SITE SPECIFIC DNA DAMAGE AND GENOMIC INSTABILITY IN MAMM

环境中存在的物质对DNA的损伤常常直接或间接地导致DNA链断裂。一些环境损害的生物学后果，如电离辐射，似乎主要来自于引入染色体的DNA链断裂。其他环境因素，如紫外线和引入碱基修饰的因素，作为DNA修复过程中的中间物，可能间接导致DNA链断裂和间隙。染色体双链断裂对细胞具有潜在的致命性。如果不进行修复，断裂会导致染色体片段或整个染色体的丢失；如果修复不当，它们会导致基因信息的重排。在哺乳动物细胞中，通过同源和非同源（非法）重组修复这种损伤的过程定义不清，而且很难研究，因为很少有方法以受控的方式引入位点特异性DNA损伤；而且很少有明确的基因组位点可以用于研究特定位点的DNA损伤。本研究利用一种完善的哺乳动物细胞遗传系统，即仓鼠腺嘌呤磷酸核糖基转移酶（APRT）基因，对其进行修饰，研究同源重组和非法重组。本研究的具体目的是：1)表征哺乳动物细胞中位点特异性DNA链断裂的物理过程。通过在染色体APRT基因中引入引入链断裂的蛋白质的识别位点以及这些蛋白质在修饰细胞系中的表达，将引入位点特异性双链和单链断裂。DNA链断裂过程中的中间产物将通过PCR和Southern分析进行检测。2)更好地理解DNA链断裂的遗传后果。DNA链断裂对周围序列突变的影响将通过在接近APRT的可选择标记中选择突变和通过DNA测序对突变进行表征来确定。3)研究结构特异性核酸酶ERCC1/XPF、XPG和FEN-1在DNA结构加工和基因组不稳定性中的作用。

DNA damage by agents present in the environment often leads directly, or indirectly, to DNA strand breaks. The biological consequences of some environmental insults, such as ionizing radiation, seem to derive primarily from DNA strand breaks introduced into chromosomes. Other environmental agents, such as ultraviolet light and agents that introduce base modifications, may indirectly lead to DNA strand breaks and gaps as intermediates in the DNA repair process. Double-strand breaks in chromosomes are potentially lethal to the cell. If they are not repaired, breaks can lead to the loss of chromosome segments or whole chromosomes; if misrepaired, they can lead to the rearrangement of genetic information. The processes responsible for the repair of this damage in mammalian cells by homologous and nonhomologous (illegitimate) recombination are poorly defined and difficult to study because there are few ways to introduce site-specific DNA damage in a controlled fashion; and there are few well-characterized genomic sites that are useful for studying site-specific DNA damage. The proposed research uses a well- established mammalian cell genetic system, the hamster adenine phosphoribosyltransferase (APRT) gene, that has been modified to study homologous and illegitimate recombination. The specific aims of the research are: 1) To characterize the physical processing of site-specific DNA strand breaks in mammalian cells. Site-specific double- and single-strand breaks will be introduced by introducing recognition sites for proteins that introduce strand breaks into the chromosomal APRT gene and expression of those proteins in the modified cell lines. Intermediates in the processing of DNA strand breaks will be detected using PCR and Southern analysis. 2) To better understand the genetic consequences of DNA strand breaks. The influence of DNA strand breaks on mutagenesis in surrounding sequences will be determined by selection for mutations in selectable markers close to APRT and characterization of the mutations by DNA sequencing. 3) To investigate the role of the structure-specific nucleases, ERCC1/XPF, XPG, and FEN-1 on processing DNA structures and genomic instability.