SITE SPECIFIC DNA DAMAGE AND GENOMIC INSTABILITY IN MAMM

环境中存在的试剂对DNA的破坏通常直接或间接地导致DNA链断裂。一些环境侮辱的生物学后果，如电离辐射，似乎主要源于引入染色体的DNA链断裂。其他环境因素，如紫外线和引入碱基修饰的试剂，可能间接导致DNA链断裂和缺口，作为DNA修复过程的中间产物。染色体的双链断裂对细胞具有潜在的致命性。如果不修复，断裂可能会导致染色体片段或整个染色体的丢失；如果修复不当，可能会导致遗传信息的重新排列。在哺乳动物细胞中，负责通过同源和非同源(不合法)重组修复这种损伤的过程定义不清，很难研究，因为几乎没有方法以受控的方式引入位点特异性DNA损伤；也没有几个特征良好的基因组位点对研究位点特异性DNA损伤有用。这项拟议的研究使用了一个成熟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，该基因已被修改，以研究同源和非法重组。这项研究的具体目的是：1)表征哺乳动物细胞中特定部位DNA链断裂的物理过程。通过为将链断裂引入染色体aprt基因的蛋白质引入识别位点，并在修饰的细胞系中表达这些蛋白质，将引入特定部位的双链和单链断裂。DNA链断裂过程中的中间体将使用聚合酶链式反应和Southern分析进行检测。2)更好地了解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对靠近APRT的可选择标记中的突变进行选择和通过DNA测序来表征突变来确定。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.