Recruitment of Rad10 in Double-strand Break Repair

说明（由申请方提供）：电离辐射和化学制剂会诱导DNA链断裂，从而导致突变和染色体改变，从而导致癌症。针对DNA链断裂的主要生物防御之一是双链断裂修复（DSBR），这是修复DNA链断裂的复杂生物途径家族。DSBR的一些模式可以进行DNA序列的完全恢复，而不会导致遗传信息的丢失，但其他模式会导致显着的染色体丢失。DSBR的分子基础正在由DNA修复领域的研究人员阐明，但关于生化要求，途径的时机，甚至如何选择最合适的途径仍然存在许多问题，因为它们在修复染色体时并不都同样有效而不丢失遗传信息。在这个询问的保护伞下，是关于参与某些途径但不参与其他途径的蛋白质的定时和招募机制的更具体的问题。该建议的具体目标是确定在进化保守的真核模型系统中，酵母，S。酿酒酵母：1）Rad 10募集到双链断裂（DSB）的模式是否根据DSB位点侧翼的DNA序列同源性的程度和接近程度而改变，2）DSB修复的“单链退火”（SSA）模式中的Rad 10募集是否依赖于SAW 1基因，以及Rad 10和Saw 1蛋白是否共定位于SSA修复位点和3）Rad 10向DSB位点的募集是否依赖于基因SLX 4以及Rad 10和Slx 4蛋白是否共定位于DSB位点。这些目标将主要使用创新的荧光显微镜实验进行研究，其中链断裂将在活酵母细胞中特异性地诱导，DNA修复位置将通过活细胞的荧光成像进行监测。将在含有野生型和突变基因的适当组合并改变DSB侧翼DNA序列的菌株中比较荧光标记蛋白的行为。将采用基于质粒的DNA损伤诱导/修复试验研究荧光标记或突变基因的功能表征，并通过Southern杂交进行分析。这些实验将解决重要的问题，有关的生化要求的时间和招聘的Rad 10在几个关键的模式/背景下DSBR和描绘的差异，Rad 10是如何招募到DSB网站。癌症和衰老的分子基础是一个重要的研究领域，以推进临床策略，最大限度地减少人类的痛苦。更详细地了解细胞修复DNA的生物化学将有助于为药物寻找新的药物靶点，从而最大限度地降低癌症风险和与衰老相关的疾病。 公共卫生相关性：这项拟议中的研究将增加我们对一种复杂的生物学途径的调节机制的理解，这种生物学途径可以防止癌症和衰老，称为双链断裂修复。这项工作的结果可能有助于制定癌症预防策略和方法，通过预防一些衰老症状来减少老年人的痛苦。

DESCRIPTION (provided by applicant): Ionizing radiation and chemical agents induce strand breaks in DNA, which give rise to mutations and chromosomal alterations that can cause cancer. One of the chief biological defenses against DNA strand breaks is Double-Strand Break Repair (DSBR), a complicated family of biologic pathways that repairs DNA strand breaks. Some modes of DSBR can proceed with full restoration of the DNA sequence and no resulting loss of genetic information, but others result in significant chromosomal loss. The molecular underpinnings of DSBR are being elucidated by researchers in the DNA Repair field, but many questions remain regarding biochemical requirements, timing of the pathways, and even how the most appropriate pathway is selected, given that they are not all equally effective in repairing chromosomes without loss of genetic information. Under this umbrella of interrogation are more specific questions regarding the mechanism for timing and recruitment of proteins that participate in some pathways but not others. The specific aims of this proposal are to determine in an evolutionarily-conserved eukaryotic model system, the yeast, S. cerevisiae: 1) whether patterns of Rad10 recruitment to Double-Strand Breaks (DSBs) are altered depending on the extent and proximity of DNA sequence homology flanking the DSB site, 2) whether Rad10 recruitment in the "single-strand annealing" (SSA) mode of DSB repair is dependent on the SAW1 gene, and whether Rad10 and Saw1 proteins colocalize at SSA repair sites and 3) whether Rad10 recruitment to DSB sites is dependent on the gene SLX4 and whether Rad10 and Slx4 proteins colocalize at DSB sites. These aims will be investigated primarily using innovative fluorescence microscopy experiments in which strand breaks will be induced site-specifically in live yeast cells and DNA repair locations will be monitored by fluorescence imaging of the live cells. The behavior of the fluorescently labeled proteins will be compared in strains containing the appropriate combinations of wild-type and mutant genes and varying the DNA sequences flanking the DSBs. Characterization of the functionality of fluorescently-labeled or mutant genes will be investigated with a plasmid-based DNA damage induction/ repair assay which will be analyzed by Southern hybridization. These experiments will address important questions regarding the biochemical requirements bearing on the timing and recruitment of Rad10 in several key modes/contexts of DSBR and delineate the differences in how Rad10 is recruited to DSB sites. The molecular basis for cancer and aging is an important area of research in order to advance clinical strategies to will minimize human suffering. A more detailed understanding of the biochemistry by which cells repair DNA will aid in finding new drug targets for pharmaceuticals that might minimize cancer risk and the ailments associated with aging. PUBLIC HEALTH RELEVANCE: The proposed research will increase our understanding of the mechanism of regulation of a complex biological pathway that protects against cancer and aging known as Double-strand Break Repair. Results of this work may aid in developing cancer prevention strategies and methods to reduce suffering in the elderly by preventing some of the symptoms of aging.