Dissecting BRCA1-PALB2 Activity in DNA Repair and Development

项目摘要/摘要 本意见书是为家长基金R01GM135293：《解剖BRCA1-PALB2》颁发的补充奖 DNA修复和发育方面的活动“。家长奖助金目前处于第一年，这使得这一年成为 是时候升级我们的设备了。蛋白质对双链DNA断裂(DSB)的定位可以是 通过核焦点的存在来量化。核内病灶的数量通常指示一个 特定的蛋白质或修复途径是有功能的。典型的焦点显示为下面的同质球体 传统的宽视场或共焦显微镜。所提供的信息仅限于焦点是否 出席或缺席。相比之下，超分辨率(SR)显微镜提供了高分辨率的成像，这可以 揭示DSB侧翼染色质拓扑的详细见解。进行SR显微镜检查的能力将 大大增强了可以从当前R01资助的项目中获得的知识。不只是在 了解突变对病灶形成的影响，对DNA损伤机制的新见解 修复机制以及染色质拓扑和DNA修复之间的相互作用可以被揭示。在父级中 格兰特，我们的目标是揭示BRCA1招募PALB2到DNA损伤部位的机制。我们 建议研究BRCA1和PALB2突变对DNA修复中心的影响。然而，SR显微镜 有可能揭示更多，而不仅仅是焦点的存在或不存在，例如对 BRCA1-PALB2的纳米结构，无论是独立的还是重叠的纳米结构域，空间 差异，以及BRCA1-PALB2拓扑相对于额外的同源重组(HR)蛋白。 此外，BRCA1和PALB2患者突变的影响以前没有被检查过 对DSB-染色质拓扑结构和纳米结构的影响。SR显微镜的使用将提供超分辨率 洞察，并最终提高我们对BRCA1和PALB2突变如何影响人类健康的理解。

PROJECT SUMMARY/ABSTRACT This submission is for a supplement award for the parent grant R01GM135293: “Dissecting BRCA1-PALB2 activity in DNA repair and development”. The parent grant is currently in its first year, making this the perfect time to upgrade our equipment. The localization of proteins to double stranded DNA breaks (DSBs) can be quantified by the presence of nuclear foci. The number of foci within a nucleus is often indicative of whether a particular protein or repair pathway is functional. A typical focus appears as a homogenous sphere under conventional widefield or confocal microscopy. The information provided does not go beyond whether foci are present or absent. In contrast, super-resolution (SR) microscopy provides high-resolution imagining, which can reveal detailed insights into the DSB-flanking chromatin topology. The ability to perform SR microscopy would greatly enhance the knowledge that can be gained from the current R01-funded project. Not only in understanding the effects of mutations on foci formation, new insights into the mechanics of the DNA damage repair machinery and the interplay between chromatin topology and DNA repair can be revealed. In the parent grant, we aim to uncover the mechanisms by which BRCA1 recruits PALB2 to sites of DNA damage. We proposed to examine the effects of BRCA1 and PALB2 mutations on DNA repair foci. However, SR microscopy has the potential to reveal so much more, beyond simply the presence or absence of foci, such as insights into the nanostructure of BRCA1-PALB2, whether there are separate or overlapping nanodomains, spatial differences, and the BRCA1-PALB2 topology relative to additional homologous recombination (HR) proteins. Moreover, the effects of BRCA1 and PALB2 patient mutations have previously not been examined for their effects on DSB-chromatin topology and nanostructure. The use of SR microscopy will provide super-resolution insight, and ultimately improve our understanding of how BRCA1 and PALB2 mutations impact human health.