Regulation of Cip-type CDK inhibitor turnover during the cell cycle and in response to DNA damage

In Xenopus, three different CDK inhibitors (CKIs) have been identified, p27Xic1 (Xic1), p16Xic2 (Xic2) and p17Xic3 (Xic3). p16Xic2 and p17Xic3 which share homology with p21Cip1 and p27 Kip1 respectively, are tissue specifically expressed and are developmentally regulated. Xic1 shares functional and sequence homology with both p21 Cip1 and p27Kip1 and contains both the N-terminal CDK binding domain and a C-terminal PCNA binding domain called the "PIP-box". Using Xenopus egg extracts, studies have shown that during DNA replication Xic1 is recruited to the site of replication in a PCNA dependent manner and is targeted for ubiquitin mediated degradation during DNA polymerase switching. However, no studies have been done to understand the regulation of Xic1 during PCNA dependent repair processes. The biochemically tractable Xenopus egg extract system which is competent to support both CKI proteolysis and PCNA dependent repair processes such as NER provides an ideal system to study the events of Xic1 regulation during PCNA dependent repair processes.;Using the Xenopus egg extract system, my in vitro studies have shown that during the NER process Xic1 is ubiquitinated and degraded in a PCNA dependent and CDK cyclin independent manner. My studies suggested that Xic1 turnover during NER requires the loading of PCNA onto UV damaged DNA and binding of PCNA to DNA polymerase delta. My results indicated that both loading of polymerase delta and its enzymatic activity at the sites of repair was required to trigger Xic1 turnover during NER. My studies also showed that the C-terminal residues 161-190 of Xic1 are necessary and sufficient for this NER mediated Xic1 proteolysis. I have also identified the Cul4A-DDB1Cdt2 (CRL4Cdt2) ubiquitin ligase as the potential E3 that mediates Xic1 turnover during NER. My studies indicated that not only Xic1 but also p16Xic2 which shares homology with p21Cip1 was also targeted for degradation during NER in a PCNA dependent manner suggesting that this mechanism of PCNA and CRL4Cdt2 mediated turnover is conserved among all Cip-type CDK inhibitors. Additionally, my studies have also contributed to the identification and characterization of CRL4Cdt2 as the E3 ligase for Xic1 during DNA replication in Xenopus egg extracts.;While my studies begin to describe how Xic1 is degraded in a DNA, PCNA and CRL4Cdt2 dependent manner during DNA replication and DNA repair, to date, no studies have addressed how protein turnover by the PCNA and CRL4Cdt2-dependent pathway might be regulated. My studies described how mitotic phosphorylation of Xic1 negatively regulates its turnover by PCNA/CRL4Cdt2 during the transition from mitosis to interphase. During mitosis, Xic1 is phosphorylated at six CDK consensus sites by CDK1-Cyclin B kinase and is stabilized. Upon exit from mitosis and entry into interphase, Xic1 gets dephosphorylated by a PP2A like phosphatase and is targeted for degradation during DNA replication and repair. I have identified the residue T172 as the critical mitotic phosphorylation site of Xic1 that prevents its turnover during the M to S phase transition. My studies show that phosphorylation of Xic1 at T172 inhibited both its binding to PCNA and its CRL4Cdt2 mediated degradation. These studies indicated that PCNA/CRL4Cdt2 mediated degradation could be regulated during the cell cycle by phosphorylation of its substrates.;Overall my work on the CKI regulation during DNA replication and repair lead to the conclusion that regulated proteolysis of Xic1 occurs during NER repair process which may be required for efficient removal of damaged DNA. My studies show for the first time that during NER, Xic1 is degraded in a trimeric PCNA dependent manner. My work also identified CRL4Cdt2 as the ubiquitin ligase for Xic1 turnover during DNA replication and repair in Xenopus egg extracts. My studies were also the first to show that phosphorylation of a substrate may be an important means to negatively regulate cell cycle dependent proteolysis by PCNA-CRL4Cdt2 pathway. (Abstract shortened by UMI.)...