Repair of localized DNA damage

未能有效地应对复制应激被认为是发育缺陷、早衰综合征和肿瘤发展的关键因素。链间交联(ICL)是一种特别危险的DNA损伤，因为它们被认为是复制的绝对阻碍，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，也是一些化疗药物治疗的结果。补骨脂素是一种具有光活性的DNA链间交联剂，已在临床上使用多年。我们已经合成并证明了抗原连接的补骨脂素的活性。与化合物孵育的细胞中特定的亚核区域的激光光激活导致了局部的交联键。在修复熟练和缺陷的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在交联链修复部位的招募情况。ICL在两个周期的过程中修复。在第一个周期中，一条DNA链与另一条解偶联。在第二个循环中，通过常规的核苷酸切除修复去除剩余的加成碱基(仍然是交联的)。除了公认的S期修复外，是否能在G1期修复还存在不确定性。我们已经证明，在细胞周期的G1期，交联链被修复，这一过程依赖于NER功能。XPC蛋白被迅速募集到交联点和单加合物位点。然而，XPE损伤结合络合物被迅速地招募到单加合物中，并缓慢地被招募到交联物中。XPE复合体的募集取决于XPC的活性和修复合成。我们的结果支持这样一种情况，即XPE复合体不识别交联键，但在第一个修复周期完成后，当剩余的单加成碱基被迫离开螺旋时，XPE复合体被招募。XPE建筑群的启用标志着第一个修复周期的完成和第二个修复周期的开始。 我们应用这种抗原标记补骨脂素技术来研究FANCD2在ICL修复中的作用。FANCD2是Fanconi贫血途径的中心蛋白。这一途径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。该途径在对复制胁迫的反应中起着关键作用。我们发现FANCD2以两种模式被吸收到激光局域的补骨脂素交联剂中。一种依赖于众所周知的DNA损伤反应途径的组成成分，另一种依赖于DNA修复功能。只有在后一条通路中，FANCD2才参与ICL的修复。FANCD2与ICL的结合需要进入DNA修复途径。不依赖复制的FA蛋白在ICL中的募集需要泛素连接酶RNF8和新发现的泛素结合蛋白FAAP20的活性。这种FAAP20-RNF8亚基级联对于细胞抵抗诸如链间交联物施加的基因组压力是重要的。了解Fanconi通路缺陷的性质将为开发有效的治疗该疾病的方法提供基础。 我们还表征了FAN1的招募和对ICL修复的贡献，FAN1是一种最近发现的与FANCD2相关的核酸酶。在缺乏FA途径的生物体中，也发现了这种缺乏FA蛋白相互作用的蛋白质结构域的这种蛋白质的祖先版本。目前认为，FAN1向ICL的招募取决于FANCD2。然而，我们发现这种蛋白以FANCD2独立的方式迅速招募到ICL中。还有第二波积累，部分依赖于与FANCD2的关联。在S阶段的复制工厂中也发现了这种蛋白质，没有任何DNA损伤。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。它还参与了无压力复制。与FA途径的关联似乎反映了FAN1对应激复制的反应。FAN1应该被视为一种进化为对DNA新陈代谢的许多不同方面做出贡献的酶。 为了研究复制分叉与ICL的相遇，我们开发了一种新的单分子方法来可视化这些事件。我们已经将在DNA纤维上显示复制区的成熟程序与对单个抗原标记的补骨脂素ICL的免疫量子点检测相结合。我们观察到单叉和双叉相撞，以及在叉状相遇远端ICL一侧的一种意想不到的DNA合成模式。我们将此称为复制遍历。Fanconi贫血易位酶FANCM是横行模式所必需的。然而，这些事件不依赖于Fanconi贫血核心复合体蛋白。由于这些通路出现在脊椎动物谱系中，而FANCM则发现于古生代，我们认为这些穿越通路是在应对主要复制挑战的早期进化而来的。我们预计，可视化遇到主要障碍的叉形相遇的新技术将有助于研究用诱导ICL的化疗药物治疗的肿瘤细胞中的复制。

Failure to respond effectively to replication stress is recognized as a key contributor to developmental defects, premature aging syndromes, and the development of neoplasias. Interstrand crosslinks (ICLs) are particularly dangerous DNA lesions as they are considered 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. ICLs are repaired in a two cycle process. In the first cycle one DNA strand is uncoupled from the other. In the second cycle the remaining adducted (and still crosslinked) base is removed via conventional Nucleotide Excision Repair. 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 antigen tagged psoralen technology to an examination of the function of FANCD2 in ICL repair. FANCD2 is is the central protein 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 laser localized psoralen crosslinks in two modes. One is dependent on components of the well known DNA Damage Response pathway, while the other is dependent on DNA repair functions. Only in the latter pathway does FANCD2 contribute to ICL repair. Entry into the DNA repair pathway is required for the association of FANCD2 with the ICL. 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. In order to study the encounter of replication forks with ICLs we have developed a novel single molecule approach for visualizing these events. We have combined well established procedures for displaying replication tracts on DNA fibers with immuno-quantum dot detection of individual antigen tagged psoralen ICLs. We observe single and double fork collisions as well as an unanticipated pattern of DNA synthesis on the side of the ICL distal to the fork encounter. We termed this replication traverse. The Fanconi Anemia translocase, FANCM, is required for the traverse patterns. However, these events are independent of the Fanconi Anemia core complex proteins. As these appear in vertebrate lineages, while FANCM is found in Archaea, we propose that the traverse pathways evolved early in response to major replication challenges. We anticipate that the new technology for visualizing fork encounters with major impediments will be useful for studying replication in tumor cells treated with chemotherapy drugs that induce ICLs.