人类BRCA2蛋白与DNA结合的首个单分子显微可视化
![Direct imaging of BRCA2 binding to RPA-coated ssDNA on single molecules of gapped λ DNA. (A) Schematic of experimental approach combining fluorescence microscopy, a microfluidic flow cell, and optical trapping, as well as the micromanipulation used to capture and image BRCA2 on individual DNA molecules. (B) Illustration of the gapped λ DNA generated through in vitro recombination of circular ssDNA with an engineered λ DNA. (C) Schematic of experimental protocol: Each molecule of gapped λ DNA was captured and micromanipulated between two beads held in separately controllable optical traps. The molecule was moved between solutions in a six-channel flow cell and successively incubated in a solution containing BRCA2. (D) Cartoon and microscopic image of a single molecule of gapped λ DNA (Left, stained with YOYO-1, cyan) that was destained and then successively incubated with BRCA2 (5 nM) plus α-BRCA2 and α-IgGAF546. Montage shows BRCA2 (magenta) binding to the gapped λ DNA at increasing time intervals. (E) Cartoon representation of the gapped λ DNA between two beads (Top) and histogram (Middle) of binding positions of BRCA2 (number of foci, N = 60). Each data point is also plotted as a single tick (Bottom) where the semi-transparent box represents the SE associated with assigning position owing to the optical resolution of the microscope. Gray bars represent the 10 to 90th percentile range of the 5′- and 3′-termined junctions (N = 98). Credit: Proceedings of the National Academy of Sciences (2023). DOI: 10.1073/pnas.2221971120 首次单分子显微镜显示了全长的人类BRCA2蛋白与DNA的结合](https://scx1.b-cdn.net/csz/news/800a/2023/first-single-molecule.jpg)
耶鲁大学癌症中心和加州大学戴维斯分校的研究人员使用自建的倒置显微镜和激光光镊来捕获DNA,首次在单分子水平上创建了全长的人类BRCA2蛋白的可视化。
乳腺癌易感基因BRCA2的突变会显著增加一个人一生中患癌症的风险。大约每400人中就有一人携带BRCA基因突变,这在可遗传癌症中占了很大比例。这项研究发表在3月28日的《美国医学杂志》上美国国家科学院院刊.
“如果你携带BRCA突变基因,你患乳腺癌的风险就会非常高卵巢癌,也适用于男性,前列腺和胰腺癌耶鲁大学癌症中心成员、该论文的合著者、耶鲁大学医学院放射治疗学副教授瑞安·詹森博士说。
“当在患者身上发现BRCA2突变时,我们在分子水平上了解这一点至关重要。如果BRCA2蛋白不能完成它的工作,为什么这种失败会导致癌症?我的实验室完全投入到了解BRCA2蛋白的各个方面。”
BRCA2基因为制造一种蛋白质提供了指令,这种蛋白质充当肿瘤抑制基因.肿瘤抑制蛋白有助于防止细胞生长和分裂过快或不受控制。Jensen博士说:“这个实验的目的是为了更好地理解BRCA2蛋白是如何一次一个分子地与DNA结合并相互作用的。这种‘视觉生物化学’方法类似于坐在剧院里,观看蛋白质如何与DNA实时结合和相互作用的电影。”
“BRCA2是一种DNA修复蛋白,所以它能修复DNA损伤。具体来说,DNA中的双链断裂,可以通过人类细胞中的几种不同途径修复。BRCA2尤其在同源重组途径中起作用,”詹森博士说。
“通过弄清楚BRCA2是如何在分子水平上这将为我们提供更多信息,以制定新的策略,有朝一日可以帮助那些对BRCA突变没有反应或在当前标准护理疗法下复发的携带者。我们掌握的知识越多越好,”詹森博士说。
更多信息:Jason C. Bell等,BRCA2伴侣RAD51到rpa包裹的ssDNA单分子,美国国家科学院院刊(2023)。DOI: 10.1073 / pnas.2221971120