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Supplemental Information:

Materials and Methods:

For the in vivo ubiquitylation studies, cells were harvested directly in Boiling buffer (1% SDS, 1mM EDTA, 2mM Na3VO4 in PBS) and boiled for 5 minutes. Samples were passed 3X through 26G needles and boiled for an additional 3 minutes. The lysates were then chilled on ice for three minutes and centrifuged for 5 minutes at RT and the supernatant transferred to clean tubes. The samples were then mixed with one volume of Immunomix mixture (1% TX100, 1% SDS, 0.5% Deoxycolic acid, 1% BSA, 1mM EDTA, 2mM Na3VO4 in PBS) and incubated at 4oC with either 50 ul of 50 % slurry flag agarose (Sigma) equilibrated with PBS for 5 hours or with anti-HA monoclonal mouse antibody (12CA5) for 4 hours followed by the addition of 50 ul of 50 % slurry protein G agarose (Amersham) for additional hour. Immunocomplexes where washed 3X with immunomix buffer and 2X with 0.1 % PBS and boiled for 5 minutes in protein sample buffer. Ubiquitylated proteins (anti-HA) or flag-tagged (anti-flag) protein conjugates were then separated by SDS-PAGE electrophoresis and transferred to nitrocellulose membranes and the blots were probed with anti p21 antibodies to detect ubiquitylated p21 proteins.

For the in vitro ubiquitylation reactions, to purify the CRL4Cdt2 enzyme complex, 293T cells were transiently transfected with myc- tagged Cul4A, myc-tagged DDB1, Flag-tagged Roc1/Rbx1 and flag-tagged Cdt2 mammalian expression plasmids. 48 hours after transfection, cells were lysed in NP-40 lysis Buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% NP-40, and 50 mM NAF) for 30 minutes on ice. The cleared lysates were then input in NP-40 immunoprecipitation buffer with either anti-flag agarose (50 ul of 50% slurry pre-equilibrated with NP-40 buffer) for 5 hours, or with anti-myc monoclonal antibody (9E10) overnight at 4oC followed by addition of 50 ul of protein G agarose (50% slurry pre-equilibrated with NP-40 Buffers) for another hour. Immunocomplexes were washed 4X in NP-40 lysis buffer and 2X with ligase buffer (25 mM Tris-HCl (pH 7.5), 50 mM NaCl, 1mM EDTA, 0.01% NP-40 and 10% Glycerol). To prepare the p21 substrate, 293T cells were transiently transfected with flag-p21 expressing plasmid for 48 hours. Cells were washed 2X with ice cold PBS and lysed with RIPA lysis buffer (50 mM Tris-HCl (pH 8), 150 mM NACl, 1% NP-40, 0.5% DOC and 0.1% SDS) and immunoprecipitated with anti-flag agarose (pre-equilibrated with RIPA buffer) for 5 hours at 4oC. Anti-flag immunocomplexes were washed 3X with RIPA buffer and 2X with ligase Buffer (above). For p21 ubiquitylation assay, the immunoprecipitated CRL4-DDB1-Roc1-Cdt2 (anti-flag or anti-myc) complexes were mixed with flag-p21 immunocomplexes on flag agarose and the mixtures were input in ubiquitylation reaction (30 ul) containing the following: 50 mM Tris-HCl (pH 7.4), 5mM MgCl2, 2 mM NAF, 10 nM Okadaic acid, 2 mM ATP, 0.6 mM DTT, 12 ug bovine ubiquitin (Sigma), 1 ug Flag-human ubiquitin (Sigma), 60 ng E1 (Boston Biochem), 300 ng E2 (hUbc5c-Boston Biochem), 5 ug myc peptide (Sigma) and 5 ug flag peptide (Sigma). The ubiquitylation reactions were incubated at 37oC for one hour and terminated by boiling in 10 ul of protein sample buffer. Proteins were then resolved by electrophoreses on an SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted with anti-p21 antibody to detect ubiquitylated p21.

Legends For Supplementary Figures:

Figure S1: p21 protein is degraded via a proteasome-dependent mechanism in UV irradiated cells in a dose dependent manner. A). Western blot analysis of protein extracts of non-irradiated or UV irradiated U2OS cells. The data demonstrate that in U2OS cells, p21 is rapidly degraded within one hour of UV irradiation and that treatment of the cells with the 26S proteasome inhibitor MG132 blocks the degradation. LC; a cross-reacting band that serves as an internal loading control. B). UV irradiation induces the degradation of p21 in U2OS cells in a dose dependent manner and is maximal at 120 J/m2 as demonstrated by the Western blot analysis with anti-p21 antibodies. C). Northern blot analysis (left panel) to test the effect of UV irradiation on the steady state p21 mRNA levels. The transcript level of GAPDH is also shown for comparison and normalization. The right panel shows the amount of p21 mRNA in non-irradiated and irradiated U2OS relative to GAPDH mRNA. The data demonstrate that UV irradiation (50 J/m2 for 6 hrs) does not significantly affect the steady state levels of p21 mRNA.

Figure S2: Depletion of human cells of the DDB1 or the DCAF Cdt2, but not an irrelevant DCAF (DDB2) inhibits UV-induced degradation of p21 and is not due to secondary effects on the cell cycle. A). Targeted depletion of DDB1 inhibits UV-induced degradation of p21. Western blot of lysates of HCT116p53-/- transfected with either control oligo (si-GL2), or two different siRNA oligos targeting DDB1 (si-DDB1-1, si-DDB1-2) and irradiated with UV (50J/m2) for six hours prior to harvesting. The anti-DDB1 blot shows significant reduction of DDB1 by both si-DDB1 oligos. The anti-p21 blots (short and longer exposure to show endogenous p21 protein in control transfected cells) demonstrate that p21 is stabilized and is resistant to UV induced degradation in cells depleted of DDB1. LC; a cross-reacting band in the anti-DDB1 blot serving as a loading control. B). Depletion of Cdt2 by siRNA specifically inhibits UV-induced degradation of p21. HCT116 p53-/- cells were transfected with siRNA targeting the ORF (si-Cdt2-1) or the 3’UTR (si-Cdt2-2) of Cdt2 for 48 hours and irradiated with UV (50J/m2) for six hours prior to harvest. The anti-Cdt2 blot indicates that Cdt2 is efficiently down-regulated by both siRNA oligos and that UV-induced degradation of p21 is inhibited. *; a cross-reacting band in the anti-Cdt2 blot. C). siRNA against an irrelevant DCAF, the DDB1 binding protein DDB2, has no effect on p21 degradation following UV. HCT116p53-/- cells were transfected with siRNA targeting DDB2 and the effect of UV irradiation on p21 was analyzed as in B). The anti-DDB2 blot demonstrates the efficient silencing of DDB2 in cells transfected with si-DDB2 oligos. A cross-reacting band in the anti-p21 blot serves as loading control. The results demonstrate that while siRNA against Cdt2 suppressed UV induced degradation of p21, the down-regulation of DDB2, an irrelevant DCAF, has no effect. D). Down-regulation of Cul4, Cdt2 or DDB1 has no effect on the cell cycle progression in HCT116 p53-/-. Cells were transfected with siRNA against a control oligo (si-GL2), Cul4 (si-Cul4A/B), Cdt2 (si-Cdt2), or DDB1 (si-DDB1) for 48 hours, fixed and stained with propidium iodide. Cell cycle profiles were subsequently analyzed by FACS.

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