Repair of localized DNA damage

修复局部DNA损伤

• 批准号: 10003713
• 负责人: Michael Seidman
• 金额: $ 88.57万
• 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/10003713
• 关键词: Aging; Antigens; Archaea; Binding; Binding Proteins; Cell Cycle; Cell Respiration; Cells; Chemicals; Clinical; Complex; Crosslinker; DNA; DNA Damage; DNA Repair; DNA Repair Pathway; DNA biosynthesis; DNA lesion; DNA replication fork; Dangerousness; Defect; Detection; Development; Disease; Distal; Enzymes; Event; Failure; Fanconi Anemia pathway; Fanconi anemia protein; Fanconi' s Anemia; Fiber; G1 Phase; Genomics; Incubated; Individual; Laser Microscopy; Lasers; Lesion; Life; Link; Metabolism; Monitor; Nature; Neoplasms; Nucleotide Excision Repair; Organ; Organism; Pathway interactions; Pattern; Pharmaceutical Preparations; Play; Postpartum Period; Premature aging syndrome; Procedures; Process; Proteins; Psoralens; Quantum Dots; Resistance; Role; S Phase; Side; Site; Stress; Structural Protein; Technology; Tissues; Ubiquitin; Uncertainty; adduct; base; biological adaptation to stress; chemotherapy; crosslink; design; effective therapy; experimental study; in utero; interest; neoplastic cell; new technology; novel; novel strategies; nuclease; photoactivation; protein complex; protein structure; recruit; repaired; replication stress; response; single molecule; translocase; ubiquitin ligase

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. Notably, non ubiquitinated FANCD2 also is required for traverse and functions in the same pathway as FANCM. FANCM associates with a subset of replisomes that encounter the block. These replisomes lose one of the key components of the replication apparatus as a result of binding FANCM. Consequently there is a FANCM dependent remodeling of the replisome as the result of replication challenge. 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.

不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生的关键因素。链间交联（ICL）是特别危险的DNA损伤，因为它们被认为是复制的绝对障碍，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。ICL在两个循环过程中修复。在第一个循环中，一条DNA链与另一条DNA链解偶联。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合（并且仍然交联）碱基。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。 我们已经将这种抗原标记的peptide技术应用于FANCD 2在ICL修复中的功能的检查。FANCD 2是范可尼贫血通路中的中心蛋白。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被招募到激光本地化的peptien交联在两种模式。一种依赖于众所周知的DNA损伤反应途径的组分，而另一种依赖于DNA修复功能。只有在后一种途径中，FANCD 2才有助于ICL修复。进入DNA修复途径是FANCD 2与ICL结合所必需的。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。 为了研究复制叉与ICLs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。我们已经结合了完善的程序显示复制道的DNA纤维与免疫量子点检测个别抗原标记的peptide ICLs。我们观察到单叉和双叉碰撞，以及一个意想不到的模式的DNA合成的ICL远端的叉遇到的一面。我们称之为复制遍历。范可尼贫血易位酶，FANCM，是需要的遍历模式。然而，这些事件独立于范可尼贫血核心复合物蛋白。由于这些出现在脊椎动物谱系中，而FANCM发现于马，我们提出，横向通路的演变早在主要的复制挑战。值得注意的是，非泛素化的FANCD 2也是穿越所需的，并且在与FANCM相同的途径中发挥作用。 FANCM与遇到阻断的复制体的子集相关联。由于结合FANCM，这些复制体失去了复制装置的关键组件之一。因此，作为复制挑战的结果，存在复制体的FANCM依赖性重塑。 我们预计，可视化叉子遇到主要障碍的新技术将有助于研究用诱导ICLs的化疗药物治疗的肿瘤细胞中的复制。