DNA Repair In Cancer And Senescence

癌症和衰老中的 DNA 修复

• 批准号: 6668731
• 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/6668731
• 关键词: DNA damage; DNA repair; DNA replication; Werner's syndrome; aging; developmental genetics; enzyme activity; genetic recombination; genetic transcription; helicase; human tissue; neoplasm /cancer; neoplasm /cancer genetics

Summary of work: Werner's 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 have made 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 seen in cell extracts in vitro. The WRNp has helicase activity and will unwind small and large DNA duplex constructs. It will also unwind unusual DNA structures such as triple helices and DNA forks. We are comparing the Werner helicase activity to that of another helicase, Bloom, which is mutated in Bloom syndrome. Both WRNp and Bloom interact physically and functionally with another protein, 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 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. The WRN exonuclease interacts both physically and functionally with the Ku heterodimer protein, which is involved in DNA double strand break repair. Recently, we have discovered a number of new functional and physical protein interactions with Werner protein. They include and interaction with Flap-endonuclease 1, which is involved in Base Excision DNA Repair and replication; and p53, involved with apoptosis and signal transduction. WRNp plays and important role in this pathways as we also find interactions with other proteins involved in this process. We have also detected a physical and functional interaction between WRNp and the telomeric binding protein, TRF2, suggesting that at least some of the WRN protein in the nucleus has a function at telomere ends. 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.

工作总结：维尔纳综合征（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。我们已经制备了纯化的 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看到。 WRNp 具有解旋酶活性，可以解开小型和大型 DNA 双链体构建体。它还会解开不寻常的 DNA 结构，例如三螺旋和 DNA 叉。我们正在将维尔纳解旋酶的活性与另一种解旋酶布卢姆（Bloom）的活性进行比较，布卢姆解旋酶在布卢姆综合征中发生突变。 WRNp 和 Bloom 都与另一种蛋白质（复制蛋白 A）在物理和功能上相互作用，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 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。 WRN 核酸外切酶与 Ku 异二聚体蛋白在物理和功能上相互作用，后者参与 DNA 双链断裂修复。最近，我们发现了许多新的功能和物理蛋白质与沃纳蛋白的相互作用。它们包括与 Flap-核酸内切酶 1 的相互作用，Flap-核酸内切酶 1 参与碱基切除 DNA 修复和复制；和p53，参与细胞凋亡和信号转导。 WRNp 在此途径中发挥着重要作用，因为我们还发现了与此过程中涉及的其他蛋白质的相互作用。我们还检测到 WRNp 和端粒结合蛋白 TRF2 之间的物理和功能相互作用，这表明细胞核中至少有一些 WRN 蛋白在端粒末端具有功能。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。