Replication fork reestablishment across a DNA interstrand crosslink

 描述(申请人提供)：DNA链间交联在某些抗肿瘤药物和环境毒素的作用中起关键作用，也在以范可尼贫血(FA)为代表的内源性DNA损伤的病理生物学中发挥关键作用。DNA交联型癌症化疗药物，包括丝裂霉素C、氮芥和铂化合物，利用活跃生长的癌细胞对交联剂的更高敏感性。FA是一种罕见的遗传性疾病，由调控复制相关的链间DNA交联链(ICL)移除的基因突变引起。通过连接两条DNA链，ICL既能抑制DNA复制，又能抑制转录。未能清除ICL病变最终会导致细胞死亡。此外，遗传性乳腺癌和卵巢癌是在FA/BRCA ICL修复途径的BRCA基因存在遗传缺陷的个体中触发的。因此，揭示ICL修复机制对于改进癌症化疗、了解人类遗传病和遗传性癌症倾向是非常关键的。长期以来，人们一直认为，当复制复合体遇到ICL时，复制分叉的进展总是被抑制，并且只有在ICL修复之后，分叉进展才会恢复。然而，我们最近在复制叉上进行的ICL修复实验产生了一个意想不到的结果，这与这一广泛认可的假设不一致。我们的结果表明在ICL下游形成了一个新的DNA复制分叉，此外，还表明最近发现的人类PrimPol聚合酶在这一事件中发挥了关键作用。这一革命性的新假说的确认和PrimPol在这一过程中的重要作用的展示有望为我们理解哺乳动物ICL修复的机制开辟重要的新途径。为了实现这一目标，将单个ICL插入到与宿主细胞复制同步的、在人类细胞中单向复制的质粒中。从细胞中回收子代质粒，并分析修复事件，作为新的叉形形成的标志。通过RNA干扰技术降低PrimPol的活性，评估了PrimPol在新叉子形成中的作用。为了进行详细的研究，PrimPol基因被一种新开发的基因敲除技术破坏，敲除细胞用PrimPol突变体补充。这些目标的实现将有助于癌症化疗药物的改进和对与ICL修复缺陷相关的人类遗传病的充分了解。

 DESCRIPTION (provided by applicant): DNA interstrand crosslinking plays a critical role in the action of certain anti-tumor agents and environmental toxins and also in the pathobiology of endogenous DNA damage as represented by Fanconi anemia (FA). DNA crosslinking cancer chemotherapeutics, including mitomycin C, nitrogen mustards, and platinum compounds, utilize the greater sensitivity of actively growing cancer cells to crosslinking agents. FA, a rare inheritd genetic disease, is caused by mutations in genes regulating replication-associated removal of interstrand DNA crosslinks (ICLs). By linking two DNA strands, ICLs inhibit both DNA replication and transcription. Failure to remove ICL lesions ultimately leads to cell death. Moreover, hereditary breast and ovarian cancers are triggered in individuals with genetic defects in the BRCA genes of FA/BRCA ICL repair pathway. Therefore, it is very critical to reveal the ICL repair mechanism to improve cancer chemotherapy and understand human genetic disease and hereditary cancer proneness. It has long been believed that progression of a replication fork is invariably inhibited when a replication complex encounters an ICL and that fork progression resumes only after the ICL is repaired. However, our recent experiments on ICL repair taking place at a replication fork produced an unexpected result, which is inconsistent with this widely endorsed hypothesis. Our results suggest the formation of a new DNA replication fork downstream of ICL and, additionally, indicate that the recently discovered human Primase-Polymerase, PRIMPOL, plays a critical role in this event. Confirmation of this revolutionary new hypothesis and the demonstration of PRIMPOL's essential role in this process are expected to open important new avenues in our understanding of the mechanism of mammalian ICL repair. To achieve this goal, a single ICL is inserted into plasmid that replicates in one direction in human cells synchronously with the replication of host cells. Progeny plasmids are recovered from cells and analyzed for repair events, which serve as a marker for a new fork formation. The role for PRIMPOL in the formation of a new fork is evaluated by knocking down its activity by the RNA interference technology. For detailed studies, the PRIMPOL gene is destroyed by a newly developed gene knockout technology, and knockout cells are complemented with PRIMPOL mutants. Achievements of these aims will contribute to the improvement of cancer chemotherapeutic drugs and the full understanding of the human genetic diseases associated with a defect in ICL repair.