DNA REPAIR IN CANCER AND SENESCENCE

癌症和衰老中的 DNA 修复

• 批准号: 6288735
• 负责人: VILHELM A. BOHR
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6288735
• 关键词: DNA damage; DNA directed RNA polymerase; DNA repair; Werner's syndrome; aging; biomarker; cell line; genetic transcription; helicase; human genetic material tag; phenotype; premature aging; protein purification; transfection

Summary of work: Werners Syndrome (WS) is a homozygous recessive disease characterized by early onset of many characteristics of normal aging, such as wrinkling of the skin, graying of the hair, cataracts, diabetes, and osteoporosis. The symptoms of WS begin to appear around the age of puberty, and most patients die before age 50. Because of the acceleration of aging in WS, the study of this disease will hopefully shed light on the degenerative processes that occur in normal aging. Cells from WS patients grow more slowly and senescence at an earlier population doubling than age-matched normal cells, possibly because these cells appear to lose the telomeric ends of their chromosomes at an accelerated rate. In general, WS cells have a high level of genomic instability, with increased amounts of DNA deletions, insertions, and rearrangements. These effects could potentially be the result of defects in DNA repair, replication, and/or recombination, although the actual biochemical defect remains unknown. The gene that is defective in WS, the WRN gene, has recently been identified and characterized. We are making purified WRN protein for use in a number of basic and complex biochemical assays. We are using several avenues to identify and characterize the biochemical defect in WS cells. We have shown that there is a transcriptional defect in WS cells, and that this defect also can be shown in in vitro cell extracts. In in vitro transcription assays, the Werner protein (WRNp) stimulates transcription. The WRNp has helicase activity and will unwind small and large DNA duplex constructs. The helicase activity is sensitive to a compound, distamycin, which is a DNA minor groove binder. Interestingly, we find that the WRNp interacts both physically and functionally with another portein, replication protein A, which plays major roles in DNA repair and in replication. WRNp does not readily recognize DNA damage and it binds more efficiently to single stranded than double stranded DNA. The WRNp also has another enzymatic activity, a 3-5 exonuclease function. We observe that the exonuclease enzyme is blocked by some forms of DNA damage on the substrate DNA, but not by others. Our ongoing and future studies will be directed towards elucidation of the causes of the accelerated aging phenotype in WS, with hope that this knowledge can also be applied to our current understanding of both the aging of cells and organisms in general. - Aging, Werner syndrome, DNA repair, recombination, DNA replication, transcription

工作总结：沃纳氏综合症（WS）是一种纯合子隐性遗传疾病，其特征是早期出现许多正常衰老特征，如皮肤起皱、头发花白、白内障、糖尿病和骨质疏松症。 WS的症状在青春期左右开始出现，大多数患者在50岁之前死亡。由于WS的衰老速度加快，对这种疾病的研究有望揭示正常衰老过程中发生的退行性过程。与年龄匹配的正常细胞相比，WS 患者的细胞生长更慢，并且在群体倍增更早的情况下衰老，可能是因为这些细胞似乎以更快的速度失去染色体端粒末端。一般来说，WS 细胞具有高度的基因组不稳定性，DNA 缺失、插入和重排数量增加。这些影响可能是 DNA 修复、复制和/或重组缺陷的结果，尽管实际的生化缺陷仍然未知。 WS 中存在缺陷的基因，即 WRN 基因，最近已被鉴定和表征。我们正在制备纯化的 WRN 蛋白，用于许多基本和复杂的生化测定。我们正在使用多种途径来识别和表征 WS 细胞的生化缺陷。我们已经证明 WS 细胞存在转录缺陷，并且这种缺陷也可以在体外细胞提取物中显示出来。在体外转录测定中，Werner 蛋白 (WRNp) 会刺激转录。 WRNp 具有解旋酶活性，可以解开小型和大型 DNA 双链体构建体。解旋酶活性对化合物偏端霉素敏感，偏端霉素是一种 DNA 小沟结合剂。有趣的是，我们发现 WRNp 在物理和功能上与另一种蛋白质（复制蛋白 A）相互作用，后者在 DNA 修复和复制中发挥着重要作用。 WRNp 不易识别 DNA 损伤，并且与单链 DNA 的结合比与双链 DNA 的结合更有效。 WRNp 还具有另一种酶活性，即 3-5 核酸外切酶功能。我们观察到核酸外切酶会被底物 DNA 上某些形式的 DNA 损伤所阻断，但不会被其他形式的损伤所阻断。我们正在进行和未来的研究将致力于阐明 WS 加速衰老表型的原因，希望这些知识也可以应用于我们目前对细胞和生物体衰老的理解。 - 衰老、维尔纳综合症、DNA修复、重组、DNA复制、转录