DNA Replication Fork: Pausing, Recombination and Disease

DNA 复制叉：暂停、重组和疾病

• 批准号: 6740173
• 负责人: JOHN J BISSLER
• 金额: $ 22.45万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6740173
• 关键词: DNA replication; DNA replication origin; cell line; gene mutation; genetic mapping; genetic recombination; laboratory mouse; loss of heterozygosity; nucleic acid repetitive sequence; polycystic kidney; renal tubule; transfection; tuberous sclerosis; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): Patients with tuberous sclerosis complex and autosomal dominant polycystic kidney disease most often are born with anatomically normal kidneys but develop significant renal involvement as they age. The abnormal tissues in these diseases are associated with the loss of heterozygosity (LOH) such that only the defective allele is present at the disease locus. Although both diseases have a second associated gene, the TSC2 and PKD1 genes cause a more severe phenotype and are more often found in patients with new mutations. We postulate that the disease severity is related to the fact that these adjacent genes are in an unstable region of chromosome 16. We present evidence that inverted Alu repeats and polypurine.polypyrimidine tracts from these genes appear to be associated with deletions, and block the human replication fork. In addition, the polypurine.polypyrimidine tract, under conditions that favor alternative secondary structure formation, can spontaneously initiate replication. Such replication initiation and termination phenomenon have both developmental and mutagenic implications. The ultimate goal of our reseach is to retard disease onset and progression by delaying the second somatic mutation leading to the LOH in the TSC2 and PKD1 genes. We hypothesize that alternative DNA secondary structures in the TSC2 and PKD1 genes promote mutagenesis through their effects on DNA replication. The proposed studies investigate the DNA structural characteristics of the Pu.Py tracts and inverted Alu repeats using 2-dimensional gel and melting curve analyses. Using repair deficient cell lines, we will also determine the replication proteins involved. Using a stable-transfection system, we will measure the ability of the sequences to stall replication and to induce recombination. The potential to initiate the human replication fork will also be studied in a well characterized system. By understanding the effects of these sequences on the fidelity of DNA replication, therapeutic inroads into delaying disease onset can be made.

描述（由申请人提供）：患有结节性硬化症和常染色体显性多囊肾病的患者通常出生时肾脏解剖结构正常，但随着年龄的增长，肾脏会出现明显的受累。这些疾病中的异常组织与杂合性缺失（LOH）相关，因此疾病位点上仅存在缺陷等位基因。尽管这两种疾病都有第二个相关基因，但 TSC2 和 PKD1 基因会导致更严重的表型，并且更常见于有新突变的患者。我们假设疾病的严重程度与这些相邻基因位于 16 号染色体不稳定区域的事实有关。我们提供的证据表明，这些基因的反向 Alu 重复序列和聚嘌呤。聚嘧啶束似乎与缺失相关，并阻止人类复制叉。此外，多聚嘌呤.多聚嘧啶束在有利于替代二级结构形成的条件下可以自发地启动复制。这种复制起始和终止现象具有发育和诱变的意义。 我们研究的最终目标是通过延迟导致 TSC2 和 PKD1 基因 LOH 的第二个体细胞突变来延缓疾病的发生和进展。我们假设 TSC2 和 PKD1 基因中的替代 DNA 二级结构通过影响 DNA 复制来促进诱变。拟议的研究使用二维凝胶和熔解曲线分析研究了 Pu.Py 束和反向 Alu 重复序列的 DNA 结构特征。使用修复缺陷细胞系，我们还将确定涉及的复制蛋白。使用稳定转染系统，我们将测量序列阻止复制和诱导重组的能力。还将在一个充分表征的系统中研究启动人类复制叉的潜力。通过了解这些序列对 DNA 复制保真度的影响，可以在延缓疾病发作方面取得进展。