1. Volume 15, Issue 4, Pages 621-634 (August 2004)
The p53-Induced Oncogenic Phosphatase PPM1D Interacts with Uracil DNA Glycosylase and Suppresses Base Excision Repair 
Xiongbin Lu, Dora Bocangel, Bonnie Nannenga, Hiroshi Yamaguchi, Ettore Appella, Lawrence A. Donehower 
Molecular Cell 
Volume 15, Issue 4, Pages (August 2004)
DOI: /j.molcel

2. Figure 1 Human and Mouse PPM1D Interact with UNG2
(A) Two-hybrid experiment shows human PPM1D-UNG2 interaction. Positive bacterial cotransformants indicative of PPM1D interactors on a carbenicillin-containing plate are shown. In the upper dish, Sector 3 expresses UNG2. Sectors 4, 5, and 6 express three other interacting proteins. Sectors 1 and 2 are positive and negative controls, respectively. The lower dish shows the same cotransformants growing on the X-gal indicator plate without carbenicillin. Only positive colonies turn blue.
(B) PPM1D in vitro interactions with UNG2. A FLAG-tagged version of human PPM1D was in vitro transcribed and translated in the presence of 35S-methionine and mixed with similarly prepared lysates containing either luciferase or UNG2. Some lysates were immunoprecipitated with the FLAG antibody and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography.
(C) In vitro binding of UNG2 to wild-type and mutant forms of PPM1D. In vitro translated lysates containing wild-type UNG2 were mixed with V5-tagged wild-type or mutant murine PPM1D and immunoprecipitated with V5 antibody.
(D) UNG2 binds to PPM1D in Saos-2 cells and U2OS cells. Saos-2 and U2OS cell lines were mock transfected or transfected with a human PPM1D-FLAG expression construct and protein lysates prepared from the transfected cells 48 hr later. Immunoprecipitations were performed with FLAG antibody followed by Western blotting with a UNG2 antibody.
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2 PPM1D Suppresses BER
(A) PPM1D null fibroblasts show increased BER activity following transfection with a damaged DNA marker plasmid. A luciferase expression construct treated with heat and acid for 6 hr was transfected into PPM1D+/+, PPM1D+/−, and PPM1D−/− MEFs. Luciferase activity as an index of BER activity in the transfected cells was measured 24 hr after transfection.
(B) In vitro BER activity in PPM1D+/+, PPM1D+/−, and PPM1D−/− MEF nuclear extracts. Undamaged pUC18 plasmid DNA was used as a negative control. pGL3-CMV treated with heat and acid for 45 min was incubated with nuclear extracts and 32P-dGTP. The upper left panel shows linearized plasmid DNA separated by 1% agarose gel electrophoresis. Incorporation of 32P-dGTP into damaged pGL3-CMV plasmid DNA is shown in the lower left panel. Quantitation of BER activity in PPM1D+/+, PPM1D+/−, and PPM1D−/− MEF extracts is shown in the right panel.
(C) p53-independent suppression of BER activity in Saos-2 cells by human and murine PPM1D. Expression constructs of human FLAG-tagged wild-type human PPM1D and murine wild-type V5-tagged PPM1D cDNAs and empty vector DNAs (control) were transfected into p53 null Saos-2 cells after a 6 hr heat and acid treatment. Luciferase activities were measured in the transfected cell lysates (left panel). Lysates from the transfected and mock transfected cells were subjected to Western blot analysis with the indicated FLAG or V5 antibody (right panel).
(D) Mutant forms of murine PPM1D fail to suppress BER in Saos-2 cells. Human and murine wild-type and mutant murine V5-tagged PPM1D expression constructs were transfected into Saos-2 cells along with acid and heat treated luciferase expression vectors and monitored for luciferase activity. Luciferase constructs were acid/heat treated for 45 min (left panel) or 6 hr (middle panel). In the right panel, expression levels of the wild-type and mutant PPM1D proteins in the transfected cells are indicated.
(E) Reduction of PPM1D mRNA by PPM1D siRNA results in increased BER. Saos-2 and U2OS cells were transfected with PPM1D siRNA or control RNA and a damaged luciferase expression construct. Forty eight hours post-transfection, lysates were prepared from the transfected cells and luciferase assays performed (left panel). White bars represent control siRNA transfected cells and black bars represent PPM1D siRNA transfected cells. Total RNA was purified from transfected cells 48 hr post-transfection and equal amounts of RNA from each line were subjected to RT-PCR with PPM1D-specific primers, followed by visualization of the PCR products (right panel).
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3 Effects of PPM1D on Uracil BER In Vitro
(A) Effect of increasing PPM1D on uracil-incision activity. Saos-2 cells were transfected with increasing amounts of PPM1D cDNA and lysates tested for uracil incision. Incision assays were performed by allowing Saos-2 nuclear extracts to react with a 32P-labeled uracil- or cytosine-containing 51-mer duplex DNA. Bar diagram summarizes quantitation of at least three independent experiments ± standard error.
(B) Effects of wild-type and mutant PPM1D on uracil-incision activity. Saos-2 cells were transfected with empty vector, cDNA for wild-type (WT), point mutant, or C-terminal truncation (376-T) variants of PPM1D. Controls (lanes 1 and 2), empty vector (lane 3), WT PPM1D (lane 4), and mutant PPM1D (lanes 5–8) are indicated. Bar diagram shows quantitation of three independent experiments ± standard error.
(C) Increased in vitro uracil BER incision activity in PPM1D null cells. Nuclear extracts of MEFs from PPM1D+/+, PPM1D+/−, or PPM1D−/− mice were tested for uracil incision activity as in (A) and (B) (top panel). Bar diagram (bottom panel) summarizes results of at least three independent experiments.
(D) In vitro uracil-BER incorporation assay. Nuclear extracts from Saos-2 cells transfected with plasmids containing an empty vector (lanes 2 and 3) or wild-type PPM1D cDNA (lane 4) were isolated and allowed to react with a nonlabeled uracil-containing 51-mer duplex DNA in standard repair synthesis reactions containing labeled [α-32P]-dCTP. Lane 1 is a no lysate negative control.
(E) Bar diagram summarizing results for 32P-dCTP incorporation into uracil-containing templates after incubation with nuclear extracts from Saos-2 cells transfected with empty vector, WT PPM1D, and point mutant or a C-terminal truncation mutants of PPM1D. Bar diagram summarizes results of at least three independent experiments ± standard error.
(F) In vitro incorporation assay using uracil-containing templates incubated with nuclear extracts from wild-type and PPM1D-deficient MEFs. The incorporation assay was performed as in (D) (upper panel). The lower panel quantitates the results of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4 PPM1D In Vitro Phosphatase Assays on UNG2 Phosphopeptides
(A) List of phosphopeptides tested in the in vitro phosphatase assay. Control phosphopeptides 1 and 2 are from p38 MAP kinase and are known to be dephosphorylated by PPM1D. The remaining phosphopeptides are derived from UNG2. Phosphothreonines and phosphotyrosines are indicated in bold.
(B) Three phosphopeptides from two regions of UNG2 are dephosphorylated in vitro by recombinant PPM1D. An in vitro phosphatase assay that detects free phosphate released from the fifteen p38 and UNG2 phosphopeptides incubated with recombinant PPM1D was used to determine relative phosphatase activities. Numbers along the X axis refer to the phosphopeptides in (A). Gray bars show the phosphatase activity of PPM1D on p38 phosphopeptides, while black bars show PPM1D activity on UNG2 phosphopeptides.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5 PPM1D Suppresses UNG2 Phosphorylation at Threonine 6
(A) UNG2 phosphorylation in U2OS cells transfected with PPM1D cDNA. Western blot analysis of nuclear extracts from U2OS cells transfected with empty vector, or with increasing amounts of WT PPM1D cDNA, using antibodies that recognize phosphorylated threonine at positions 6 (left panel), or position 126 (right panel). Results were standardized by Western blot with UNG2 and β-actin antibodies (lower panels). Bar diagram summarizes results of at least three independent experiments ± standard error.
(B) In vitro phosphatase assays on intact phospho-UNG2 protein by purified PPM1D-FLAG. Immunoprecipitated UNG2 proteins from UV-radiated HCT116 cells were dephosphorylated in vitro by increasing (0–300 ng) amounts of purified FLAG-PPM1D. After the assay, the reaction mix was immunoblotted and the level of threonine 6 phosphorylated UNG2 was detected by the UNG2(p6T) phospho-specific antibody.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
UNG2-Associated Uracil Incision Activity Is Increased by UV-Induced Phosphorylation and Is Decreased by PPM1D Dephosphorylation
(A) UV treatment increases UNG2 phosphorylation and uracil incision activity. Nuclear extracts from unirradiated (lane 3) or UV-irradiated (lanes 4–8) Saos-2 cells were incubated with a uracil-containing 32P-end-labeled 51 bp duplex. Prior to incubation, some extracts were precleared by treatment with antibodies to UNG2 protein (lane 5) or to phospho-specific UNG2 6pT (lane 6) or 126pT (lane 7) or both phospho-specific antibodies (lane 8). Controls were uracil duplexes not incubated with lysates (lane 1) and non-uracil containing duplexes incubated with lysates (lane 2). Following incubations, the labeled duplex DNAs were subjected to polyacrylamide gel electrophoresis and visualized by autoradiography. Formation of a 25-mer band indicated incision adjacent to the uracil moiety. The relative incision results were quantitated by phosphorimaging and presented in graph form in the second panel. An equal portion of each nuclear extract was also subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with antibodies to UNG2, phospho-specific UNG2 6pT, PPM1D, and β-actin (lower four panels).
(B) PPM1D dephosphorylates UNG2 and reduces its uracil-associated incision activity. Reaction conditions are similar to those in (A) and extract treatments are indicated above each lane. Purified PPM1D, either intact (lanes 5 and 8) or inactivated (lane 6), were added to the extracts. Extracts were from cells treated with UV, except those in lanes 2 and 3, and extracts in lanes 7 and 8 were pre-cleared with UNG2 antibody. Controls in lanes 1 and 2 are as in (A), as are quantitations (second panel) and UNG2, UNG2(p6T), and β-actin protein amounts (lower three panels).
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Model for PPM1D Activities in Regulating p53 and BER after UV Irradiation
UV irradiation induces cyclobutane pyrimidine dimer formation. Cytosines in these dimers have very high rates of deamination and formation of uracils. After dimer bond removal, the remaining uracils are targets for UNG2 activity. UV damage also causes activation of the ATR and p38 MAP kinases. These kinases phosphorylate p53, contributing to p53 activation. In turn, p53 promotes BER activity. BER repairosomes that include UNG2 repair the damage. We hypothesize that UV-activated MKKs, while targeting p38, may also phosphorylate UNG2, increasing its activity. After initiation of BER, p53 transcriptionally upregulates PPM1D. Increased PPM1D protein eventually results in dephosphorylation and inhibition of p38 MAP kinase. Decreased p38 activity reduces p53 phosphorylation and destabilizes it. The increased PPM1D may repress uracil-BER through binding and dephosphorylation of UNG2 at threonine 6. BER repression by PPM1D helps revert DNA repair activity to basal levels after elimination of DNA damage. Solid lines indicate immediate responses to UV and dashed lines indicate more delayed responses.
Molecular Cell  , DOI: ( /j.molcel )