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首页> 外文学位 >Proteomic and transcriptional analysis of components of the DNA replication and repair machinery in mouse embryonic stem cells.
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Proteomic and transcriptional analysis of components of the DNA replication and repair machinery in mouse embryonic stem cells.

机译：小鼠胚胎干细胞中DNA复制和修复机制的组成部分的蛋白质组和转录分析。

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摘要

DNA replication timing, chromatin state and transcriptional activity of a genomic locus are tightly correlated and undergo vast changes during cell fate changes underlying differentiation and reprogramming processes. It has been proposed that the correlation of replication timing and transcriptional activity is mediated by the interaction of pre-replication complex (preRC) components with the transcription machinery. We addressed this question by investigating the influence of preRC components on transcriptional activity in embryonic stem cells (ESCs) by depleting components of the preRC. Interestingly, we did not find a global change of transcriptional activity. In addition, we tested the interaction of the preRC with the COMPASS chromatin modification complex. Contrary to results in the yeast system, we found no interaction between preRC components and the COMPASS complex in mouse ESCs do, and thus the recruitment of the preRC to origins of replication via this interacton is unlikely. As the replication fork clamp proliferacting cell nuclear antigen (PCNA) is conveniently located at the replication fork and has a plethora of interaction partners, including a number of chromatin-modifying enzymes, PCNA might mediate the replication of chromatin states by recruiting chromatin-modifying enzymes to the replication fork at appropriate times during S-phase. We addressed this hypothesis by determining the PCNA interactome in unsynchronized ESCs as well as ESCs specifically in early or late S-phase, respectively, and identified a number of chromatin-modifying complexes as PCNA interactors some of which interacted specifically in early or late S-phase, supporting our model. Interestingly, among the PCNA interaction partners identified, we found the annealing helicase Zinc finger Ran domain containing protein 3 (Zranb3), which we found to be particularly highly expressed in ESC. We further investigated the function of Zranb3 and identified the MutSalpha complex of the DNA mismatch repair machinery (MMR), a process especially upregulated in ESC, as interactor of Zranb3. Given the interaction of Zranb3 with the MutSalpha complex as well as PCNA and its high levels in ESCs, we suggest a model in which MutSalpha; recruits PCNA to sites of MMR and both are then bound by Zranb3 to repair DNA lesions.
机译：基因组基因座的DNA复制时间，染色质状态和转录活性紧密相关，并在分化和重编程过程中的细胞命运变化期间发生巨大变化。已经提出，复制时机与转录活性的相关性是由复制前复合物（preRC）组分与转录机制的相互作用介导的。我们通过研究preRC成分的消耗来研究preRC成分对胚胎干细胞（ESCs）转录活性的影响，从而解决了这个问题。有趣的是，我们没有发现转录活性的整体变化。此外，我们测试了preRC与COMPASS染色质修饰复合物的相互作用。与酵母系统中的结果相反，我们发现在小鼠ESC中preRC组分与COMPASS复合物之间没有相互作用，因此不太可能通过该相互作用子将preRC募集到复制起点。由于复制叉钳位增殖细胞核抗原（PCNA）方便地位于复制叉处并且具有过多的相互作用伙伴，包括许多染色质修饰酶，因此PCNA可能通过募集染色质修饰酶来介导染色质状态的复制。在S阶段的适当时间连接到复制叉。我们通过确定未同步的ESC中的PCNA相互作用组以及分别在S期早期或晚期的ESC确定PCNA相互作用组，并鉴定了许多染色质修饰复合物作为PCNA相互作用物，其中一些在S期早期或晚期进行了特异性相互作用。阶段，支持我们的模型。有趣的是，在确定的PCNA相互作用伙伴中，我们发现了包含蛋白质3（Zranb3）的退火解旋酶锌指Ran结构域，我们发现它在ESC中表达特别高。我们进一步研究了Zranb3的功能，并确定了DNA错配修复机制（MMR）的MutSalpha复合体，该过程在ESC中特别上调，是Zranb3的相互作用因子。考虑到Zranb3与MutSalpha复合物以及PCNA的相互作用以及其在ESC中的高水平，我们建议建立一个模型，其中MutSalpha;将PCNA募集到MMR的位置，然后将两者都与Zranb3结合以修复DNA损伤。

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  • 作者

    Sellami, Nadia.;

  • 作者单位

    University of California, Los Angeles.;

  • 授予单位 University of California, Los Angeles.;
  • 学科 Biology Molecular.;Biology Cell.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 164 p.
  • 总页数 164
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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