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Activation of ubiquitin-dependent DNA damage bypass is mediated by replication protein a.

Davies AA, Huttner D, Daigaku Y, Chen S, Ulrich HD - Mol. Cell (2008)

Bottom Line: The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation.Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro.Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms EN6 3LD, UK.

ABSTRACT
Replicative DNA damage bypass, mediated by the ubiquitylation of the sliding clamp protein PCNA, facilitates the survival of a cell in the presence of genotoxic agents, but it can also promote genomic instability by damage-induced mutagenesis. We show here that PCNA ubiquitylation in budding yeast is activated independently of the replication-dependent S phase checkpoint but by similar conditions involving the accumulation of single-stranded DNA at stalled replication intermediates. The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation. We found that RPA directly interacts with the ubiquitin ligase responsible for the modification of PCNA, Rad18, both in yeast and in mammalian cells. Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro. Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.

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Effects of the Cell Cycle and RAD18 Overexpression on PCNA Ubiquitylation(A) Cell-cycle dependence of PCNA modification. Cells arrested in G1, S, and G2 phase were treated with 0.02% MMS for 90 min where indicated, and modifications of HisPCNA, isolated under denaturing conditions, were detected by western blot. DNA contents were monitored by flow cytometry (FACS). Asynchronous cells (AS) were processed in parallel.(B) Effects of RAD18 overexpression on PCNA modification throughout the cell cycle. Cells were treated and analyzed as in (A). Note that, in this panel, monoubiquitylated PCNA is abundant enough to be detected by the anti-ubiquitin antibody, which recognizes this form very poorly (Hoege et al., 2002). Rad18 was detected in total cell extracts.
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fig1: Effects of the Cell Cycle and RAD18 Overexpression on PCNA Ubiquitylation(A) Cell-cycle dependence of PCNA modification. Cells arrested in G1, S, and G2 phase were treated with 0.02% MMS for 90 min where indicated, and modifications of HisPCNA, isolated under denaturing conditions, were detected by western blot. DNA contents were monitored by flow cytometry (FACS). Asynchronous cells (AS) were processed in parallel.(B) Effects of RAD18 overexpression on PCNA modification throughout the cell cycle. Cells were treated and analyzed as in (A). Note that, in this panel, monoubiquitylated PCNA is abundant enough to be detected by the anti-ubiquitin antibody, which recognizes this form very poorly (Hoege et al., 2002). Rad18 was detected in total cell extracts.

Mentions: In order to characterize the conditions required for PCNA ubiquitylation in S. cerevisiae, we examined possible contributions of the DNA damage checkpoint and cell-cycle regulation. We found that, in budding yeast, as in X. laevis egg extracts and in S. pombe (Chang et al., 2006; Frampton et al., 2006), ubiquitin-dependent DNA damage tolerance and checkpoint signaling operate independently (see Figure S1 available online). Given the importance of ubiquitylated PCNA for replicative lesion bypass, the modification is expected to be most relevant during S phase. In fact, consistent with our previous findings (Papouli et al., 2005) and with the situation in mammalian cells (Kannouche et al., 2004), arrest in S phase with hydroxyurea (HU), which causes replication fork stalling by nucleotide depletion without directly damaging DNA, is sufficient to trigger PCNA modification (Figure 1A). In contrast, ubiquitylated PCNA was not detected in G1- or G2-arrested cells even after treatment with DNA-damaging agents. This indicates that, even in asynchronous populations, all detectable PCNA ubiquitylation arises from S phase cells.


Activation of ubiquitin-dependent DNA damage bypass is mediated by replication protein a.

Davies AA, Huttner D, Daigaku Y, Chen S, Ulrich HD - Mol. Cell (2008)

Effects of the Cell Cycle and RAD18 Overexpression on PCNA Ubiquitylation(A) Cell-cycle dependence of PCNA modification. Cells arrested in G1, S, and G2 phase were treated with 0.02% MMS for 90 min where indicated, and modifications of HisPCNA, isolated under denaturing conditions, were detected by western blot. DNA contents were monitored by flow cytometry (FACS). Asynchronous cells (AS) were processed in parallel.(B) Effects of RAD18 overexpression on PCNA modification throughout the cell cycle. Cells were treated and analyzed as in (A). Note that, in this panel, monoubiquitylated PCNA is abundant enough to be detected by the anti-ubiquitin antibody, which recognizes this form very poorly (Hoege et al., 2002). Rad18 was detected in total cell extracts.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2507760&req=5

fig1: Effects of the Cell Cycle and RAD18 Overexpression on PCNA Ubiquitylation(A) Cell-cycle dependence of PCNA modification. Cells arrested in G1, S, and G2 phase were treated with 0.02% MMS for 90 min where indicated, and modifications of HisPCNA, isolated under denaturing conditions, were detected by western blot. DNA contents were monitored by flow cytometry (FACS). Asynchronous cells (AS) were processed in parallel.(B) Effects of RAD18 overexpression on PCNA modification throughout the cell cycle. Cells were treated and analyzed as in (A). Note that, in this panel, monoubiquitylated PCNA is abundant enough to be detected by the anti-ubiquitin antibody, which recognizes this form very poorly (Hoege et al., 2002). Rad18 was detected in total cell extracts.
Mentions: In order to characterize the conditions required for PCNA ubiquitylation in S. cerevisiae, we examined possible contributions of the DNA damage checkpoint and cell-cycle regulation. We found that, in budding yeast, as in X. laevis egg extracts and in S. pombe (Chang et al., 2006; Frampton et al., 2006), ubiquitin-dependent DNA damage tolerance and checkpoint signaling operate independently (see Figure S1 available online). Given the importance of ubiquitylated PCNA for replicative lesion bypass, the modification is expected to be most relevant during S phase. In fact, consistent with our previous findings (Papouli et al., 2005) and with the situation in mammalian cells (Kannouche et al., 2004), arrest in S phase with hydroxyurea (HU), which causes replication fork stalling by nucleotide depletion without directly damaging DNA, is sufficient to trigger PCNA modification (Figure 1A). In contrast, ubiquitylated PCNA was not detected in G1- or G2-arrested cells even after treatment with DNA-damaging agents. This indicates that, even in asynchronous populations, all detectable PCNA ubiquitylation arises from S phase cells.

Bottom Line: The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation.Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro.Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK London Research Institute, Clare Hall Laboratories, Blanche Lane, South Mimms EN6 3LD, UK.

ABSTRACT
Replicative DNA damage bypass, mediated by the ubiquitylation of the sliding clamp protein PCNA, facilitates the survival of a cell in the presence of genotoxic agents, but it can also promote genomic instability by damage-induced mutagenesis. We show here that PCNA ubiquitylation in budding yeast is activated independently of the replication-dependent S phase checkpoint but by similar conditions involving the accumulation of single-stranded DNA at stalled replication intermediates. The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation. We found that RPA directly interacts with the ubiquitin ligase responsible for the modification of PCNA, Rad18, both in yeast and in mammalian cells. Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro. Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.

Show MeSH
Related in: MedlinePlus