Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification
Expression of the ubiquitin-conjugating DNA repair enzymes HHR6A and B suggests a role in spermatogenesis and chromatin modification
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DOI：
10.1006/dbio.1996.0011
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发表时间：
1996-01-10
影响因子：
2.7
通讯作者：
Hoeijmakers, JHJ
中科院分区：
文献类型：
--
作者：
Koken, MHM;Hoogerbrugge, JW;Hoeijmakers, JHJ
RAD6是泛素结合（E2）酶家族的成员，在酿酒酵母的所谓“N规则”蛋白质分解途径中发挥作用。在体外，该蛋白质可以将一个或多个泛素（U B）部分连接到组蛋白H2 A和B上，并触发它们的E3依赖性降解。Rad6突变体显示出显著的多效性表型，暗示该蛋白质参与DNA损伤诱导的诱变、复制后修复、逆转录转座抑制和孢子形成。RAD6转录强烈诱导紫外线照射和减数分裂，这表明它是一个损伤诱导的反应途径的一部分，它是参与减数分裂重组。据推测，该蛋白质通过调节染色质结构发挥其功能。在此之前，我们已经克隆了该基因的两个人类同源物（命名为HHR6A和HHR6B），并证明它们部分补充了酵母缺陷。在这里，我们提出了一个详细的表征，他们的表达在转录本和蛋白质水平。这两个HHR6蛋白，解决了二维免疫印迹分析，在所有哺乳动物组织和细胞类型的检查，表明这两个基因的功能和组成型表达。虽然这些蛋白质是高度保守的，但酵母中存在的紫外线诱导并没有被保留，这表明酵母和哺乳动物之间在损伤反应方面存在重要差异。HHR6转录物或蛋白质在热休克后和细胞周期期间没有改变，表明这些蛋白质不参与应激反应或细胞周期调节。在睾丸中发现HHR 6转录本和蛋白水平升高。增强HHR6的表达并不符合减数分裂重组，但与组蛋白的过渡蛋白的替代。免疫组织化学显示HHR6蛋白位于细胞核中，与染色质的功能联系一致。电子显微镜结合免疫金标记揭示了HHR6在常染色质区域的优先定位，表明该蛋白与转录活性区域相关。我们的研究结果支持这样的想法，这两个HHR6基因有重叠的，与染色质构象的组成性功能，他们有一个特定的作用，在精子发生，涉及UB介导的组蛋白降解。(C)出版社：Academic Press，Inc.
RAD6, a member of the expanding family of ubiquitin-conjugating (E2) enzymes, functions in the so-called ''N-rule'' protein breakdown pathway of Saccharomyces cerevisiae. In vitro, the protein can attach one or multiple ubiquitin (Ub) moieties to histones H2A and B and trigger their E3-dependent degradation. Rad6 mutants display a remarkably pleiotropic phenotype, implicating the protein in DNA damage-induced mutagenesis, postreplication repair, repression of retrotransposition, and sporulation. RAD6 transcription is strongly induced upon UV exposure and in meiosis, suggesting that it is part of a damage-induced response pathway and that it is involved in meiotic recombination. It is postulated that the protein exerts its functions by modulating chromatin structure. Previously, we have cloned two human homologs of this gene (designated HHR6A and HHR6B) and demonstrated that they partially complement the yeast defect. Here we present a detailed characterisation of their expression at the transcript and protein levels. Both HHR6 proteins, resolved by 2-dimensional immunoblot analysis, are expressed in all mammalian tissues and cell types examined, indicating that both genes are functional and constitutively expressed. Although the proteins are highly conserved, the UV induction present in yeast is not preserved, pointing to important differences in damage response between yeast and mammals. Absence of alterations in HHR6 transcripts or protein upon heat shock and during the cell cycle suggests that the proteins are not involved in stress response or cell cycle regulation. Elevated levels of HHR6 transcripts and proteins were found in testis. Enhanced HHR6 expression did not coincide with meiotic recombination but with the replacement of histones by transition proteins. Immunohistochemistry demonstrated that the HHR6 proteins are located in the nucleus, consistent with a functional link with chromatin. Electron microscopy combined with immunogold labeling revealed a preferential localisation of HHR6 in euchromatin areas, suggesting that the protein is associated with transcriptionally active regions. Our findings support the idea that both HHR6 genes have overlapping, constitutive functions related to chromatin conformation and that they have a specific role in spermatogenesis, involving Ub-mediated histone degradation. (C) 1996 Academic Press, Inc.