Department of Cell and Developmental Biology and Department of Biotechnology and Biophysics, University of Wuerzburg, Biocenter, Am Hubland, D-97074 Wuerzburg, Germany.
Nucleic Acids Res. 2011 Sep 1;39(16):7124-33. doi: 10.1093/nar/gkr396. Epub 2011 May 19.
Binding of proteins to DNA is usually considered 1D with one protein bound to one DNA molecule. In principle, proteins with multiple DNA binding domains could also bind to and thereby cross-link different DNA molecules. We have investigated this possibility using high-mobility group A1 (HMGA1) proteins, which are architectural elements of chromatin and are involved in the regulation of multiple DNA-dependent processes. Using direct stochastic optical reconstruction microscopy (dSTORM), we could show that overexpression of HMGA1a-eGFP in Cos-7 cells leads to chromatin aggregation. To investigate if HMGA1a is directly responsible for this chromatin compaction we developed a DNA cross-linking assay. We were able to show for the first time that HMGA1a can cross-link DNA directly. Detailed analysis using point mutated proteins revealed a novel DNA cross-linking domain. Electron microscopy indicates that HMGA1 proteins are able to create DNA loops and supercoils in linearized DNA confirming the cross-linking ability of HMGA1a. This capacity has profound implications for the spatial organization of DNA in the cell nucleus and suggests cross-linking activities for additional nuclear proteins.
蛋白质与 DNA 的结合通常被认为是一维的，即一个蛋白质结合到一个 DNA 分子上。原则上，具有多个 DNA 结合结构域的蛋白质也可以结合并交联不同的 DNA 分子。我们使用高迁移率族蛋白 A1（HMGA1）蛋白研究了这种可能性，HMGA1 蛋白是染色质的结构元件，参与多种 DNA 依赖过程的调控。使用直接随机光学重建显微镜（dSTORM），我们可以证明在 Cos-7 细胞中过表达 HMGA1a-eGFP 会导致染色质聚集。为了研究 HMGA1a 是否直接导致这种染色质紧缩，我们开发了一种 DNA 交联测定法。我们首次表明 HMGA1a 可以直接交联 DNA。使用点突变蛋白的详细分析揭示了一个新的 DNA 交联结构域。电子显微镜表明，HMGA1 蛋白能够在线性化 DNA 中形成 DNA 环和超螺旋，证实了 HMGA1a 的交联能力。这种能力对细胞核中 DNA 的空间组织具有深远的影响，并暗示其他核蛋白具有交联活性。
