DNA Repair In Cancer And Senescence

工作概述：Werner综合征(WS)是一种纯合子隐性疾病，以许多正常衰老特征的早期发病为特征，如皮肤皱纹、头发灰白、白内障、糖尿病和骨质疏松。WS的症状开始出现在青春期年龄，大多数患者在50岁之前死亡。由于WS的衰老加速，对这种疾病的研究将有望揭示正常衰老中发生的退行性过程。WS患者的细胞生长更慢，衰老的群体比年龄匹配的正常细胞早一倍，可能是因为这些细胞似乎以加速的速度丢失了它们染色体的端粒末端。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/或重组缺陷的结果，尽管实际的生化缺陷仍不清楚。在WS中存在缺陷的基因，WRN基因，最近已经被发现和鉴定。我们已经制备了纯化的WRN蛋白，用于一些基本和复杂的生化分析。我们正在使用几种方法来鉴定和表征WS细胞的生化缺陷。我们已经证明在WS细胞中存在转录缺陷，并且在体外细胞提取液中也可以看到这种缺陷。WRNp具有解旋酶活性，并将解开大小DNA双链结构。它还将解开不寻常的DNA结构，如三螺旋和DNA叉状结构。我们正在将Werner解旋酶的活性与在Bloom综合征中突变的另一种解旋酶Bloom的活性进行比较。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修复和复制中发挥重要作用。WRNp不容易识别DNA损伤，它与单链DNA的结合比双链DNA更有效。WRNp还有另一种酶活性，即3-5‘核酸外切酶功能。我们观察到，核酸外切酶被底物DNA上的某些形式的DNA损伤所阻断，但不被其他形式的DNA损伤所阻断。WRN核酸外切酶与参与DNA双链断裂修复的Ku异源二聚体蛋白在物理和功能上都相互作用。最近，我们发现了一些新的蛋白质与Werner蛋白的功能和物理相互作用。它们包括参与碱基切除DNA修复和复制的翻盖核酸内切酶1和参与细胞凋亡和信号转导的p53。WRNp在这一途径中发挥着重要作用，我们还发现与参与这一过程的其他蛋白质相互作用。我们还检测到WRNp与端粒结合蛋白TRF2之间的物理和功能相互作用，表明细胞核中至少有部分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的加速衰老表型的原因，希望这一知识也可以应用于我们目前对细胞和生物体衰老的理解。

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.