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Cell cycle-regulated human DNA helicase B in DNA damage response.

机译：细胞周期调节的人类DNA解旋酶B在DNA损伤反应中。

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摘要

DNA helicases play essential roles in DNA replication, repair and recombination pathways. DNA helicase B has been identified in two mammalian species, mouse and human. The human DNA helicase B (HDHB) cDNA was cloned by homology to the mouse gene. The predicted protein is 1087 residues in length and contains conserved motifs characteristic of helicase superfamily 1, which are strikingly similar to those of E. coli RecD and T4 Dda proteins.; Previously, we have found that a dominant negative point mutant HDHB (MutB) could block DNA replication. To analyze the subcellular localization of wt HDHB, GFP-HDHB fusion proteins were transiently expressed. In G1 phase cells, the fusion protein localized in DNA damage nuclear foci, while in S phase cells it localized in the cytoplasm, suggesting the existence of cell cycle-dependent localization signal(s) in the protein. Mutational analysis suggests that there is a functional subcellular localization domain in the extreme C-terminus of HDHB. We have also found that HDHB is phosphorylated in vivo in a cell cycle-dependent pattern, and that phosphorylation of ser 967 regulates the subcellular localization of the protein. In vitro, cyclin dependent kinase (CDK) phosphorylated purified recombinant HDHB on the same sites phosphorylated in vivo. Consistent with this, we found the subcellular localization of endogenous HDHB is also cell cycle-dependent.; Tagged HDHB co-localizes in nuclear foci with endogenous Mre11, NBS1, Rad50, ATM, and RPA, and co-immunoprecipitates with Mre11 and ATM. Cells expressing GFP-HDHB, but not helicase-deficient HDHB, contain single-stranded DNA (ssDNA) in vivo and pan-nuclear gammaH2AX. These observations suggested that unregulated processing of endogenous DNA damage by over-expressed HDHB promotes damage signaling.; To examine whether endogenous HDHB participates in damage signaling, stable HDHB-knockdown cell lines were generated. HDHB-depleted cells are sensitive to ionizing radiation and display a significant delay in radiation-induced phosphorylation of NBS1, H2AX, and Chk2, particularly in G1 phase. NBS1 and RPA focus formation after irradiation is also delayed in HDHB-depleted cells. We suggest that endogenous HDHB associates with MRN complexes at sites of damage, generating ssDNA that amplifies damage signals.
机译：DNA解旋酶在DNA复制，修复和重组途径中起重要作用。 DNA解旋酶B已在两种哺乳动物，小鼠和人类中得到鉴定。通过与小鼠基因的同源性克隆人DNA解旋酶B（HDHB）cDNA。所预测的蛋白质长度为1087个残基，并包含解旋酶超家族1的保守基序，这些基序与大肠杆菌RecD和T4 Dda蛋白极为相似。以前，我们发现显性负点突变体HDHB（MutB）可以阻止DNA复制。为了分析野生型HDHB的亚细胞定位，GFP-HDHB融合蛋白被瞬时表达。在G1期细胞中，位于DNA中的融合蛋白会损伤核灶，而在S期细胞中，它会位于细胞质中，提示该蛋白中存在依赖于细胞周期的定位信号。突变分析表明，HDHB的极端C端有一个功能性亚细胞定位域。我们还发现，HDHB在体内以细胞周期依赖性模式被磷酸化，而ser 967的磷酸化调节蛋白质的亚细胞定位。在体外，细胞周期蛋白依赖性激酶（CDK）在体内磷酸化的相同位点上磷酸化了纯化的重组HDHB。与此相一致，我们发现内源性HDHB的亚细胞定位也与细胞周期有关。标记的HDHB与内源性Mre11，NBS1，Rad50，ATM和RPA共定位于核灶中，并与Mre11和ATM共免疫沉淀。表达GFP-HDHB而不表达解旋酶缺陷的HDHB的细胞在体内含有单链DNA（ssDNA）和泛核gammaH2AX。这些观察结果表明，过度表达的HDHB对内源性DNA损伤的无调控处理会促进损伤信号传导。为了检查内源性HDHB是否参与损伤信号传导，产生了稳定的HDHB-knockdown细胞系。耗尽HDHB的细胞对电离辐射敏感，并且在辐射诱导的NBS1，H2AX和Chk2磷酸化中表现出明显的延迟，特别是在G1期。在HDHB耗尽的细胞中，辐射后NBS1和RPA焦点形成也被延迟。我们建议内源性HDHB与MRN复合物在损伤位点缔合，产生可放大损伤信号的ssDNA。

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  • 作者

    Gu, Jinming.;

  • 作者单位

    Vanderbilt University.;

  • 授予单位 Vanderbilt University.;
  • 学科 Biology Molecular.; Biology Cell.
  • 学位 Ph.D.
  • 年度 2005
  • 页码 109 p.
  • 总页数 109
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 分子遗传学;细胞生物学;
  • 关键词

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