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Role of SUMO modification of human PCNA at stalled replication fork.

Gali H, Juhasz S, Morocz M, Hajdu I, Fatyol K, Szukacsov V, Burkovics P, Haracska L - Nucleic Acids Res. (2012)

Bottom Line: We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out.Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions.These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.

ABSTRACT
DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions. These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.

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In vitro SUMO modification of human PCNA. (A) in vitro SUMOylation reaction of human PCNA (40 nM) was carried out in the presence of purified SAE1/2 (10 nM), Ubc9 (100 nM), RFC (10 nM) nicked PUC19 plasmid DNA (2 nM) and either GST-SUMO1, or GST-SUMO2, or GST-SUMO3, or SUMO1, or SUMO2, or SUMO3 (500 nM) at 37°C for 60 min. Samples containing unmodified and SUMOylated PCNA were separated on 10% denaturing polyacrylamide gel and visualized by western blot using anti-PCNA antibody. Structure of human PCNA from the front (B) and side (C) views; surface lysine residues are represented by red spheres (K117, K138, K164, K168, K181, K190, K240, K248 and K254). PCNA structures showing the surface lysine residues were generated using the PyMOL version 0.96 by DeLano scientific (http.//www.pymolsourceforge.net). (D) Wild-type and lysine point-mutant PCNA samples were subjected to in vitro SUMOylation reaction as described above. (E) In vitro SUMOylation and ubiquitylation reactions of PCNA were compared in the absence or presence of combinations of RFC and nicked plasmid DNA as indicated.
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gks256-F2: In vitro SUMO modification of human PCNA. (A) in vitro SUMOylation reaction of human PCNA (40 nM) was carried out in the presence of purified SAE1/2 (10 nM), Ubc9 (100 nM), RFC (10 nM) nicked PUC19 plasmid DNA (2 nM) and either GST-SUMO1, or GST-SUMO2, or GST-SUMO3, or SUMO1, or SUMO2, or SUMO3 (500 nM) at 37°C for 60 min. Samples containing unmodified and SUMOylated PCNA were separated on 10% denaturing polyacrylamide gel and visualized by western blot using anti-PCNA antibody. Structure of human PCNA from the front (B) and side (C) views; surface lysine residues are represented by red spheres (K117, K138, K164, K168, K181, K190, K240, K248 and K254). PCNA structures showing the surface lysine residues were generated using the PyMOL version 0.96 by DeLano scientific (http.//www.pymolsourceforge.net). (D) Wild-type and lysine point-mutant PCNA samples were subjected to in vitro SUMOylation reaction as described above. (E) In vitro SUMOylation and ubiquitylation reactions of PCNA were compared in the absence or presence of combinations of RFC and nicked plasmid DNA as indicated.

Mentions: To further characterize, we set up an in vitro PCNA SUMOylation system using purified E1 SUMO-activating enzyme (SAE1/SAE2), E2 SUMO-conjugating enzyme (Ubc9), various E3 SUMO-ligases (Pias1, Pias2, Pias3 and Pias4) and all three SUMO isoforms (SUMO1, SUMO2 and SUMO3; Figure 2 and Supplementary Figure S2). Consistently with the in vivo findings, we established that out of the three SUMO isoforms, only SUMO1 could be efficiently conjugated to PCNA (Figure 2A) and that the lysine 164 residue of PCNA was one of the main SUMO attachment site (Figure 2D). In addition, using surface lysine mutant PCNAs, we managed to identify the K254 residue as a second SUMOylation site in PCNA that locates in a consensus ΨKxE SUMO attachment site, where Ψ is an aliphatic residue (Figures 2D). Consistently with alternate lysine modifications that we concluded from our in vivo experiments, at higher enzyme concentrations, new SUMO-PCNA shifts also became apparent, particularly for certain lysine mutants (Supplementary Figure S2A). Strikingly, PCNA SUMOylation was dependent on replication factor C (Figure 2E), but did not require any of the four PIAS E3 ligases (Supplementary Figure S2B). Moreover, comparing PCNA SUMOylation with ubiquitylation in the presence of RFC, nicked plasmid DNA or their combination revealed that in contrast to ubiquitylation, which requires RFC-dependent loading of PCNA onto DNA, PCNA SUMOylation was dependent on RFC but not DNA (Figures 2E and Supplementary Figure S2C). Thus, interaction between PCNA and RFC is a prerequisite for PCNA SUMOylation presumably by exposing residues in PCNA or giving access for Ubc9.Figure 2.


Role of SUMO modification of human PCNA at stalled replication fork.

Gali H, Juhasz S, Morocz M, Hajdu I, Fatyol K, Szukacsov V, Burkovics P, Haracska L - Nucleic Acids Res. (2012)

In vitro SUMO modification of human PCNA. (A) in vitro SUMOylation reaction of human PCNA (40 nM) was carried out in the presence of purified SAE1/2 (10 nM), Ubc9 (100 nM), RFC (10 nM) nicked PUC19 plasmid DNA (2 nM) and either GST-SUMO1, or GST-SUMO2, or GST-SUMO3, or SUMO1, or SUMO2, or SUMO3 (500 nM) at 37°C for 60 min. Samples containing unmodified and SUMOylated PCNA were separated on 10% denaturing polyacrylamide gel and visualized by western blot using anti-PCNA antibody. Structure of human PCNA from the front (B) and side (C) views; surface lysine residues are represented by red spheres (K117, K138, K164, K168, K181, K190, K240, K248 and K254). PCNA structures showing the surface lysine residues were generated using the PyMOL version 0.96 by DeLano scientific (http.//www.pymolsourceforge.net). (D) Wild-type and lysine point-mutant PCNA samples were subjected to in vitro SUMOylation reaction as described above. (E) In vitro SUMOylation and ubiquitylation reactions of PCNA were compared in the absence or presence of combinations of RFC and nicked plasmid DNA as indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3401441&req=5

gks256-F2: In vitro SUMO modification of human PCNA. (A) in vitro SUMOylation reaction of human PCNA (40 nM) was carried out in the presence of purified SAE1/2 (10 nM), Ubc9 (100 nM), RFC (10 nM) nicked PUC19 plasmid DNA (2 nM) and either GST-SUMO1, or GST-SUMO2, or GST-SUMO3, or SUMO1, or SUMO2, or SUMO3 (500 nM) at 37°C for 60 min. Samples containing unmodified and SUMOylated PCNA were separated on 10% denaturing polyacrylamide gel and visualized by western blot using anti-PCNA antibody. Structure of human PCNA from the front (B) and side (C) views; surface lysine residues are represented by red spheres (K117, K138, K164, K168, K181, K190, K240, K248 and K254). PCNA structures showing the surface lysine residues were generated using the PyMOL version 0.96 by DeLano scientific (http.//www.pymolsourceforge.net). (D) Wild-type and lysine point-mutant PCNA samples were subjected to in vitro SUMOylation reaction as described above. (E) In vitro SUMOylation and ubiquitylation reactions of PCNA were compared in the absence or presence of combinations of RFC and nicked plasmid DNA as indicated.
Mentions: To further characterize, we set up an in vitro PCNA SUMOylation system using purified E1 SUMO-activating enzyme (SAE1/SAE2), E2 SUMO-conjugating enzyme (Ubc9), various E3 SUMO-ligases (Pias1, Pias2, Pias3 and Pias4) and all three SUMO isoforms (SUMO1, SUMO2 and SUMO3; Figure 2 and Supplementary Figure S2). Consistently with the in vivo findings, we established that out of the three SUMO isoforms, only SUMO1 could be efficiently conjugated to PCNA (Figure 2A) and that the lysine 164 residue of PCNA was one of the main SUMO attachment site (Figure 2D). In addition, using surface lysine mutant PCNAs, we managed to identify the K254 residue as a second SUMOylation site in PCNA that locates in a consensus ΨKxE SUMO attachment site, where Ψ is an aliphatic residue (Figures 2D). Consistently with alternate lysine modifications that we concluded from our in vivo experiments, at higher enzyme concentrations, new SUMO-PCNA shifts also became apparent, particularly for certain lysine mutants (Supplementary Figure S2A). Strikingly, PCNA SUMOylation was dependent on replication factor C (Figure 2E), but did not require any of the four PIAS E3 ligases (Supplementary Figure S2B). Moreover, comparing PCNA SUMOylation with ubiquitylation in the presence of RFC, nicked plasmid DNA or their combination revealed that in contrast to ubiquitylation, which requires RFC-dependent loading of PCNA onto DNA, PCNA SUMOylation was dependent on RFC but not DNA (Figures 2E and Supplementary Figure S2C). Thus, interaction between PCNA and RFC is a prerequisite for PCNA SUMOylation presumably by exposing residues in PCNA or giving access for Ubc9.Figure 2.

Bottom Line: We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out.Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions.These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary.

ABSTRACT
DNA double-strand breaks (DSBs) can be generated not only by reactive agents but also as a result of replication fork collapse at unrepaired DNA lesions. Whereas ubiquitylation of proliferating cell nuclear antigen (PCNA) facilitates damage bypass, modification of yeast PCNA by small ubiquitin-like modifier (SUMO) controls recombination by providing access for the Srs2 helicase to disrupt Rad51 nucleoprotein filaments. However, in human cells, the roles of PCNA SUMOylation have not been explored. Here, we characterize the modification of human PCNA by SUMO in vivo as well as in vitro. We establish that human PCNA can be SUMOylated at multiple sites including its highly conserved K164 residue and that SUMO modification is facilitated by replication factor C (RFC). We also show that expression of SUMOylation site PCNA mutants leads to increased DSB formation in the Rad18(-/-) cell line where the effect of Rad18-dependent K164 PCNA ubiquitylation can be ruled out. Moreover, expression of PCNA-SUMO1 fusion prevents DSB formation as well as inhibits recombination if replication stalls at DNA lesions. These findings suggest the importance of SUMO modification of human PCNA in preventing replication fork collapse to DSB and providing genome stability.

Show MeSH
Related in: MedlinePlus