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首页> 外文学位 >Regulation of Cip-type CDK inhibitor turnover during the cell cycle and in response to DNA damage.
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Regulation of Cip-type CDK inhibitor turnover during the cell cycle and in response to DNA damage.

机译：Cip型CDK抑制剂在细胞周期中以及对DNA损伤的响应中的调节。

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摘要

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.)
机译：在非洲爪蟾中，已鉴定出三种不同的CDK抑制剂（CKI）：p27Xic1（Xic1），p16Xic2（Xic2）和p17Xic3（Xic3）。分别与p21Cip1和p27 Kip1具有同源性的p16Xic2和p17Xic3被组织特异性表达并受到发育调控。 Xic1与p21 Cip1和p27Kip1共享功能和序列同源性，并且包含N端CDK结合域和C端PCNA结合域，称为“ PIP-box”。使用非洲爪蟾卵提取物，研究表明，在DNA复制过程中，Xic1以PCNA依赖性方式被募集到复制位点，并在DNA聚合酶转换过程中靶向泛素介导的降解。但是，尚未进行任何研究来了解PCNA依赖性修复过程中Xic1的调节。能够支持CKI蛋白水解和PCNA依赖性修复过程（如NER）的生化易于处理的非洲爪蟾卵提取物系统，为研究PCNA依赖性修复过程中Xic1调控的事件提供了理想的系统。体外研究表明，在NER过程中，Xic1以PCNA依赖性和CDK细胞周期蛋白非依赖性方式泛素化并降解。我的研究表明，NER期间的Xic1周转需要将PCNA加载到紫外线损伤的DNA上，并使PCNA与DNA聚合酶δ结合。我的结果表明，在NER期间触发Xic1周转需要聚合酶δ的加载及其在修复位点的酶活性。我的研究还表明，Xic1的C末端残基161-190对于这种NER介导的Xic1蛋白水解是必要和充分的。我还确定了Cul4A-DDB1Cdt2（CRL4Cdt2）泛素连接酶是在NER期间介导Xic1转换的潜在E3。我的研究表明，不仅Xic1，而且与p21Cip1具有同源性的p16Xic2也以PCNA依赖性方式靶向于NER期间的降解，这表明PCNA和CRL4Cdt2介导的周转机制在所有Cip型CDK抑制剂中均得以保留。此外，我的研究还有助于鉴定和鉴定CRL4Cdt2作为非洲爪蟾卵提取物中DNA复制过程中Xic1的E3连接酶。;虽然我的研究开始描述Xic1如何在DNA，PCNA和CRL4Cdt2依赖性方式下降解DNA。复制和DNA修复，迄今为止，尚无研究探讨如何调节PCNA和CRL4Cdt2依赖性途径的蛋白质更新。我的研究描述了Xic1的有丝分裂磷酸化如何在PCNA / CRL4Cdt2从有丝分裂过渡到间期的过程中负调控其更新。在有丝分裂期间，Xic1在六个CDK共有位点被CDK1-Cyclin B激酶磷酸化并稳定。从有丝分裂退出并进入相间期后，Xic1被PP2A（如磷酸酶）去磷酸化，并成为DNA复制和修复过程中降解的目标。我已将残基T172鉴定为Xic1的关键有丝分裂磷酸化位点，该位点可防止其在M到S相转变期间的更新。我的研究表明，Xic1在T172处的磷酸化抑制了它与PCNA的结合以及CRL4Cdt2介导的降解。这些研究表明PCNA / CRL4Cdt2介导的降解可以在细胞周期中通过其底物的磷酸化来调节。;总体上，我在DNA复制和修复过程中对CKI调节的研究得出结论，Xic1的蛋白水解发生在NER修复过程中，可能需要有效去除受损的DNA。我的研究首次表明，在NER期间，Xic1以三聚PCNA依赖性方式降解。我的工作还确定CRL4Cdt2是Xenopus卵提取物中DNA复制和修复过程中Xic1周转的泛素连接酶。我的研究还首次表明底物的磷酸化可能是通过PCNA-CRL4Cdt2途径负调控细胞周期依赖性蛋白水解的重要手段。 （摘要由UMI缩短。）

著录项

  • 作者

    Budhavarapu, Varija N.;

  • 作者单位

    The University of Texas Health Science Center at San Antonio.;

  • 授予单位 The University of Texas Health Science Center at San Antonio.;
  • 学科 Biology Molecular.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 237 p.
  • 总页数 237
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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