DNA Replication Fork: Pausing, Recombination and Disease

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的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。这些疾病中的异常组织与杂合性缺失（LOH）相关，因此只有缺陷等位基因存在于疾病位点。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，并且更常在新突变的患者中发现。我们推测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，反向Alu重复和多嘌呤。来自这些基因的多嘧啶束似乎与缺失有关，并阻断了人类复制叉。此外，还有聚嘌呤。多嘧啶通道，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双重意义。

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.