Repair of localized DNA damage

不能有效地应对复制应激被认为是发育缺陷的关键因素，是早衰综合征的基础，也是肿瘤形成的刺激因素。链间交联是特别危险的DNA损伤，因为它们是复制的绝对障碍，因此是复制应激反应的主要挑战。它们被认为是氧化代谢的产物，也是某些化疗药物治疗的结果。光活性DNA链间交联剂已在临床上使用多年。我们已经合成了抗原连接的肽，并证明了其活性。 在与化合物孵育的细胞中，激光光活化确定的亚核区域导致局部交联。 在修复熟练和缺陷细胞中监测这些加合物的修复。我们正在使用这种方法来跟踪蛋白质进入交联修复位点的募集。在两个循环过程中修复链间交联。在第一个循环中，在交联基底的任一侧切割股线。在第二个循环中，通过常规NER除去剩余的加合（并且仍然交联）碱。除了已确立的S期修复外，G1期是否会发生修复还存在不确定性。我们已经表明，在依赖于NER功能的过程中，交联在细胞周期的G1期被修复。XPC蛋白被迅速募集到交联和单加合物的位点。 然而，XPE损伤结合复合物被迅速募集为单加合物，并缓慢地募集为交联物。募集的XPE复合物依赖于XPC活性，和修复合成。我们的研究结果支持这样一种情况，即XPE复合物不识别交联，但在完成第一个修复周期后，当剩余的单加合碱基被迫离开螺旋时，XPE复合物被招募。XPE复合物的募集是第一个修复周期完成和第二个修复周期开始的标志。 我们已经将该技术应用于FANCD 2在ICL修复中的功能的检查。 FANCD 2蛋白是范可尼贫血途径中的中心节点。在这一途径中存在缺陷的个体会遭受严重的发育缺陷，并在产后生活中表现出过早衰老的迹象。该通路在复制应激反应中起着关键作用。 我们发现，FANCD 2被招募到多个刺激双链断裂的细胞周期独立的方式;激光定位peptide交联的细胞周期独立的;以及S期特异性方案。FA蛋白向ICL的非复制依赖性募集需要RNF 8的活性，RNF 8是一种泛素连接酶，以及一种新发现的泛素结合蛋白FAAP 20。这种FAAP 20-RNF 8泛素级联对于细胞对基因组应激（例如由链间交联施加的）的抗性是重要的。了解Fanconi通路中缺陷的性质将为开发针对这种疾病的有效疗法提供基础。 我们还表征了FAN 1的募集和对ICL修复的贡献，FAN 1是最近发现的与FANCD 2相关的核酸酶。 在也缺乏FA途径的生物体中发现了这种缺乏参与FA蛋白相互作用的蛋白质结构域的蛋白质的祖先版本。目前认为，FAN 1向ICL的募集依赖于FANCD 2。然而，我们已经发现，这种蛋白质以FANCD 2独立的方式快速募集到ICL。还有第二波积累，部分依赖于与FANCD 2的关联。在没有任何DNA损伤的情况下，该蛋白也存在于S期的复制工厂中。这些和其他实验的结果表明，祖先蛋白在整个细胞周期中参与了对DNA损伤的快速，多相反应。它也参与无压力复制。与FA途径的关联似乎反映了FAN 1对应激复制的反应。FAN 1应该被视为一种酶，它进化到有助于DNA代谢的许多不同方面。

Failure to respond effectively to replication stress is recognized as a key contributor to developmental defects, the basis of premature aging syndromes, and a stimulus for the development of neoplasia. Interstrand crosslinks are particularly dangerous DNA lesions as they are absolute blocks to replication, and thus a major challenge to the replication stress response. They are believed to occur as a product of oxidative metabolism, and are also a consequence of treatment with some chemotherapy drugs. Psoralens are photoactive DNA interstrand crosslinkers that have been used clinically for many years. We have synthesized, and demonstrated the activity of, antigen linked psoralens. Laser photoactivation of defined subnuclear regions in cells incubated with the compounds resulted in localized crosslinks. Repair of these adducts was monitored in repair proficient and deficient cells. We are using this approach to follow the recruitment of proteins into sites of crosslink repair. Interstrand crosslinks are repaired in a two cycle process. In the first cycle on strand is incised on either side of the crosslinked base. In the second cycle the remaining adducted (and still crosslinked) base is removed via conventional NER. There is uncertainty as to whether repair can occur in G1 phase, in addition to the well established S phase repair. We have shown that crosslinks are repaired in the G1 phase of the cell cycle, in a process that is dependent on NER functions. XPC protein was rapidly recruited to sites of crosslinks and monoadduct. However, the XPE damage binding complex was recruited rapidly to monoadducts and slowly to crosslinks. Recruitment of the XPE complex was dependent on XPC activity, and repair synthesis. Our results support a scenario in which the XPE complex does not recognize the crosslink, but is recruited when the remaining monoadducted base is forced out of the helix after the completion of the first repair cycle. The recruitment of the XPE complex is a marker of completion of the first repair cycle and the start of the second. We have applied this technology to an examination of the function of FANCD2 in ICL repair. The FANCD2 protein is the central node in the Fanconi Anemia pathway. Individuals with deficiencies in this pathway suffer severe developmental defects, and show signs of premature aging during postpartum life. The pathway plays a key role in the response to replication stress. We find that FANCD2 is recruited to multiple stimuli-double strand breaks in a cell cycle independent manner; laser localized psoralen crosslinks independent of cell cycle; as well as an S phase specific scheme. Replication independent recruitment of FA proteins to ICLs requires the activity of RNF8, a ubiquitin ligase, and a newly discovered ubiquitin binding protein-FAAP20. This FAAP20-RNF8 ubquitin cascade is important for cellular resistance to genomic stress such as imposed by interstrand crosslinks. Understanding the nature of defects in the Fanconi pathway will provide the basis for developing effective therapies for this disorder. We have also characterized the recruitment and contribution to ICL repair of FAN1, a recently discovered nuclease that associates with FANCD2. Ancestral versions of this protein that lack the protein structural domain involved in FA protein interactions are found in organisms that also lack the FA pathway. It is currently believed that FAN1 recruitment to ICLs is dependent on FANCD2. However we have found that this protein is rapidly recruited to ICLs, in a FANCD2 independent manner. There is a second wave of accumulation that is partially dependent on the association with FANCD2. The protein is also found in replication factories in S phase in the absence of any DNA damage. The results of these and other experiments indicate that the ancestral protein participates in a rapid, multiphasic, response to DNA damage throughout the cell cycle. It also is involved in unstressed replication. The association with the FA pathway appears to reflect the response of FAN1 to stressed replication. FAN1 should be seen as a enzyme that evolved to contribute to many different aspects of DNA metabolism.