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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 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.
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fig6: 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.

Mentions: To investigate what step in replication was being affected by the loss of lysine 164 modifications, we used alkaline agarose gels to determine the length of the nascent strands of DNA when either wild-type or mutant PCNA was added to the extract. Replication assays were performed under normal conditions and samples were then treated with a high pH buffer to separate the strands. Samples were run on a gel under basic conditions and dried, and signal was measured with a phosphoimager (Fig. 6 A). Using National Institutes of Health Image software, a line was placed in the center of each lane and the pixel intensity at each point along the line was measured. The pixel intensity at a point is an indication of how many molecules of that length were generated during replication. By comparing the gel to a DNA ladder we were able to plot pixel intensity versus DNA fragment size (Fig. 6 B). As shown in Fig. 6 (A and B), the addition of rPCNA (K164R) to the extracts reduces the length of nascent DNA strands relative to rPCNA wild type. One possible explanation of this result is that in the presence of rPCNA (K164R) some replication forks are abandoned, whereas others progress normally. To test this hypothesis we repeated this experiment but added 1 mM of cold dATP after 25 min, which allowed us to watch the progression of only those replication forks that had already initiated DNA synthesis. As seen in Fig. 6 C, it is clear that high molecular mass strands are formed at a slower rate in the presence of K164R, indicating that K164R PCNA does not induce a significant amount of irreversible fork abandonment. We conclude that PCNA modification is required for replication elongation to occur with maximal efficiency on undamaged chromosomes. The fact that modification of lysine 164 is not required for replication of ssDNA, but facilitates chromatin replication, suggests that monoubiquitylation of PCNA may play a role in unwinding the DNA or in relaxing the chromatin structure to allow for proper replication fork progression.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171325&req=5

fig6: 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.
Mentions: To investigate what step in replication was being affected by the loss of lysine 164 modifications, we used alkaline agarose gels to determine the length of the nascent strands of DNA when either wild-type or mutant PCNA was added to the extract. Replication assays were performed under normal conditions and samples were then treated with a high pH buffer to separate the strands. Samples were run on a gel under basic conditions and dried, and signal was measured with a phosphoimager (Fig. 6 A). Using National Institutes of Health Image software, a line was placed in the center of each lane and the pixel intensity at each point along the line was measured. The pixel intensity at a point is an indication of how many molecules of that length were generated during replication. By comparing the gel to a DNA ladder we were able to plot pixel intensity versus DNA fragment size (Fig. 6 B). As shown in Fig. 6 (A and B), the addition of rPCNA (K164R) to the extracts reduces the length of nascent DNA strands relative to rPCNA wild type. One possible explanation of this result is that in the presence of rPCNA (K164R) some replication forks are abandoned, whereas others progress normally. To test this hypothesis we repeated this experiment but added 1 mM of cold dATP after 25 min, which allowed us to watch the progression of only those replication forks that had already initiated DNA synthesis. As seen in Fig. 6 C, it is clear that high molecular mass strands are formed at a slower rate in the presence of K164R, indicating that K164R PCNA does not induce a significant amount of irreversible fork abandonment. We conclude that PCNA modification is required for replication elongation to occur with maximal efficiency on undamaged chromosomes. The fact that modification of lysine 164 is not required for replication of ssDNA, but facilitates chromatin replication, suggests that monoubiquitylation of PCNA may play a role in unwinding the DNA or in relaxing the chromatin structure to allow for proper replication fork progression.

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