Molecular Studies Of Protein-DNA Interactions

蛋白质-DNA 相互作用的分子研究

• 批准号: 7337469
• 负责人: Peggy Hsieh
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7337469
• 关键词: Molecular; Studies; Protein; DNA; Interactions

We seek to understand at a molecular level the various ways by which an organism maintains the integrity of its genome while accommodating the need for genetic diversity. Our research efforts currently focus on a highly conserved DNA repair pathway, DNA mismatch repair (MMR). MMR, exemplified by the E. coli methyl-directed MMR pathway, targets base pair mismatches that arise through DNA replication errors, homologous recombination and DNA damage. Inactivation of MMR results in a large increase in the rate of spontaneous mutation and is associated with both sporadic and hereditary cancers. We are currently investigating molecular mechanisms of DNA damage signaling mediated by mammalian MMR proteins. SN1 alkylating agents commonly used in chemotherapy produce cytoxic O6-methylguanine residues. Cell killing results from cell cycle arrest and apoptosis triggered by DNA damage recognition by MMR proteins MutSa and MutLa. We have developed an in vitro system in which we can study the molecular mechanism underlying activation of the ATR kinase signaling cascade in response to DNA alkylation. We have shown that MutSa and MutLa recruit ATR to sites of cytoxic O6-methylguanine-thymidine mispairs resulting in activation of the ATR kinase activity and phosphorylation of cell cycle checkpoint proteins such as Chk1 and SMC1. Other DNA lesions are currently being examined. In collaboration with Dr. Dorothy Erie, we are using atomic force microscopy (AFM) to examine the conformations of protein-DNA complexes. Measurement of fractional occupancies at specific locations on a mismatched DNA allowed us to determine the binding affinity, specificity and stoichiometry of MutS bound to mismatched DNAs. Mutant MMR proteins are being utilized to identify key aspects of mismatch recognition.

我们试图在分子水平上了解生物体在适应遗传多样性需要的同时保持其基因组完整性的各种方式。我们的研究工作目前集中在一个高度保守的DNA修复途径，DNA错配修复（MMR）。MMR以E. coli甲基介导的MMR途径，靶向通过DNA复制错误、同源重组和DNA损伤引起的碱基对错配。MMR失活导致自发突变率大幅增加，并与散发性和遗传性癌症相关。 我们目前正在研究哺乳动物MMR蛋白介导的DNA损伤信号传导的分子机制。化疗中常用的SN 1烷化剂会产生细胞毒性O 6-甲基鸟嘌呤残基。细胞杀伤是由MMR蛋白MutSa和MutLa识别DNA损伤引发的细胞周期停滞和细胞凋亡引起的。我们已经开发了一种体外系统，在该系统中，我们可以研究响应于DNA烷基化的ATR激酶信号级联激活的分子机制。我们已经表明，MutSa和MutLa招募ATR的细胞毒性O 6-甲基鸟嘌呤-胸苷错配的网站，导致激活的ATR激酶活性和磷酸化的细胞周期检查点蛋白，如Chk 1和SMC 1。目前正在检查其他DNA损伤。在多萝西伊利博士的合作下，我们正在使用原子力显微镜（AFM）来检查蛋白质-DNA复合物的构象。在错配DNA上的特定位置处的分数占据率的测量使我们能够确定MutS与错配DNA结合的结合亲和力、特异性和化学计量。突变的MMR蛋白被用于识别错配识别的关键方面。