DNA Replication Fork: Pausing, Recombination and Disease

DNA 复制叉：暂停、重组和疾病

• 批准号: 6576325
• 负责人: JOHN J BISSLER
• 金额: $ 30.63万
• 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/6576325
• 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）：结节性硬化症和常染色体显性多囊肾病患者通常出生时肾脏解剖正常，但随着年龄的增长，肾脏受累严重。这些疾病中的异常组织与杂合性缺失（洛）相关，使得在疾病基因座处仅存在缺陷等位基因。虽然这两种疾病都有第二个相关基因，但TSC2和PKD1基因导致更严重的表型，并且更常见于新突变的患者。我们假设疾病的严重程度与这些相邻基因位于16号染色体的不稳定区域有关。我们提出的证据表明，从这些基因的反向Alu重复序列和聚嘌呤。聚嘧啶束似乎与缺失，并阻止人类复制叉。此外，多嘌呤、多嘧啶段在有利于形成替代性二级结构的条件下，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致突变的意义。 本研究的最终目的是通过延缓TSC 2和PKD 1基因中导致洛缺失的第二个体细胞突变来延缓疾病的发生和发展。我们推测，替代的DNA二级结构中的TSC 2和PKD 1基因通过其对DNA复制的影响促进诱变。本研究利用二维凝胶和熔解曲线分析研究了Pu.Py片段和反向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，我们将测量序列停止复制和诱导重组的能力。还将在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，可以在延迟疾病发作方面取得治疗进展。