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.

工作概述：沃纳综合征（Werner’s Syndrome， WS）是一种纯合隐性疾病，其特征是许多正常衰老的特征早发，如皮肤起皱、头发变白、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中衰老的加速，对这种疾病的研究有望揭示正常衰老中发生的退行性过程。与年龄匹配的正常细胞相比，来自WS患者的细胞生长更慢，衰老的时间更早，数量增加了一倍，可能是因为这些细胞似乎以更快的速度失去了染色体的端粒末端。一般来说，WS细胞具有高度的基因组不稳定性，DNA缺失、插入和重排的数量增加。这些影响可能是DNA修复、复制和/或重组缺陷的潜在结果，尽管实际的生化缺陷尚不清楚。在WS中有缺陷的基因，WRN基因，最近已经被鉴定和表征。我们已经制备了纯化的WRN蛋白，用于许多基础和复杂的生化分析。我们正在使用几种途径来识别和表征WS细胞中的生化缺陷。我们已经证明WS细胞中存在转录缺陷，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，可以解开大小DNA双链结构。它还将解开不寻常的DNA结构，如三螺旋和DNA分叉。我们正在比较Werner解旋酶和另一种解旋酶Bloom的活性，Bloom在Bloom综合征中发生了突变。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，复制蛋白A在DNA修复和复制中起主要作用。WRNp不易识别DNA损伤，它与单链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'外切酶功能。我们观察到外切酶酶被底物DNA上某些形式的DNA损伤所阻断，而不是被其他形式的DNA所阻断。WRN外切酶在物理和功能上与Ku异二聚体蛋白相互作用，参与DNA双链断裂修复。近年来，我们发现了许多新的功能和物理蛋白与Werner蛋白相互作用。它们包括与参与碱基切除、DNA修复和复制的flap -内切酶1相互作用；p53，参与细胞凋亡和信号转导。WRNp在这一途径中发挥着重要作用，因为我们也发现了与这一过程中涉及的其他蛋白质的相互作用。我们还发现了WRNp与端粒结合蛋白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。我们正在进行的和未来的研究将致力于阐明WS中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。