Human DNA ligase I interacts with and is targeted for degradation by the DCAF7 specificity factor of the CUL4-DDB1 ubiquitin ligase complex

The synthesis, processing and joining of Okazaki fragments during DNA replication is complex, requiring the sequential action of a large number of proteins. Proliferating cell nuclear antigen, a DNA sliding clamp, interacts with and co-ordinates the activity of several DNA replication proteins, including the enzymes FEN-1 and DNA ligase I that complete the processing and joining of Okazaki fragments, respectively. While it is evident that maintaining the appropriate relative stoichiometry of FEN-1 and DNA ligase I, which compete for binding to proliferating cell nuclear antigen, is critical to prevent genomic instability, little is known about how the steady state levels of DNA replication proteins are regulated, in particular the proteolytic mechanisms involved in their turnover. Since DNA ligase I has been reported to be ubiquitylated,

quiescent cells are induced to proliferate (4). In addition, LigI physically and functionally interacts with two key DNA replication protein complexes, proliferating cell nuclear antigen (PCNA) and replication factor C (RFC), and co-localizes with these factors in replication foci during S phase (5)(6)(7)(8). Finally, the human ligI mutant human cell line 46BR.1G1 has a defect in joining Okazaki fragments that is complemented by expression of wild type LigI (6,9).
The 46BR.1G1 cell line was established from a patient with growth retardation, sunlight sensitivity, and severe immunodeficiency (10). This individual was a compound heterozygote with one allele (Glu566 to Lys) encoding an inactive polypeptide and the other (Arg771 to Trp) encoding a version of LigI with about 20-fold less activity than wild type LigI (9). Notably, knock-in mice expressing a mouse version of LigI equivalent to the Arg771 to Trp version of human LigI exhibited genome instability and cancer predisposition in addition to a defect in joining Okazaki fragments, (11). Thus, the LIG1 gene is a member of the caretaker class of tumor suppressor genes with LigI-deficiency resulting in an increased incidence of cancer.
Interestingly, elevated levels of LigI also cause genomic instability with overexpression of LigI causing trinucleotide repeat instability in human cells (12). Similar studies in Saccharomyces cerevisiae showed that overexpression of the yeast LigI homolog, Cdc9, not only resulted in increased instability of trinucleotide repeat sequences but also increased mitotic recombination with this effect being dependent on the interaction of Cdc9 with PCNA, not Cdc9 catalytic activity (13). Furthermore, we and others have shown that LigI is frequently overexpressed in tumor biopsies and cancer cell lines (14,15). Together, these observations suggest that overexpression of LigI may contribute to the genomic instability that drives tumor progression and the acquisition of resistance to chemotherapeutics.
The PCNA-interacting protein, flap endonuclease 1 (FEN-1) that processes the 5' end of Okazaki fragments during the replication of the lagging strand is also overexpressed in a variety of cancers (16). Similar to LigI, both reduced FEN-1 activity and increased steady state levels of FEN-1 result in genomic instability in human cells (17,18). While these studies indicate that maintaining the appropriate relative levels of DNA replication proteins, in particular PCNA-interacting proteins involved in Okazaki fragment synthesis, processing and ligation, is critical for genomic stability, relatively little is know about the posttranslational mechanisms that regulate the steady state levels of DNA replication proteins. Although proteomic studies have shown that LigI is ubiquitylated (19-21), neither the proteins responsible for LigI ubiquitylation nor the role(s) of ubiquitylation in regulating the cellular functions of LigI have been described. In this study, we show that the steady state levels of LigI are markedly reduced in response to inhibition of cell proliferation and that LigI is also degraded by the proteasome in a ubiquitin-dependent manner. Interestingly, LigI is targeted for degradation by a different E3 ubiquitin ligase activity than the one implicated in the ubiquitylation and degradation of FEN-1 (18). Specifically we have shown that, LigI is targeted for degradation by the Cul4-DDB1 complex with specificity conferred by the DDB1and Cul4-associated factor (DCAF) DCAF7 that is also known as Han11 (22).

Periodic expression of LigI during the cell cycle.
Previously it was shown that the steady state levels of LigI mRNA were markedly reduced in nonproliferating cells compared with dividing cells and that the levels of LigI mRNA increased when cells were stimulated to proliferate (4). In accord with these observations, the steady state levels of LigI mRNA increased when confluent MCF10A cells derived from normal breast epithelium were plated at a lower density reaching a maximum after about 15 hours (Fig. 1a). Interestingly, the LigI mRNA levels declined and then peaked again at 27 hours as the cell population progressed through the first cell cycle and into the next one, indicating that the LIG1 gene is transcribed periodically during the cell cycle as well as in response to proliferation (Fig. 1a). The peak of LigI mRNA preceded the peak of S phase cells by about 6 hours (Fig. 1a). Changes in the steady state levels of LigI and FEN-1 proteins followed a similar pattern. The protein levels began to rise as cells entered the first S phase and continued to rise until the next peak of S phase cells before declining (Fig. 1b). In contrast, the levels of proliferating cell nuclear antigen (PCNA), which plays a critical role in coordinating lagging strand DNA synthesis (23,24), remained relatively constant under these conditions (Fig. 1b). To determine the contribution of proteolytic degradation to the steady state levels of LigI, FEN-1 and PCNA, asynchronous MCF10A cells were treated with cycloheximide to inhibit protein synthesis. While the steady state levels of PCNA remained relatively constant under these conditions, LigI and FEN-1 had half-lives of about 9 hours and 12 hours, respectively ( Fig. 2a and b).
Degradation of LigI by the proteasome in response to inhibition of cell proliferation and DNA damage. To confirm that growth inhibition results in a reduction in the level of LigI, we examined the effect of serum starvation and contact inhibition on the steady state levels of LigI mRNA and protein in MCF10A cells. As expected, the steady state levels of both LigI mRNA and protein were reduced after serum starvation for 24 hours (Fig. 3a) and contact inhibition for 24 hours (Fig. 3b). Under these conditions, the levels of FEN-1 and PCNA did not change (data not shown). In the non-dividing cells, the reduction of LigI protein levels (about 70-80%) was greater than the reduction of LigI mRNA (45-65%), suggesting that proteolytic degradation of LigI protein may be increased and/or translation of LigI mRNA decreased in non-dividing cells. Notably, levels of LigI protein but not mRNA were maintained in the growth-inhibited cells in the presence of the proteasome inhibitors, MG132 (Fig. 3a, lane 3) and epoxomicin (Fig. 3b, lane 3). In accord with a published study (25), we observed that treatment with the DNA damaging agent, doxorubicin, also caused a reduction in LigI protein levels (Fig. 3a, lane 4). Once again the reduction in LigI protein but not mRNA was prevented by co-incubation with the proteasome inhibitor MG132 (Fig. 3a, lane 5). The effect of doxorubicin on LigI levels is likely to be due to cell cycle arrest mediated by DNA damage-activated cell cycle checkpoints (25).
Serum-starvation of fibroblast cell lines from normal individuals that had been immortalized either with telomerase (HCA-Ltrt, Fig. 4a, left panel) or SV40 T antigen (GM00847, Fig. 4a, right panel) also resulted in a reduction in the steady state levels of LigI that was prevented by a proteasome inhibitor. Since these results indicate that the proteasome plays an important role in determining the steady state levels of LigI, we asked whether LigI was ubiquitylated in GM00847 cells that were transiently transfected with a plasmid expressing HA-tagged ubiquitin. Only low levels of LigI ubiquitylation were detected in LigI immunoprecipitates from extracts of asynchronous cell populations even in the presence of a proteasome inhibitor (Fig. 4b, lanes 2 and 3). As expected, there were higher levels of LigI ubiquitylation in LigI immunoprecipitates from extracts of serum-starved cells and these levels were further increased by the inclusion of the proteasome inhibitor epoxomicin during serum starvation (Fig. 4b, lanes 4 and 5).

Identification of LigI ubiquitylation sites and association with the Cul4-DDB1 E3 ligase.
To map the sites of ubiquitylation, anti-FLAG immunoprecipitates from extracts of 293T cells that stably express FLAG-tagged human LigI were analyzed by mass spectrometry for peptides with the di-Gly modification that is characteristic of ubiquitylation peptides (19-21). The peptide coverage for LigI in these experiments was greater than 90%. Lysine 376 (K376, Fig. 5a) was definitively identified as a major site of ubiquitylation site by mass spectrometry and, Lysine 79 (K79, Fig. 5b), and Lysine 192 (K192, Fig. 5c) were identified as possible ubiquitylation sites.
To examine the role of these lysine residues, K79, K192 and K376 and another lysine residue, K226, identified as a LigI ubiquitylation site in other studies (19,20), we constructed FLAG-tagged versions of LigI in which one or more of the lysine residues was replaced with an arginine and then isolated stable derivatives of SV40-immortlaized GM00847 cells that stably express the FLAG-tagged mutant versions of LigI at levels about 2-times higher than endogenous LigI. While none of the single amino acid substitutions significantly impacted protein stability, a mutant version of LigI, in which all four of the lysine residues were replaced with arginine (K79R, K192R, K226R, K376R) was more resistant to serum-starvation induced degradation (~50% after 24 hours) compared with endogenous wild type LigI (~25% after 24 hours) indicating that ubiquitylation of these lysine residues contributes to LigI degradation by the proteasome (Fig. 5d). Since the levels of the mutant version of LigI are reduced in response to serum starvation, it is likely that additional lysine residues within LigI are also ubiquitylated.
FLAG immunoprecipitates were also examined by SILAC-based mass spectrometry for associated E3 ubiquitin ligases that may be involved in the ubiquitylation of LigI. Compared with parental 293T cells, higher levels of the Cul4 and DDB1 subunits of the core Cul4-DDB1 complex and DCAF7, a specificity factor for Cul4-DDB1 complex, were detected in FLAGimmunoprecipitates from derivatives of 293T cells expressing FLAG-LigI ( Table 1), suggesting that LigI associates with the Cul4-DDB1 complex. The co-immunoprecipitation of DDB1 and DCAF7 with FLAG-tagged LigI was confirmed by immunoblotting ( Fig. 6a). In reciprocal experiments, FLAG-tagged LigI and the DDB1 subunit of the Cul4-DDB1 complex were coimmunoprecipitated by a Cul4 antibody. Notably, the amount FLAG-tagged LigI coimmunoprecipitated with Cul4 increased in extracts from serum-starved cells (Fig. 6b, compare lanes 4 and 5) and, as expected, this was further increased by co-incubation with a proteasome inhibitor (Fig. 6b, lane 3). The increased association of LigI with the Cul4-DDB1 E3 ligase in serum-starved cells suggests that the capacity or rate of proteasome-mediated degradation may be limiting under these conditions.

DCAF7 interacts with and targets LigI for degradation
To determine whether DCAF7 is the specificity factor that targets LigI for ubquitylation by the Cul4-DDB1 E3 ligase and subsequent degradation by the proteasome, we initially carried out pull down experiments with purified proteins. As shown in Figure 7a, DCAF7 bound specifically to nickel beads liganded by histagged LigI. Next we asked whether depletion of DCAF7 alters LigI for degradation in vivo. GM00847 cells were stably transfected with either a DCAF7-specific shRNA or a modified control shRNA construct that differs in two nucleotides from the DCAF7 shRNA. Knockdown of DCAF7 levels by about 70% reduced the degradation of LigI in response to serum starvation (Fig. 7b).
Finally, we asked whether the Cul4 complex immunoprecipitated from GM00847 cells could ubiquitylate LigI in vitro in the presence of human ubiquitin activating enzyme E1, a cocktail of ubiquitin conjugating E2 enzymes and biotinylated ubiquitin. Low levels of LigI ubiquitylation were detected in assays with the Cul4 immunoprecipitate that presumably contains low levels of DCAF7 (Fig. 7c,

DISCUSSION
While there is compelling evidence showing that alterations in the relative stoichiometry of proteins involved in DNA replication result in genomic instability (13,18), the proteolytic mechanisms that regulate the steady state levels of DNA replication proteins are relatively unexplored. Since proteomic studies have shown that LigI is ubiquitylated (19-21), we examined the role of ubiquitin-dependent proteolysis in determining the steady state levels of LigI. In this study, we have shown that LigI is ubiquitylated by a member of the cullin-RING finger E3 ligase family, Cul4-DDB1 and then degraded by the proteasome. Cullin-RING finger E3 ligases, which are composed of one of 7 cullins plus an adaptor and substrate receptor, are the largest family of ubiquitin ligases in eukaryotes (26). The complexes formed by cullins, CUL4A and CUL4B, with DDB1 have been implicated in several different aspects of genome maintenance, including the repair of DNA damage caused by UV-light (27-29), replication licensing (30-32), DNA damage-and stress-activated signaling (33,34), replication-associated nucleosome assembly (35) and Polδ subunit structure (36,37). Furthermore, mutations in CUL4B have been identified in humans with mental retardation, macrocephaly and peripheral neuropathy with cell lines from these patients exhibiting defects in the repair of camptothecin-induced DNA single-strand breaks (38). The substrate specificity of Cul4-DDB1 complexes is determined by substrate receptor proteins that interact with DDB1 (39). Here we have demonstrated that the Cul4-DDB1 substrate receptor DCAF7 directly interacts with LigI and targets it for ubiquitylation by the CUL4-DDB1 E3 ligase complex in vitro. Previous studies have shown that DCAF7, also known as HAN11 and WDR68, interacts with and negatively regulates the signaling activity of several protein kinases, including DYRK1A, DYRK1B, HIPK2 and MEKK1 (22,40,41). In these instances, it is not known whether DCAF7 is acting as a scaffolding protein for the kinases or is targeting them for degradation. Notably, depletion of cellular DCAF7 increases the stability of LigI, confirming that DCAF7 plays a key role in determining the steady state levels of LigI.
When cells are induced to proliferate, expression of both the FEN-1 and LIG1 genes is increased (4,42). Furthermore, our studies revealed periodic changes in expression of the LIG1 gene in synchronized, proliferating human cells, indicating that transcription of the LIG1 gene is also cell cycle-regulated. Thus, the regulation of human LIG1 gene expression is more similar to that of the functionally homologous CDC9 gene of S. cerevisiae than the Sch. pombe CDC17gene (43). Since the level of LigI protein correlates with the mRNA levels, it appears that, in cycling cells, changes in the steady state levels of LigI are primarily determined by transcription rates rather than by proteolysis. The similar changes in the steady state levels of LigI and FEN-1 proteins as cells enter and progress through S phase suggest that that expression of the FEN-1 and LIG1 genes are co-ordinately regulated in a cell cycledependent manner.
Previously it has been demonstrated that FEN-1 is ubiquitylated by the UBE1/UBE2/PRP19 E3 ligase during the G2/M phase of the cell cycle and that this ubiquitylation is regulated by sequential post-translational modifications of FEN-1 (18). Our studies identifying the Cul4-DDB1 E3 ligase as the major enzyme directing the proteolytic degradation of LigI reveal that two distinct mechanisms are utilized to maintain the relative steady state levels of these PCNA-interacting enzymes that catalyze the last two steps of Okazaki fragment processing and joining. While it appears that degradation of LigI is enhanced in non-dividing cells, further studies are needed to determine whether the ubiquitylation of LigI by the Cul4-DDB1-DCAF7 complex varies during the cell cycle and in response to inhibition of proliferation and whether cell cycle-dependent phosphorylation of LigI regulates its ubiquitylation (44,45) The efficient and accurate replication of DNA requires a high degree of co-ordination among a large number of proteins. This is particularly evident for lagging strand DNA synthesis where protein-protein interactions with PCNA co-ordinate the sequential actions of the enzymes that synthesize, process and join Okazaki fragments (46). This mechanism, which involves transient interactions with overlapping binding sites on the PCNA trimer, is prone to disruption by changes in the relative stoichiometry of the PCNA-interacting proteins (12,13,18). Although the expression of many of the genes encoding DNA replication proteins are co-ordinately regulated, our studies reveal that different mechanisms control the turnover of these proteins. While this increases the complexity of the network of pathways that regulate the relative stoichiometry of DNA replication proteins, it appears to be designed to integrate DNA replication with other aspects of the cell cycle since FEN-1 degradation is linked to the degradation of cyclin B (18). It is possible that alterations in the proteolytic mechanisms that target LigI and FEN-1 may underlie the frequently elevated steady state levels of these proteins observed in cancers. This may constitute an unexplored source of genomic instability in cancer.

Determination of protein half-life.
Half-life experiments employing cycloheximide, an inhibitor of protein synthesis, were performed as described previously (19). The levels of FEN-1, LigI and PCNA were detected by immunoblotting and quantitated using Image J software (NIH).

Detection of LigI ubiquitylation in vivo.
GM00847 cells were transiently transfected with a plasmid encoding HA-tagged ubiquitin using Lipofectamine (Invitrogen) according to the manufacturer's instructions. After 24 h, the culture medium was replaced with medium lacking serum and incubation continued for 24 h.
Where indicated, a proteasome inhibitor, either epoxomicin (0.2 µM, Millipore) or MG132 (20 µM, Sigma) was added to the culture medium for the final 8 h of the 24 h incubation. Cells were lysed in IP lysis buffer (20 mM Tris-HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 5% glycerol, 1% Triton X-100, containing a protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktails 2 and 3 (Sigma) according to the manufacturers recommendations. Clarified lysates (20 mg) were incubated with anti-LigI antibody (1:500) for 1 h at 4 o C prior to overnight the incubation with 20 µl protein A beads (GE Healthcare). After washing 5 times with IP lysis buffer, bound proteins were eluted from the beads with SDS-PAGE sample buffer and then separated by SDS-PAGE. Ubiquitylated proteins were by guest on March 24, 2020 http://www.jbc.org/ Downloaded from detected by immunoblotting with anti-HA antibody.

Identification of LigI ubiquitylation sites.
To identify ubiquitylated lysine residues in LigI, we selected a derivative of the 293T cell line that stably expresses FLAG-tagged wild-type LigI at levels about 2-times higher than endogenous LigI. FLAG-tagged LigI was immunoprecipitated from clarified extracts (1 mg) of the 293T cell line (1 x 10 8 cells) using anti-FLAG M2 affinity beads (Sigma) as described above. Proteins retained on the beads were reduced, alkylated and then digested with trypsin. The resulting peptides were analyzed by nano HPLC-coupled LTQ Orbitrap mass spectrometer (Thermo Scientific) as described previously (52).
Raw data were searched against the human database with SEQUEST algorithm built in Bioworks (v3.3.1 SP1, Thermo Scientific). A variable modification of lysine with mass shift of +114.04Da was set to identify the ubiquitylation sites.
Other modifications included a fixed modification of carbamidomethyl cysteine and a variable modification of oxidized methionine.
Identification of LigI associated proteins by mass spectrometry. 293T cells that stably express FLAG-tagged LIG1 were metabolically labeled with 13 C 2 15 N 6 -L-lysine and 13 C 4 15 N 6 -L-arginine (53). Nuclear extracts were prepared from labeled 293T cells expressing FLAG-tagged hLigI and unlabeled parental 293T cells as described previously (54). Equal amounts of the labeled and unlabeled nuclear extracts were mixed and then incubated with anti-FLAG M2 affinity beads (Sigma) as described above. Proteins were eluted from the beads with 3XFLAG peptide (Sigma) and then digested with trypsin using Filter Aided Sample Preparation (FASP) procedure (55). The resulting peptides were desalted on a Sep-Pak C18 cartridge (Waters) and dried using a SpeedVac (Thermo Scientific). After further fractionation using stop and go extraction (STAGE) tips made in-house with strong anion exchange (SAX) disk and desalting with a STAGE tip containing three layers of C18 membrane disks (3M) (56,57), peptide solutions were dried and stored at -80˚C prior to analysis by nano HPLC-coupled LTQ Orbitrap mass spectrometer (Thermo Scientific). A database search was carried out as described previously to identify the proteins from which the tryptic peptides were derived (52). The ratios of peptides from labeled and non-labeled cells to parental cells were calculated using IsoQuant software (58).

Generation of cell lines that stably expression tagged versions of LigI.
A pVP-Flag5 plasmid encoding an N-terminal FLAG-tagged version of LigI (6,7,51) was mutated by site-directed mutagenesis using the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The mutations altered the coding sequence such that lysine residues 79, 192, 226, and 376 were replaced with arginine residues either singly or all together. After verification of the nucleotide sequence by DNA sequencing, N-terminally FLAG-tagged wild-type LigI (WT) and FLAG-tagged mutant LigI cDNAs were subcloned into the mammalian expression vector pRC/RSV. After selection for G418resistance, single colonies were isolated and evaluated for the level of tagged LigI expression relative to endogenous LigI using FLAG and LigI antibodies. Cell lines that stably express FLAGtagged mutant versions of LigI at levels about 2times higher than endogenous LigI were selected for analysis.

Immunoprecipitation.
A total of 4 x 10 8 cells were harvested, and lysed in 1 ml of 10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2.5 mM MgCl 2 , and 0.5% Triton X-100 containing protease and phosphatase inhibitors. After centrifugation, the supernatant was incubated at 4°C overnight with a 100 µl 50% suspension of protein A/G-Sepharose (Amersham Biosciences) coupled with 2 µl of anti-FLAG M2 antibody. The beads were washed extensively with the buffer described above prior to the elution of bound proteins with SDS-sample buffer. After separation by SDS-PAGE, eluted proteins were identified by immunoblotting.
Proteins. Plasmids encoding either GST or GST-DCAF7 were transformed into E. coli BL21(DE3) cells. GST and GST-DCAF7 proteins were purified from cell extracts by glutathione-Sepharose 4B affinity column chromatography (GE Healthcare). Wild-type and mutant LigI cDNAs were subcloned into the bacterial expression vector pET28a. After expression in E. coli BL21(DE3) cells, the N-terminally His-tagged versions of hLigI were purified by HisTrap and HiTrap Q (GE Healthcare Life Sciences) column chromatography. Purified proteins fractions were separated by SDS-PAGE prior to Coomassie blue staining.
Pull-down assay. Purified His-tagged LigI (1 µg) was incubated with either GST (2 µg) or GST-DCAF7 (2 µg) in 300 µl of buffer containing 50 mM NaH 2 PO 4 (pH 7.4), 250 mM NaCl, and 10 mM imidazole for 1 hour on ice, followed by the addition of 15 µl of Ni-NTA agarose beads (Qiagen) with rotation for 1 hour at 4 °C. The beads were collected by centrifugation and then washed five times with 500 µl of 50 mM NaH 2 PO 4 (pH 7.4), 250 mM NaCl and 30 mM imidazole. After resuspension in 2 x SDS-PAGE sample buffer, the beads were incubated at 100°C for 5 min. Eluted proteins were separated by SDS-PAGE prior to detection both by staining with Coomassie Blue and immunblotting with GST and LigI antibodies, Generation of cell lines that stably express DCAF7 shRNA. GM00847 fibroblast cells were transfected with plasmids encoding DCAF7 shRNA or a scrambled shRNA (22) using the Lipofectamine transfection reagent (Invitrogen) according to the manufacturer's directions. After selection for resistance to puromycin, single colonies were isolated. The steady state levels of DCAF7 and hLigI proteins in these clones were determined by immunoblotting with antibodies against hLigI and DCAF7.
In vitro ubiquitylation assay hLigI ubiquitylation in vitro was performed using the ubiquitilylation kit (Enzo Life Science) according to the manufacturer's directions. Briefly, purified Histagged versions of wild type and mutant versions of LigI (1 µM) were incubated as indicated with E1 and E2 enzymes, biotinylated ubiquitin, 0.75 µl ubiquitin aldehyde, ATP, Cul4 complex immunoprecipitated from GM00847 cells, and purified DCAF7 (1 µM) in a final 15 µl volume at 30°C for 1 hour. Ubiquitylated LigI protein was detected by immunoblotting using the Pierce Far-Western Blot Kit for Biotinylated Protein (Thermofisher) according to the manufacturer's directions.
Statistical analysis. Data represent the average ± SD at least from three independent experiments. Statistical comparisons were performed with Student's two-tailed paired t test and analysis of variance (ANOVA). Values of p < 0.01 were considered statistically significant.     Anti-FLAG immunoprcipitates from nuclear extracts of labeled 293T cells expressing FLAG-tagged LigI mixed with nuclear extracts of unlabeled parental 293T cells were digested with trypsin and tryptic peptides identified by mass spectrometry as described in Materials and Methods. MS/MS spectra of peptides bearing ubiquitylation of K376 (a), K79 (b) and K192 (c) in LigI. K # , ubiquitylation site. R', 13 C 6 15 N 4 -arginine. Red peaks correspond to b ions (masses of fragments generated from the peptide N-terminus) and blue peaks correspond to y ions (masses of fragments generated from the peptide C-terminus). d. Asynchronously proliferating GM00087 cells expressing the FLAG-tagged K79R K192R K226R K376R version of LigI were serum starved for the indicated times. FLAG-tagged and endogenous versions of LigI were detected by immunoblotting with the LigI antibody. Figure 6. Association of LigI with the Cul4-DDB1-DCAF7 complex. a. Lane 1, 293T cell lysate, 5 µg (10% input). Lane 2, protein A/G beads only and Lane 3, protein A/G beads plus LigI. 293T cell lysate incubated with; lane 4, protein A/G beads; lane 5, LigI antibody followed by protein A/G beads; lane 6, LigI antibody plus 10 μg/ml ethidium bromide followed by protein A/G beads. DDB1 and DCAF7 proteins detected by immunoblotting. b. Input lysates (5 µg, 10% of input) and Cul4 immunoprecipitates from GM00847 cells that were; lanes 1 and 4, untreated; lanes 2 and 5, serum starved for 8 hours; lanes 3 and 6, serum starved for 8 hours in the presence of 0.2 µM epoximicin for 8 hours. Cul4, DDB1 and LigI proteins were detected by immunoblotting.  5); GST and his-tagged LigI (lanes 2 and 6); GST-DCAF7 (lanes 3 and 7) and GST-DCAF7 and his-tagged LigI (lanes 4 and 8) were incubated with nickel beads. The input lanes (1-4) contain 0.2 ug of the GST and 0.2 ug his-tagged LigI as indicated (10% of total input). Proteins retained on the beads were eluted with SDS-PAGE sample buffer and then separated by SDS-PAGE. Proteins were detected by immunoblotting with antibodies against GST, DCAF7 and LigI. b. GM00847 cells that were stably transfected with either a control shRNA (lanes 1 and 2) or a DCAF7 shRNA (lanes 3 and 4) were serum-starved or not as indicated. LigI, DCAF7 and β-actin were detected in whole cell lysates (20 µg) by immunoblotting. c. Purified wild type LigI (lanes 1, 2, 5 and 6, 1 µM) and the K79R K192R K226R K376R version (lanes 7 and 8, 1 µM) were incubated with biotinylated ubiquitin provided by ENZO Ubiquitinylation kit, a Cul4 immunoprecipitate from GM00847 cells, a purified E1 enzyme and cocktail of E2 enzymes and purified DCAF7 (1 µM) as indicated. Ubiquitylated proteins were detected by immunoblotting with Pierce Far-Western Blot Kit for Biotinylated Proteins. The ubiquitylated band indicated by the asterisk is generated by the E1 and E2 enzymes in the absence of LigI. Legend: The ratio (Ratio) of selected LigI-associated proteins in HEK293-FLAG-LIG cells versus parental HEK293 cells was derived from the ratios of their individual identified peptides (# of peptides). The standard deviation (SD) and coefficient of variation (CV) for these peptide ratios is shown.