an ATP-regeneration system, and incubated it at room temperature. After the indicated amounts of time, the chromatin was isolated from the extracts by sequential centrifugation through two sucrose cushions and resuspended in SDS sample buffer. The samples were separated by PAGE and subjected to Western blotting analysis with an antibody recognizing PCNA. Duplicate samples also contained 32P-dATP. Samples were collected at the indicated times, and DNA replication was measured as described in Materials and methods. As seen in Fig. 1 A, two slower migrating bands are detected at 30 and 60 min, coincident with the majority of nucleotide incorporation (Fig. 1, A and B). To further investigate the dependence of these bands on DNA replication we repeated the chromatin spin down, but in the presence or absence of recombinant GST-tagged geminin. Geminin inhibits the function of the essential replication factor Cdt1 and thereby blocks replication fork assembly and subsequent DNA replication. We also examined the unbound fraction of these chromatin isolations to assess whether or not the modification of PCNA was limited to the chromatin-bound PCNA. Fig. 1 C clearly demonstrates that only chromatin-bound PCNA exhibits slower migrating versions of PCNA. Also, it is clear that inhibition of replication by the addition of GST-tagged geminin prevents the appearance of the slower migrating forms of PCNA.

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