Replication fork reestablishment across a DNA interstrand crosslink

跨 DNA 链间交联的复制叉重建

• 批准号: 9032712
• 负责人: Masaaki Moriya
• 金额: $ 23.7万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9032712
• 关键词: Achievement; Antineoplastic Agents; Cell Death; Cells; Chemotherapy-Oncologic Procedure; Complement; Complementary DNA; Complex; DNA; DNA Crosslinking; DNA Crosslinking Agent; DNA Damage; DNA Interstrand Crosslinking; DNA Primase; DNA Repair; DNA biosynthesis; DNA replication fork; Defect; Development; Disease; Event; Excision; Failure; Fanconi' s Anemia; Gene Silencing; Genes; Genetic Recombination; Genetic Transcription; Goals; Hereditary Breast Carcinoma; Hereditary Disease; Hereditary Malignant Neoplasm; Human; Human Genetics; Human Genome; In Vitro; Individual; Inherited; Investigation; Knock-out; Lesion; Light; Link; Malignant Neoplasms; Malignant neoplasm of ovary; Mechlorethamine; Mitomycins; Mutation; Nucleotide Excision Repair; Pathway interactions; Patients; Pharmaceutical Preparations; Plasmids; Platinum Compounds; Play; Polymerase; Prevention therapy; Process; RNA Interference; Research; Role; Surgical incisions; Symptoms; System; Technology; Toxic Environmental Substances; XPA gene; antitumor agent; cancer cell; cancer prevention; crosslink; improved; knock-down; knockout gene; mutant; public health relevance; recombinational repair; repaired; research study; transcription activator-like effector nucleases

 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.

 描述（由申请人提供）：DNA链间交联在某些抗肿瘤药物和环境毒素的作用中以及在以范可尼贫血（FA）为代表的内源性DNA损伤的病理生物学中起关键作用。DNA交联癌症化学治疗剂，包括丝裂霉素C、氮霉素和铂化合物，利用活跃生长的癌细胞对交联剂的更大敏感性。FA是一种罕见的遗传性疾病，由调控复制相关的DNA链间交联（ICLs）去除的基因突变引起。通过连接两条DNA链，ICL抑制DNA复制和转录。未能去除ICL病变最终导致细胞死亡。此外，遗传性乳腺癌和卵巢癌在FA/BRCA ICL修复途径的BRCA基因中存在遗传缺陷的个体中触发。因此，揭示ICL的修复机制，对于改善癌症的化疗方案，了解人类遗传性疾病和遗传性癌症易感性具有重要意义。长期以来，人们一直认为，当复制复合物遇到ICL时，复制叉的进展总是受到抑制，并且只有在ICL修复后，复制叉的进展才能恢复。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。我们的研究结果表明，ICL下游形成了一个新的DNA复制叉，此外，还表明最近发现的人类引物聚合酶PRIMPOL在这一事件中起着关键作用。这一革命性的新假说的证实和PRIMPOL在这一过程中的重要作用的演示，预计将打开重要的新途径，在我们的理解哺乳动物ICL修复的机制。为了实现这一目标，将单个ICL插入质粒中，该质粒在人细胞中以一个方向复制，与宿主细胞的复制同步。从细胞中回收子代质粒并分析修复事件，其用作新叉形成的标记。通过RNA干扰技术敲低PRIMPOL的活性来评估PRIMPOL在新分叉形成中的作用。为了进行详细的研究，通过新开发的基因敲除技术破坏PRIMPOL基因，并用PRIMPOL突变体补充敲除细胞。这些目标的实现将有助于改善癌症化疗药物和充分了解与ICL修复缺陷相关的人类遗传性疾病。