The Function of Werner Syndrome Protein

Werner综合征(WS)是一种纯合子隐性疾病，其特征是早期出现许多正常衰老的特征，如皮肤皱纹、头发变白、白内障、糖尿病和骨质疏松。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。WS中存在缺陷的基因WRN已被鉴定和鉴定。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。我们正在寻求几种方法来鉴定和表征WS细胞的生化缺陷。WRN蛋白具有解旋酶活性，可以解开大大小小的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RecQ解旋酶家族中的另一种解旋酶的活性进行比较，这些解旋酶都参与维持基因组的稳定性。WRN还有另一种酶活性，即3-5‘核酸外切酶功能。我们正在寻找WRN参与的途径，并发现了一些新的功能和物理上的蛋白质与Werner蛋白质的相互作用。 我们的数据有力地表明，WRN参与了两条主要的DNA修复途径：碱基切除修复和重组。这一结论得到了蛋白质功能相互作用的生化研究和细胞生物学数据的支持。此外，我们的观察和他人的结果表明，WRN的主要功能是在端粒末端。WRN与关键的端粒蛋白如TRF1、2和POT1相互作用。 由于端粒富含G，它们在氧化应激后和正常衰老过程中积累8-oxodG。为了研究WRN、BLM和RecQ5在端粒DNA损伤时端粒维持中的作用，在8-oxodG修饰的端粒D-loop底物上评价了这些RecQ解旋酶的催化活性。我们的结果表明，WRN和BLM优先解开8-oxodG修饰的端粒D-环。此外，在这些研究中评估了POT1的DNA结合，我们发现POT1与8-oxodG修饰的端粒D-环结合有更高的亲和力，但没有显示出对8-oxodG修饰的端粒单链DNA的偏好。 此外，我们还分析了WRN与DNA PKcs在体外和体内的相互作用。众所周知，DNA PKcs在双链断裂修复中起作用。在正常情况下，端粒在细胞中不被识别为双链断裂，然而，当端粒变得非常短时，端粒末端被识别，这导致DNA PKcs的激活和双链断裂修复反应。我们的结果表明，DNA-PKcs在体外选择性地刺激WRN解旋酶，而不是WRN外切核酸酶活性。此外，我们还发现，WRN的过表达可以逆转DNA-PKcs基因敲除细胞中常见的端粒G-尾的侵蚀。这项研究首次表明WRN和DNA PKcs共同维持正常的端粒DNA长度。DNA PKcs和WRN之间的功能相互作用可能对正常衰老具有重要意义，因为细胞衰老也与端粒G尾缩短有关。 总之，这些观察结果扩展了我们之前对端粒WRNS功能的分析，并表明WRN蛋白参与了受损和未受损端粒末端的维持。 WRN也参与了DNA修复过程。具体地说，我们在体外和体内发现了WRN在氧化DNA碱基损伤的DNA修复中的作用的证据。我们最近的工作集中在WRN与NEIL1的相互作用上。NEIL1是对去除几种氧化生成的DNA损伤的反应，最著名的是甲酰胺并嘧啶类化合物。其他研究表明，NEIL1失活会导致胃癌，这强烈表明NEIL1的活性对甲酰胺基嘧啶和其他开环碱基损伤的修复至关重要。我们以前报道过NEIL1和WRN相互作用，WRN刺激了NEIL1的切开活动。这项研究被扩展到其他四个RecQ解旋酶，我们发现NEIL1：WRN的相互作用是特异的，因为没有其他RecQ解旋酶可以类似地刺激NEIL1。我们进一步证明，这种刺激需要双链DNA底物，这一发现可能很重要，因为NEIL1也可以作用于嵌入单链DNA底物的病变。这项研究是第一批在一项研究中比较和对比所有五种人类解旋酶的研究之一，通过这样的分析，我们试图更好地了解RecQ解旋酶中哪些细胞功能是独特的或共享的。

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. 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 been identified and characterized. We have made purified WRN protein for use in a number of basic and complex biochemical assays. We are pursuing several avenues to identify and characterize the biochemical defect in WS cells. WRN protein 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 helicases in the family of RecQ helicases that are all involved in the maintenance of genome stability. WRN has another enzymatic activity, a 3-5' exonuclease function. We are searching for pathways in which WRN participates and have discovered a number of new functional and physical protein interactions with Werner protein. Our data strongly suggest that WRN is involved in two of the major DNA repair pathways: base excision repair and recombination. This conclusion is supported by biochemical studies of protein functional interactions and by cell biological data. Further, our observations and results from others suggest that a major function of WRN is at the telomere ends. WRN interacts with key telomeric proteins such as TRF1 and 2 and POT1. Since telomeres are G-rich, they accumulate 8-oxodG after oxidative stress and during the normal aging process. To investigate the role that WRN, BLM, and RecQ5 might play in telomere maintenance when telomeric DNA is damaged, the catalytic activities of these RecQ helicases were evaluated on 8-oxodG-modified telomeric D-loop substrates. Our results revealed that WRN and BLM preferentially unwound 8-oxodG-modified telomeric D-loops. Additionally, POT1 DNA binding was assessed in these studies and we showed that POT1 bound with higher affinity to 8-oxodG-modified telomeric D-loops but shows no preference for 8-oxodG-modified telomeric single-stranded DNA. Additionally, we analyzed the in vitro and in vivo interactions between WRN and DNA PKcs. DNA PKcs is well known to function in double strand break repair. Normally telomeres are not recognized as double strand breaks in cells however when telemeres become critically short telomere ends are recognized and this leads to the activation of DNA PKcs and a double strand break repair response. Our results showed that DNA-PKcs selectively stimulated WRN helicase but not WRN exonuclease activity in vitro on a model telomeric D-loop model. Additionally, we showed that overexpression of WRN could reverse the erosion of the telomeric G-tails normally found in DNA-PKcs knockdown cells. This study was the first to show that WRN and DNA PKcs cooperate to maintain normal telomeric DNA length. The functional interaction between DNA PKcs and WRN may have important implications for normal aging, because cellular senescence is also associated with shorted telomeric G-tails. Together, these observations extend our previous analysis of WRNs function at the telomeres and suggest that WRN protein is involved in the maintenance of damaged and undamaged telomere ends. WRN is also involved in the DNA repair processes. Specifically, we find in vitro and in vivo evidence for a role of WRN in the DNA repair of oxidative DNA base lesions. Our recent work has focused on the interaction of WRN with NEIL1. NEIL1 is response for the removal of several oxidatively generated DNA lesions, most notable the formamidopyrimidines. Others have shown that inactivation of NEIL1 leads to gastric cancers which strongly suggest that the activities of NEIL1 are critical for repair of formamidopyrimidines and other ring opened base lesions. We previously reported that NEIL1 and WRN interacted and that WRN stimulated NEIL1 incision activity. This study was extended to the other four RecQ helicases and we show that the NEIL1:WRN interaction is specific as no other RecQ helicase can similarly stimulate NEIL1. We further show that this stimulation requires a double-stranded DNA substrate and this finding may be important because NEIL1 can also work on lesions imbedded in single stranded DNA substrates. This study was one of the first to compare and contrast all five human helicases in one study and through such analysis we seek to gain a better understanding of which cellular functions are unique or shared among the RecQ helicases.