Mechanism of Triplex DNA Damage and Repair in Skin Cells

描述(由申请人提供)：皮肤细胞必须对环境暴露和治疗剂造成的DNA损伤做出反应。一种反应，DNA修复，是细胞识别、移除和替换基因组中的损伤的过程。DNA修复缺陷是几种遗传性疾病的基础，这些疾病容易导致皮肤癌、光敏和发育缺陷。尽管随机DNA损伤的受损修复是有害的，但靶向DNA损伤并选择性地抑制某些基因的修复可能是达到治疗效果所必需的。针对特定核苷酸序列的DNA损伤剂的三链形成寡核苷酸(TFOS)已成为一种有前途的基因治疗方法。TFOS在结构上创造了非常规类型的DNA损伤，可以深入了解正常DNA修复的机制，实际上可能模拟与皮肤疾病有关的自然发生的大分子相互作用，并有可能发展为实用的皮肤基因治疗形式。该项目的总体目标是了解和操纵TFOS如何靶向补骨脂素与皮肤来源细胞间质胶原酶基因的光加合物，并确定修复这种不寻常的大分子损伤的分子基础。假设TFO和染色质结构积极地调节一种独特形式的大分子DNA损伤的传递和修复，并且存在新的细胞机制来修复这种类型的损伤。为了验证这一假说，该项目的具体目标是：1)评估TFO结构在补骨脂素光加合物损伤的传递和修复中的作用。具有修饰骨架的TFOS将被评估其靶向补骨脂素光加合物并在间质胶原酶基因中介导其修复的能力；2)确定染色质结构和基因活性在TFO靶向DNA损伤和修复中的作用。将检查一般或局部破坏染色质结构或改变转录的外源性因素是否能够影响TFO靶点损伤和修复的可及性；3)确定核苷酸切除修复在去除TFOS靶向补骨脂素加合物中的机制。将利用结构TFO变异体和DNA修复缺陷的人类细胞系来剖析核苷酸切除修复在处理不同TFO靶向的补骨脂素光加合物中的作用；4)评估细胞解旋酶在修复TFO损伤中的作用。具有不同解旋酶活性或缺陷的细胞系将被用来确定这些酶是否参与TFO介导的损伤的处理。在项目期结束时，拟议的工作应有助于我们对DNA修复机制的基本知识，并提供基本原则，通过操纵DNA损伤和修复来指导TFOS的治疗应用。

DESCRIPTION (provided by applicant): Skin cells must respond to DNA damage caused by both environmental exposure as well as therapeutic agents. One response, DNA repair, is the process by which cells recognize, remove and replace lesions in their genomes. Defects in DNA repair underlie several heritable diseases that predispose individuals to skin cancer, photosensitivity and developmental defects. Although impaired repair of random DNA damage is deleterious, targeted DNA damage and selective inhibition of its repair in certain genes can be desirable to achieve a therapeutic effect. Triplex-forming oligonucleotides (TFOs) that target a DNA damaging agent to a specific nucleotide sequence have emerged as a promising approach for gene therapy. TFOs create structurally unconventional types of DNA damage that can provide insight into the mechanisms of normal DNA repair, that may actually mimic naturally occurring macromolecular interactions that are involved in diseases of the skin, and that have potential to develop into a practical form of cutaneous gene therapy. The overall objective of the proposed project is to understand and manipulate how TFOs target psoralen photoadducts to the interstitial collagenase gene in skin derived cells, and to identify the molecular basis for repair of this unusual macromolecular damage. The hypothesis is that TFO and chromatin structures actively modulate both delivery and repair of a unique form of macromolecular DNA damage, and that novel cellular mechanisms exist to repair this type of damage. To test this hypothesis, the specific aims of the project are: 1) To assess the role of TFO structure in delivery and repair of psoralen photoadduct damage in genomic targets. TFOs with modified backbones will be assessed for their ability to target psoralen photoadducts and mediate their repair in the interstitial collagenase gene; 2) To determine the role of chromatin structure and gene activity in TFO-targeted DNA damage and repair. Exogenous agents that generally or focally disrupt chromatin structure or that alter transcription will be examined for the ability to affect accessibility of TFO target sites to damage and repair; 3) To determine the mechanism of nucleotide excision repair in removal of psoralen adducts targeted by TFOs. Structural TFO variants and human cell lines defective in DNA repair will be used to dissect the role of nucleotide excision repair in processing psoralen photoadducts targeted by different TFOs; 4) To assess the role of cellular helicases in repairing TFO lesions. Cell lines with different helicase activities or deficiencies will be used to determine if these enzymes participate in processing of TFO-mediated damage. At the conclusion of the project period, the proposed work should contribute to our fundamental knowledge of DNA repair mechanisms as well as provide basic principles to guide therapeutic applications of TFOs by manipulating DNA damage and repair.