Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Mar 14;29(5):625-36.
doi: 10.1016/j.molcel.2007.12.016.

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

Adelina A Davies  1 , Diana Huttner, Yasukazu Daigaku, Shuhua Chen, Helle D Ulrich
Affiliations

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

Adelina A Davies et al. Mol Cell. .

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
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.
Figure 2
Figure 2
Active Replication Forks Are Required for PCNA Ubiquitylation (A) Experimental strategy to prevent the formation of replication forks in the cdc7ts mutant. (B) FACS profiles of WT and cdc7ts cells subjected to the treatment outlined in (A). (C) Progression through the cell cycle in WT and cdc7ts cells as monitored by Clb2 and Sic1 levels. Note that at 37°C WT cells have completed mitosis and re-entered G1 phase at 80 min, whereas cdc7ts mutants accumulate in G2/M with abnormally high Clb2 and low Sic1 levels. The asterisk indicates a band crossreactive to the Sic1 antibody. Detection of phosphoglycerate kinase (PGK) served as a loading control. (D) Damage-induced PCNA ubiquitylation in WT and cdc7ts cells after treatment as outlined in (A). S phase samples in cdc7ts cells and in WT at 24°C were taken 40 min after release, whereas the S phase sample in the WT at 37°C was taken after 25 min due to the faster cell-cycle progression at this temperature (see [B] and [C]). (E) CDC7 is dispensable for PCNA ubiquitylation. Damage-induced PCNA modification was examined in asynchronous cultures of isogenic WT, bob1, and bob1 cdc7Δ cells.
Figure 3
Figure 3
Influence of the Type of DNA Damage on PCNA Ubiquitylation (A) PCNA ubiquitylation after treatment with HU, MMS, NQO, H2O2, UV, IR, Bleomycin (Bleo), and CPT at the indicated doses. Modified forms of PCNA were detected as described in the legend to Figure 1. (B) Sensitivities of WT cells to Bleomycin as monitored by spot assays. (C) Sensitivities of WT and rad52 cells to CPT. (D) PCNA ubiquitylation in WT versus rad52 cells. Treatment with damaging agents (50 μg/ml CPT, 0.02% MMS) and detection of the modifications was performed as above. (E) Induction of the DNA damage checkpoint after treatment with selected DNA-damaging agents. Rad53 phosphorylation, monitored by western blot analysis, was used as an indicator of checkpoint activation. (F) Quantification of ssDNA after treatment with MMS, HU, or CPT by radioactive random-primed labeling of nondenatured genomic DNA. The graph shows the average labeling efficiencies with standard deviations (in cpm) from triplicate experiments, corrected for the amount of template DNA as determined by real-time PCR.
Figure 4
Figure 4
RPA Is Required for PCNA Ubiquitylation (A) PCNA is ubiquitylated normally in the rfa1-t11 and rfa1-t33 mutants. MMS-induced PCNA ubiquitylation was detected as described in the legend to Figure 1 in WT (RFA1) and two rfa1 mutants. (B) DNA association of the mutant protein encoded by rfa1-t33 is indistinguishable from the WT protein at 25°C and 37°C. Whole-cell extracts (W) were fractionated by centrifugation into soluble (S) and chromatin-associated (C) material. Proteins were detected by western blot. The distributions of the chromatin-associated Orc6 protein and soluble PGK served as controls for the quality of fractionation. (C) Experimental strategy to deplete Rfa1td from yeast cells. In the rfa1-td strain, the construct encoding the heat-labile Rfa1td protein is controlled by a doxycycline (Dox)-repressible promoter, and UBR1, encoding the E3 responsible for degradation of Rfa1td, is induced by galactose (Gal). Rfa1td remains stable at 25°C in raffinose (Raf) medium. Temperature effects were excluded by returning the cells to 25°C without allowing resynthesis of Rfa1td before releasing them into S phase. (D) DNA replication proceeds after depletion of the Rfa1td protein. FACS samples were taken before and 50 min after release from G1 arrest following the scheme shown in (C). (E) Loss of damage-induced PCNA ubiquitylation after depletion of the Rfa1td protein. HisPCNA and its modified forms were detected as in Figure 1. Rfa1 was detected in total cell extracts. (F) Chromatin association of PCNA in RFA1 and rfa1-td. Chromatin-binding assays were performed as described in (B). Note that the Rfa1td protein remaining after depletion is entirely associated with chromatin. The degron tag is partially cleaved from the protein during extract preparation.
Figure 5
Figure 5
Interaction of Rad18 with the RPA Complex (A) Protein-protein interactions between Rad18, Rad5, and the RPA subunits Rfa1, Rfa2, and Rfa3 in the yeast two-hybrid system. The open reading frames were expressed as fusions to the Gal4 activation (AD) and DNA-binding domains (BD), and the presence of the constructs was confirmed by growth on selective medium (-LW). Positive interactions were scored by growth on plates lacking histidine (-HLW) and stronger interactions on plates lacking histidine and adenine (-AHLW). Interactions between Rad18 and Rad5 (Ulrich and Jentsch, 2000) and between the individual RPA subunits are shown as internal controls. (B) Coomassie-stained gel of recombinant HisVSVRad18 isolated in complex with untagged Rad6 from baculovirus-infected insect cells. (C) Interaction between purified recombinant RPA, covalently coupled to Sepharose, and HisVSVRad18. HisVSVRad18 retained on the RPA beads was eluted in Laemmli buffer and detected by western blotting. BSA-coupled beads served as negative control. (D) Two-hybrid analysis of the Rad18 domains interacting with Rfa1 and Rfa2. The scheme on the right indicates the RING domain, a Zinc finger (ZF), a SAP domain, and the region relevant for interaction with Rad6. Positions C48 and C190 are indicated by asterisks in the respective mutants. (E) Two-hybrid analysis of the Rfa1 domains interacting with Rad18. The scheme on the right indicates the regions relevant for ssDNA binding and holocomplex formation as well as a Zinc finger domain. The position of the rfa1-t11 mutation (Umezu et al., 1998) is indicated by an asterisk. (F) Interaction of Rad18 with the ssDNA-binding domain of Rfa1 and with Rfa2 in vitro. Purified recombinant GSTRfa1(182–421) or Rfa2, immobilized on glutathione Sepharose, was incubated with HisVSVRad18. Bound material was eluted and analyzed by western blot to detect Rad18 (upper panel) or by Coomassie staining to detect the GST constructs (lower panel). (G) In vivo interaction of Rad18 with RPA was analyzed by coimmunoprecipitation of yeast Rad186HA and Rfa19myc by using the indicated antibodies. Strains expressing untagged RAD18 or RFA1 served as negative controls for precipitation of Rad186HA and Rfa19myc, respectively. In the anti-Rfa1 precipitation, antibody was omitted as a negative control. Bands of higher molecular weight in anti-HA precipitates represent ubiquitylated Rad186HA. (H) Interaction between hRad18 and hRPA in cultured human cells. Immunoprecipitations (anti-FLAG) were performed from extracts derived from HEK293T cells transiently transfected with an expression construct for FLAGhRad18. Cells transfected with the empty vector served as negative control. Extracts were partitioned into detergent-soluble (S) and -insoluble (I) material. Note that FLAGhRad18 is monoubiquitylated in the soluble fraction, which includes the cytoplasmic material, but not in the chromatin-associated fraction.
Figure 6
Figure 6
In Vivo Association of Rad18 with ssDNA (A) Association of Rad189myc with a replication fork in HU-treated cells detected by ChIP after release from G1 arrest. Rad189myc was precipitated (anti-myc) from formaldehyde-crosslinked cells at the indicated times, and associated DNA was quantified by real-time PCNA using primers specific for an early-firing origin, ARS607 (black); a sequence 4 kbp removed from ARS607 (gray); and a late-firing origin, ARS501 (white). AS, asynchronous cells. Error bars in all panels represent combined standard deviations from three independent amplifications. (B) Rfa19myc is detectable by ChIP at sequences adjacent to an HO-induced DSB. Enrichment of sequences adjacent to the HO site (MAT) relative to those at an unrelated locus (ACT1) in immunoprecipitates (anti-myc) from crosslinked cells was followed over the indicated time using real-time PCR as in (A). ChIP assays were performed in WT (black) and rad52 mutants (white). (C) PCNA (Pol309myc) is not detectable by ChIP at the site of an HO-induced DSB in WT (black) or rad52 (white) cells. (D) Association of Rad189myc next to a DSB, analyzed as above in WT (black) and rad52 (white) cells.
Figure 7
Figure 7
Effects of DNA on the Interaction between Rad18 and RPA (A) Recruitment of HisVSVRad18 to RPA-coated ssDNA under high-salt conditions. A 75 nt 5′-biotinylated oligonucleotide was immobilized on streptavidin agarose and preincubated with increasing amounts of purified yeast RPA, and retention of HisVSVRad18 was analyzed by western blotting. RPA and HisVSVRad18 were allowed to bind in a buffer containing 15 mM KCl and 250 mM NaCl. (B) DNA binding of HisVSVRad18 under low-salt conditions. The experiment was performed exactly as in (A), except that the proteins were allowed to bind to the ssDNA at 15 mM KCl. (C) Preincubation of short oligonucleotides with GSTRfa1(182–421) at a ratio of 1:1 or 5:1 enhances interaction with Rad18. Pull-down assays were performed as described in the legend to Figure 5F. (D) Deletion (Δ) or mutation (∗) of the Rad18 SAP domain impairs PCNA ubiquitylation in vivo. The rad18 deletion mutant was complemented by the indicated alleles, and PCNA modifications were analyzed as in Figure 1. Presence of the mutant proteins was verified by immunoprecipitation and western blot (lower panel). (E) The Rad18 SAP domain is required for protection from DNA damage in vivo. Spot assays for NQO and MMS sensitivity were performed on a rad18 deletion mutant complemented by the indicated RAD18 constructs.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Belli G., Gari E., Piedrafita L., Aldea M., Herrero E. An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast. Nucleic Acids Res. 1998;26:942–947. - PMC - PubMed
    1. Bi X., Barkley L.R., Slater D.M., Tateishi S., Yamaizumi M., Ohmori H., Vaziri C. Rad18 regulates DNA polymerase κ and is required for recovery from S-phase checkpoint-mediated arrest. Mol. Cell. Biol. 2006;26:3527–3540. - PMC - PubMed
    1. Branzei D., Foiani M. The DNA damage response during DNA replication. Curr. Opin. Cell Biol. 2005;17:568–575. - PubMed
    1. Chang D.J., Lupardus P.J., Cimprich K.A. Monoubiquitination of proliferating cell nuclear antigen induced by stalled replication requires uncoupling of DNA polymerase and mini-chromosome maintenance helicase activities. J. Biol. Chem. 2006;281:32081–32088. - PubMed
    1. Chen S., Davies A.A., Sagan D., Ulrich H.D. The RING finger ATPase Rad5p of Saccharomyces cerevisiae contributes to DNA double-strand break repair in a ubiquitin-independent manner. Nucleic Acids Res. 2005;33:5878–5886. - PMC - PubMed

Publication types

MeSH terms