Ubiquitination of p27Kip1 requires physical interaction with cyclin E and probable phosphate recognition by SKP2

p27Kip1 is an essential cell cycle inhibitor of Cyclin-dependent kinases. Ubiquitin-mediated proteolysis of p27Kip1 is an important mechanism for activation of Cyclin E-Cdk2 and facilitates G1/S transition. Ubiquitination of p27 is primarily catalyzed by a multisubunit E3 ubiquitin ligase, SCF(Skp2), and requires an adapter protein Cks1. In addition, phosphorylation of p27 at Thr187 by Cyclin E and Cdk2 is also essential for triggering substrate ubiquitination. Here we investigate the molecular mechanism of p27 ubiquitination. We show that Cyclin E-Cdk2 is essential for targeting the p27 substrate to SCF(Skp2). Direct physical contact between Cyclin E but not Cdk2 and p27 is required for p27 recruitment to SCF(Skp2). In a search for positively charged amino acid residues that may be involved in recognition of the Thr187 phosphate group, we found that Arg306 of Skp2 is required for association and ubiquitination of phosphorylated p27 but dispensable for ubiquitination of unphosphorylated p21. Thus, our data unravel the molecular organization of the ubiquitination complex that catalyzes p27 ubiquitination and provide unique insights into the specificity of substrate recognition by SCF(Skp2).     

Ubiquitination of Cyclin-Dependent Kinase Inhibitor,Xic1, is Mediated by the Xenopus F-box Protein xSkp2

In the frog, Xenopus laevis, the Cip/Kip-type cyclin-dependent kinase (CDK) inhibitor, Xic1, inhibits DNA replication in interphase egg extracts through the binding of CDK2-cyclins and Proliferating Cell Nuclear Antigen (PCNA). During DNA polymerase switching in the replicating Xenopus egg extract, Xic1 is targeted for ubiquitination and degradation when localized to chromatin through its binding to PCNA. To date, the machinery responsible for Xic1 ubiquitination is unknown and although it is predicted that the E3 called SCF may mediate Xic1 ubiquitination, characterization of the SCF in Xenopus is lacking. In this study, we describe the identification and characterization of Xenopus Skp2 (xSkp2) and the role of xSkp2 in the ubiquitination of Xic1. Our results indicate that the expression of xSkp2 appears to be developmentally regulated with low protein levels found in the egg and increased levels found in the developing embryo. We also demonstrate that when ectopically expressed, a xSkp2 F-box deletion mutant inhibits the initiation of DNA replication suggesting a role for the SCF in the onset of S phase in Xenopus egg extracts. We further show that xSkp2 binds to C-terminal residues of Xic1 and when co-expressed with Skp1, promotes the proteolysis of Xic1 in the egg extract. Moreover, the xSkp2 F-box deletion mutant inhibits the DNA-dependent ubiquitination and proteolysis of Xic1 when added to the interphase egg extract. Importantly, our studies demonstrate that SCFxSkp2 supports the ubiquitination of Xic1 in a reconstituted in vitro ubiquitination assay and that this Xic1 ubiquitination does not require either CDK2-cyclins or Cks1. These studies provide the first characterization of the SCF in Xenopus and its role in the ubiquitination of CDK inhibitor, Xic1, during DNA replication initiation.     

Regulation of nuclear transport and degradation of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1

The regulation of the vertebrate cell cycle is controlled by the function of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. The Xenopus laevis kinase inhibitor, p27(Xic1) (Xic1) is a member of the p21(Cip1)/p27(Kip1)/p57(Kip2) CDK inhibitor family and inhibits CDK2-cyclin E in vitro as well as DNA replication in Xenopus egg extracts. Xic1 is targeted for degradation in interphase extracts in a manner dependent on both the ubiquitin conjugating enzyme, Cdc34, and nuclei. Here we show that ubiquitination of Xic1 occurs exclusively in the nucleus and that nuclear localization of Xic1 is necessary for its degradation. We find that Xic1 nuclear localization is independently mediated by binding to CDK2-cyclin E and by nuclear localization sequences within the C terminus of Xic1. Our results also indicate that binding of Xic1 to CDK2-cyclin E is dispensable for Xic1 ubiquitination and degradation. Moreover, we show that amino acids 180-183 of Xic1 are critical determinants of Xic1 degradation. This region of Xic1 may define a motif of Xic1 essential for recognition by the ubiquitin conjugation machinery or for binding an alternate protein required for degradation.     

Structural basis of the Cks1-dependent recognition of p27(Kip1) by the SCF(Skp2) ubiquitin ligase

The ubiquitin-mediated proteolysis of the Cdk2 inhibitor p27(Kip1) plays a central role in cell cycle progression, and enhanced degradation of p27(Kip1) is associated with many common cancers. Proteolysis of p27(Kip1) is triggered by Thr187 phosphorylation, which leads to the binding of the SCF(Skp2) (Skp1-Cul1-Rbx1-Skp2) ubiquitin ligase complex. Unlike other known SCF substrates, p27(Kip1) ubiquitination also requires the accessory protein Cks1. The crystal structure of the Skp1-Skp2-Cks1 complex bound to a p27(Kip1) phosphopeptide shows that Cks1 binds to the leucine-rich repeat (LRR) domain and C-terminal tail of Skp2, whereas p27(Kip1) binds to both Cks1 and Skp2. The phosphorylated Thr187 side chain of p27(Kip1) is recognized by a Cks1 phosphate binding site, whereas the side chain of an invariant Glu185 inserts into the interface between Skp2 and Cks1, interacting with both. The structure and biochemical data support the proposed model that Cdk2-cyclin A contributes to the recruitment of p27(Kip1) to the SCF(Skp2)-Cks1 complex.     

Ubiquitination of p21Cip1/WAF1 by SCFSkp2: substrate requirement and ubiquitination site selection

Multiple proteolytic pathways are involved in the degradation of the cyclin-dependent kinase inhibitor p21(Cip1/WAF1). Timed destruction of p21(Cip1/WAF1) plays a critical role in cell-cycle progression and cellular response to DNA damage. The SCF(Skp2) complex (consisting of Rbx1, Cul1, Skp1, and Skp2) is one of the E3 ubiquitin ligases involved in ubiquitination of p21(Cip1/WAF1). Little is known about how SCF(Skp2) recruits its substrates and selects particular acceptor lysine residues for ubiquitination. In this study, we investigated the requirements for SCF(Skp2) recognition of p21(Cip1/WAF1) and lysine residues that are ubiquitinated in vitro and inside cells. We demonstrate that ubiquitination of p21(Cip1/WAF1) requires a functional interaction between p21(Cip1/WAF1) and the cyclin E-Cdk2 complex. Mutation of both the cyclin E recruitment motif (RXL) and the Cdk2-binding motif (FNF) at the N terminus of p21(Cip1/WAF1) abolishes its ubiquitination by SCF(Skp2), while mutation of either motif alone has minimal effects, suggesting either contact is sufficient for substrate recruitment. Thus, SCF(Skp2) appears to recognize a trimeric complex consisting of cyclin E-Cdk2-p21(Cip1/WAF1). Furthermore, we show that p21(Cip1/WAF1) can be ubiquitinated at four distinct lysine residues located in the carboxyl-terminal region but not two other lysine residues in the N-terminal region. Any one of these four lysine residues can be targeted for ubiquitination in the absence of the others in vitro, and three of these four lysine residues are also ubiquitinated in vivo, suggesting that there is limited specificity in the selection of ubiquitination sites. Interestingly, mutation of the carboxyl-terminal proline to lysine enables ubiquitin conjugation at the carboxyl terminus of the substrate both in vitro and in vivo. Thus, our results highlight a unique property of the ubiquitination enzymatic reaction in that substrate ubiquitination site selection can be remarkably diverse and occur in distinct spatial areas.     

Targeted disruption of Skp2 results in accumulation of cyclin E and p27(Kip1), polyploidy and centrosome overduplication

The ubiquitin-proteasome pathway plays an important role in control of the abundance of cell cycle regulators. Mice lacking Skp2, an F-box protein and substrate recognition component of an Skp1-Cullin-F-box protein (SCF) ubiquitin ligase, were generated. Although Skp2(-/-) animals are viable, cells in the mutant mice contain markedly enlarged nuclei with polyploidy and multiple centrosomes, and show a reduced growth rate and increased apoptosis. Skp2(-/-) cells also exhibit increased accumulation of both cyclin E and p27(Kip1). The elimination of cyclin E during S and G(2) phases is impaired in Skp2(-/-) cells, resulting in loss of cyclin E periodicity. Biochemical studies showed that Skp2 interacts specifically with cyclin E and thereby promotes its ubiquitylation and degradation both in vivo and in vitro. These results suggest that specific degradation of cyclin E and p27(Kip1) is mediated by the SCF(Skp2) ubiquitin ligase complex, and that Skp2 may control chromosome replication and centrosome duplication by determining the abundance of cell cycle regulators.     

A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27Kip1.

The Cks/Suc1 proteins associate with CDK/cyclin complexes, but their precise function(s) is not well defined. Here we demonstrate that Cks1 directs the ubiquitin-mediated proteolysis of the CDK-bound substrate p27Kip1 by the protein ubiquitin ligase (E3) SCF(Skp2). Cks1 associates with the F box protein Skp2 and is essential for recognition of the p27Kip1 substrate for ubiquitination in vivo and in vitro. Using purified recombinant proteins, we reconstituted p27Kip1 ubiquitination activity and show that it is dependent on Cks1. CKS1-/- mice are abnormally small, and cells derived from them proliferate poorly, particularly under limiting mitogen conditions, possibly due to elevated levels of p27Kip1.     

Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation

Eukaryotic cells tightly control DNA replication so that replication origins fire only once during S phase within the same cell cycle. Cell cycle-regulated degradation of the replication licensing factor Cdt1 plays important roles in preventing more than one round of DNA replication per cell cycle. We have previously shown that the SCF(Skp2)-mediated ubiquitination pathway plays an important role in Cdt1 degradation. In this study, we demonstrate that human Cdt1 is a substrate of Cdk2 and Cdk4 both in vivo and in vitro. Overexpression of cyclin-dependent kinase inhibitors such as p21 and p27 dramatically suppresses the phosphorylation of Cdt1, disrupts the interaction of Cdt1 with the F-box protein Skp2, and blocks the degradation of Cdt1. Further analysis reveals that Cdt1 interacts with cyclin/cyclin-dependent kinase (Cdk) complexes through a cyclin/Cdk binding consensus site, located at the N terminus of Cdt1. A Cdt1 mutant carrying four amino acid substitutions at the Cdk binding site dramatically reduces associations with cyclin/Cdk complexes. This mutant is not phosphorylated, fails to bind Skp2 and is more stable than wild-type Cdt1. These data suggest that cyclin/Cdk-mediated Cdt1 phosphorylation is required for the association of Cdt1 with the SCF(Skp2) ubiquitin ligase and thus is important for the cell cycle dependent degradation of Cdt1 in mammalian cells.     

Human origin recognition complex large subunit is degraded by ubiquitin-mediated proteolysis after initiation of DNA replication

Eukaryotic cells possess overlapping mechanisms to ensure that DNA replication is restricted to the S phase of the cell cycle. The levels of hOrc1p, the largest subunit of the human origin recognition complex, vary during the cell division cycle. In rapidly proliferating cells, hOrc1p is expressed and targeted to chromatin as cells exit mitosis and prereplicative complexes are formed. Later, as cyclin A accumulates and cells enter S phase, hOrc1p is ubiquitinated on chromatin and then degraded. hOrc1p destruction occurs through the proteasome and is signaled in part by the SCF(Skp2) ubiquitin-ligase complex. Other hORC subunits are stable throughout the cell cycle. The regulation of hOrc1p may be an important mechanism in maintaining the ploidy in human cells.     

p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27.

Many tumorigenic processes affect cell-cycle progression by their effects on the levels of the cyclin-dependent kinase inhibitor p27(Kip1) [1,2]. The phosphorylation- and ubiquitination-dependent proteolysis of p27 is implicated in control of the G1-S transition in the cell cycle [3-6]. To determine the factors that control p27 stability, we established a cell-free extract assay that recapitulates the degradation of p27. Phosphorylation of p27 at Thr187 was essential for its degradation. Degradation was also dependent on SCF(Skp2), a protein complex implicated in targeting phosphorylated proteins for ubiquitination [7-10]. Immunodepletion of components of the complex - Cul-1, Skp1, or Skp2 - from the extract abolished p27 degradation, while addition of purified SCF(Skp2) to Skp2- depleted extract restored the capacity to degrade p27. A specific association was observed between Skp2 and a p27 carboxy-terminal peptide containing phosphorylated Thr187, but not between Skp2 and the non-phosphorylated peptide. Skp2-dependent associations between Skp1 or Cul-1 and the p27 phosphopeptide were also detected. Isolated SCF(Skp2) contained an E3 ubiquitin ligase activity towards p27. Our data thus suggest that SCF(Skp2) specifically targets p27 for degradation during cell-cycle progression.     

Cyclin D1 overexpression induces progestin resistance in T-47D breast cancer cells despite p27(Kip1) association with cyclin E-Cdk2

Long-term growth inhibition, arrest in G(1) phase and reduced activity of both cyclin D1-Cdk4 and cyclin E-Cdk2 are elicited by progestin treatment of breast cancer cells in culture. Decreased cyclin expression, induction of p18(INK4c) and increased association of the CDK inhibitors p21(WAF1/Cip1) and p27(Kip1) with cyclin E-Cdk2 have been implicated in these responses. To determine the role of decreased cyclin expression, T-47D human breast cancer cells constitutively expressing cyclin D1 or cyclin E were treated with the progestin ORG 2058. Overexpression of cyclin E had only a modest effect on growth inhibition. Although cyclin E expression was maintained during progestin treatment, cyclin E-Cdk2 activity decreased by approximately 60%. This was accompanied by p27(Kip1) association with cyclin E-Cdk2, indicating that both cyclin E down-regulation and p27(Kip1) recruitment contribute to the decrease in activity. In contrast, overexpression of cyclin D1 induced progestin resistance and cell proliferation continued despite decreased cyclin E-Cdk2 activity. Progestin treatment of cyclin D1-overexpressing cells was associated with increased p27(Kip1) association with cyclin E-Cdk2. Thus the ability of cyclin D1 to confer progestin resistance does not depend on sequestration of p27(Kip1) away from cyclin E-Cdk2, providing evidence for a critical function of cyclin D1 other than as a high-capacity "sink" for p27(Kip1). These data indicate that regulation of cyclin D1 is a critical element of progestin inhibition in breast cancer cells and suggest that breast cancers overexpressing cyclin D1 may respond poorly to progestin therapy.     

Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kip1

The transition from G1 phase to S phase of the mammalian cell cycle is controlled by many positive and negative regulators, among which cyclin E and p27Kip1, respectively, undergo the most marked changes in concentration at this transition. The abundance of both cyclin E and p27Kip1 is regulated predominantly by posttranslational mechanisms, in particular by proteolysis mediated by the ubiquitin-proteasome pathway. Cyclin E and p27Kip1 each bind to and undergo polyubiquitination by the same ubiquitin ligase, known as SCF(Skp2). The degradation of cyclin E and p27Kip1 is greatly impaired in Skp2-deficient mice, resulting in intracellular accumulation of these proteins. In this article, recent progress in characterization of the molecular mechanisms that control the proteolysis of cyclin E and p27Kip1 is reviewed.     

Degradation of p27(Kip1) at the G(0)-G(1) transition mediated by a Skp2-independent ubiquitination pathway.

Targeting of the cyclin-dependent kinase inhibitor p27(Kip1) for proteolysis has been thought to be mediated by Skp2, the F-box protein component of an SCF ubiquitin ligase complex. Degradation of p27(Kip1) at the G(0)-G(1) transition of the cell cycle has now been shown to proceed normally in Skp2(-/-) lymphocytes, whereas p27(Kip1) proteolysis during S-G(2) phases is impaired in these Skp2-deficient cells. Degradation of p27(Kip1) at the G(0)-G(1) transition was blocked by lactacystin, a specific proteasome inhibitor, suggesting that it is mediated by the ubiquitin-proteasome pathway. The first cell cycle of stimulated Skp2(-/-) lymphocytes appeared normal, but the second cycle was markedly inhibited, presumably as a result of p27(Kip1) accumulation during S-G(2) phases of the first cell cycle. Polyubiquitination of p27(Kip1) in the nucleus is dependent on Skp2 and phosphorylation of p27(Kip1) on threonine 187. However, polyubiquitination activity was also detected in the cytoplasm of Skp2(-/-) cells, even with a threonine 187 --> alanine mutant of p27(Kip1) as substrate. These results suggest that a polyubiquitination activity in the cytoplasm contributes to the early phase of p27(Kip1) degradation in a Skp2-independent manner, thereby promoting cell cycle progression from G(0) to G(1).     

Antimitogenesis linked to regulation of Skp2 gene expression

Prostacyclin has many effects in the vasculature; one of the less well understood is the ability to block cell cycle progression through G(1) phase. We previously reported that the prostacyclin mimetic, cicaprost, selectively inhibits cyclin E-cyclin-dependent kinase-2 (Cdk2), and now we show that it acts by regulating the expression of Skp2, the F-box protein that targets p27(Kip1) for ubiquitin-mediated proteolysis. First, we show that cicaprost prevents the late G(1) phase down-regulation of p27(Kip1) and that the inhibitory effect of cicaprost on cyclin E-Cdk2 activity and S phase entry is eliminated by deleting p27(Kip1). Levels of the closely related Cdk2 inhibitor, p21(Cip1), are unaffected by cicaprost. Moreover, we show that cicaprost blocks the induction of Skp2 mRNA and that ectopic expression of a Skp2 cDNA overrides the effect of cicaprost on p27(Kip1) levels and S phase entry. Our data show that inhibition of F-box protein gene expression can underlie the effect of a potent antimitogen.     

Skp2 regulates G2/M progression in a p53-dependent manner

Targeted proteasomal degradation mediated by E3 ubiquitin ligases controls cell cycle progression, and alterations in their activities likely contribute to malignant cell proliferation. S phase kinase-associated protein 2 (Skp2) is the F-box component of an E3 ubiquitin ligase complex that targets p27^Kip1 and cyclin E1 to the proteasome. In human melanoma, Skp2 is highly expressed, regulated by mutant B-RAF, and required for cell growth. We show that Skp2 depletion in melanoma cells resulted in a tetraploid cell cycle arrest. Surprisingly, co-knockdown of p27^Kip1 or cyclin E1 failed to prevent the tetraploid arrest induced by Skp2 knockdown. Enhanced Aurora A phosphorylation and repression of G2/M regulators cyclin B1, cyclin-dependent kinase 1, and cyclin A indicated a G2/early M phase arrest in Skp2-depleted cells. Furthermore, expression of nuclear localized cyclin B1 prevented tetraploid accumulation after Skp2 knockdown. The p53 status is most frequently wild type in melanoma, and the tetraploid arrest and down-regulation of G2/M regulatory genes were strongly dependent on wild-type p53 expression. In mutant p53 melanoma lines, Skp2 depletion did not induce cell cycle arrest despite up-regulation of p27^Kip1. These data indicate that elevated Skp2 expression may overcome p53-dependent cell cycle checkpoints in melanoma cells and highlight Skp2 actions that are independent of p27^Kip1 degradation.     

Cul4A and DDB1 associate with Skp2 to target p27Kip1 for proteolysis involving the COP9 signalosome

DDB1, a subunit of the damaged-DNA binding protein DDB, has been shown to function also as an adaptor for Cul4A, a member of the cullin family of E3 ubiquitin ligase. The Cul4A-DDB1 complex remains associated with the COP9 signalosome, and that interaction is conserved from fission yeast to human. Studies with fission yeast suggested a role of the Pcu4-Ddb1-signalosome complex in the proteolysis of the replication inhibitor Spd1. Here we provide evidence that the function of replication inhibitor proteolysis is conserved in the mammalian DDB1-Cul4A-signalosome complex. We show that small interfering RNA-mediated knockdown of DDB1, CSN1 (a subunit of the signalosome), and Cul4A in mammalian cells causes an accumulation of p27Kip1. Moreover, expression of DDB1 reduces the level of p27Kip1 by increasing its decay rate. The DDB1-induced proteolysis of p27Kip1 requires signalosome and Cul4A, because DDB1 failed to increase the decay rate of p27Kip1 in cells deficient in CSN1 or Cul4A. Surprisingly, the DDB1-induced proteolysis of p27Kip1 also involves Skp2, an F-box protein that allows targeting of p27Kip1 for ubiquitination by the Skp1-Cul1-F-box complex. Moreover, we provide evidence for a physical association between Cul4A, DDB1, and Skp2. We speculate that the F-box protein Skp2, in addition to utilizing Cul1-Skp1, utilizes Cul4A-DDB1 to induce proteolysis of p27Kip1.     

Bradykinin B1 receptor blocks PDGF-induced mitogenesis by prolonging ERK activation and increasing p27Kip1

The mechanism by which the bradykininB₁ receptor (B1R) inhibits platelet-derived growth factor(PDGF)-stimulated proliferation was investigated in cultured ratmesenteric arterial smooth muscle cells. The B1R agonistdes-Arg⁹-bradykinin (DABK) was found to inhibitPDGF-mediated activation of the cyclin E-cyclin-dependent kinase 2 (Cdk2) complex and to prevent hyperphosphorylation of retinoblastomaprotein. DABK did not inhibit upregulation of cyclin E expression butincreased expression of the Cdk2 inhibitor p27Kip1 and the associationof p27Kip1 with the cyclin E-Cdk2 complex. In addition, DABK inhibited the PDGF-stimulated expression of cyclin D that would otherwise siphonp27Kip1 away from inhibition of cyclin E-Cdk2. The signaling mechanismby which DABK regulated p27Kip1 was explored. DABK was found tostimulate the activity of mitogen-activated protein kinase kinase (MEK)and extracellular signal-regulated kinase (ERK) and to prolongactivation of MEK and ERK by PDGF. Inhibition of ERK activation withthe MEK inhibitors PD-98059 and U-0126 as well as the Src family kinaseinhibitor PP2 completely blocked the effect of DABK to increase p27Kip1and partially reversed the DABK-mediated inhibition of PDGF-stimulatedproliferation. These studies demonstrate that the B1R inhibitsPDGF-stimulated mitogenesis in part by prolonged activation of ERKleading to increased expression of p27Kip1.

     

p27 phosphorylation by Src regulates inhibition of cyclin E-Cdk2

The kinase inhibitor p27Kip1 regulates the G1 cell cycle phase. Here, we present data indicating that the oncogenic kinase Src regulates p27 stability through phosphorylation of p27 at tyrosine 74 and tyrosine 88. Src inhibitors increase cellular p27 stability, and Src overexpression accelerates p27 proteolysis. Src-phosphorylated p27 is shown to inhibit cyclin E-Cdk2 poorly in vitro, and Src transfection reduces p27-cyclin E-Cdk2 complexes. Our data indicate that phosphorylation by Src impairs the Cdk2 inhibitory action of p27 and reduces its steady-state binding to cyclin E-Cdk2 to facilitate cyclin E-Cdk2-dependent p27 proteolysis. Furthermore, we find that Src-activated breast cancer lines show reduced p27 and observe a correlation between Src activation and reduced nuclear p27 in 482 primary human breast cancers. Importantly, we report that in tamoxifen-resistant breast cancer cell lines, Src inhibition can increase p27 levels and restore tamoxifen sensitivity. These data provide a new rationale for Src inhibitors in cancer therapy.     

Proliferating cell nuclear antigen recruits cyclin-dependent kinase inhibitor Xic1 to DNA and couples its proteolysis to DNA polymerase switching

The Xenopus cyclin-dependent kinase (CDK) inhibitor, p27(Xic1) (Xic1), binds to CDK2-cyclins and proliferating cell nuclear antigen (PCNA), inhibits DNA synthesis in Xenopus extracts, and is targeted for ubiquitin-mediated proteolysis. Previous studies suggest that Xic1 ubiquitination and degradation are coupled to the initiation of DNA replication, but the precise timing and molecular mechanism of Xic1 proteolysis has not been determined. Here we demonstrate that Xic1 proteolysis is temporally restricted to late replication initiation following the requirements for DNA polymerase alpha-primase, replication factor C, and PCNA. Our studies also indicate that Xic1 degradation is absolutely dependent upon the binding of Xic1 to PCNA in both Xenopus egg and gastrulation stage extracts. Additionally, extracts depleted of PCNA do not support Xic1 proteolysis. Importantly, while the addition of recombinant wild-type PCNA alone restores Xic1 degradation, the addition of a PCNA mutant defective for trimer formation does not restore Xic1 proteolysis in PCNA-depleted extracts, suggesting Xic1 proteolysis requires both PCNA binding to Xic1 and the ability of PCNA to be loaded onto primed DNA by replication factor C. Taken together, our studies suggest that Xic1 is targeted for ubiquitination and degradation during DNA polymerase switching through its interaction with PCNA at a site of initiation.