SITE SPECIFIC DNA DAMAGE AND GENOMIC INSTABILITY IN MAMM

存在于环境中的试剂造成的DNA损伤通常直接或间接导致DNA链断裂。 某些环境损伤（如电离辐射）的生物学后果似乎主要来自染色体中引入的DNA链断裂。 其他环境因素，如紫外线和引入碱基修饰的因素，可能间接导致DNA链断裂和缺口，作为DNA修复过程中的中间产物。 染色体中的双链断裂对细胞是潜在的致命的。 如果它们不被修复，断裂可能导致染色体片段或整个染色体的丢失;如果错误修复，它们可能导致遗传信息的重排。 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，难以研究，因为很少有方法以受控的方式引入位点特异性DNA损伤;并且很少有表征良好的基因组位点可用于研究位点特异性DNA损伤。 拟议的研究使用了一个完善的哺乳动物细胞遗传系统，仓鼠腺嘌呤磷酸核糖转移酶（APRT）基因，该基因已被修改，以研究同源和非法重组。 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。将通过引入将链断裂引入染色体APRT基因的蛋白质的识别位点以及这些蛋白质在修饰的细胞系中的表达来引入位点特异性双链和单链断裂。 将使用PCR和Southern分析检测DNA链断裂过程中的中间产物。 2)为了更好地了解DNA链断裂的遗传后果。 DNA链断裂对周围序列中诱变的影响将通过选择接近APRT的选择标记中的突变并通过DNA测序表征突变来确定。 3)研究结构特异性核酸酶ERCC 1/XPF、XPG和FEN-1在加工DNA结构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.