Walker L J, Robson C N, Black E, Gillespie D, Hickson I D
Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, United Kingdom.
Mol Cell Biol. 1993 Sep;13(9):5370-6. doi: 10.1128/mcb.13.9.5370-5376.1993.
The DNA binding activity of the c-jun proto-oncogene product is inhibited by oxidation of a specific cysteine residue (Cys-252) in the DNA binding domain. Jun protein inactivated by oxidation of this residue can be efficiently reactivated by a factor from human cell nuclei, recently identified as a DNA repair enzyme (termed HAP1 or Ref-1). The HAP1 protein consists of a core domain, which is highly conserved in a family of prokaryotic and eukaryotic DNA repair enzymes, and a 61-amino-acid N-terminal domain absent from bacterial homologs such as Escherichia coli exonuclease III. The eukaryote-specific N-terminal domain was dispensable for the DNA repair functions of the HAP1 protein but was essential for reactivation of the DNA binding activity of oxidized Jun protein. Consistent with this finding, exonuclease III protein could not reactive Jun. A minimal 26-residue region of the N-terminal domain proximal to the core of the HAP1 enzyme was required for redox activity. By site-directed mutagenesis, cysteine 65 was identified as the redox active site in the HAP1 enzyme. In addition, it is proposed that cysteine 93 interacts with the redox active site, probably via disulfide bridge formation. It is concluded that the HAP1 protein has evolved a novel redox activation domain capable of regulating the DNA binding activity of a proto-oncogene product which is not essential for its DNA repair functions. Identification of a putative active site cysteine residue should facilitate analysis of the mechanism by which the HAP1 protein may alter the redox state of a wide range of transcription factors.
c-jun原癌基因产物的DNA结合活性受到DNA结合结构域中特定半胱氨酸残基（Cys-252）氧化的抑制。因该残基氧化而失活的Jun蛋白可被一种来自人类细胞核的因子有效重新激活，该因子最近被鉴定为一种DNA修复酶（称为HAP1或Ref-1）。HAP1蛋白由一个核心结构域和一个61个氨基酸的N端结构域组成，核心结构域在原核和真核DNA修复酶家族中高度保守，而细菌同源物如大肠杆菌核酸外切酶III则没有N端结构域。真核生物特有的N端结构域对于HAP1蛋白的DNA修复功能并非必需，但对于氧化型Jun蛋白DNA结合活性的重新激活至关重要。与这一发现一致，核酸外切酶III蛋白无法使Jun重新激活。HAP1酶核心附近的N端结构域中一个最小的26个残基区域是氧化还原活性所必需的。通过定点诱变，半胱氨酸65被确定为HAP1酶中的氧化还原活性位点。此外，有人提出半胱氨酸93可能通过形成二硫键与氧化还原活性位点相互作用。结论是，HAP1蛋白进化出了一个新的氧化还原激活结构域，该结构域能够调节一种原癌基因产物的DNA结合活性，而这对于其DNA修复功能并非必需。确定一个假定的活性位点半胱氨酸残基应有助于分析HAP1蛋白改变多种转录因子氧化还原状态的机制。
