CATCH-IT method for measuring nucleosome turnover

Direct measurement of nucleosome turnover dynamics by using co-translational incorporation of the methionine (Met) surrogate azidohomoalaine (Aha) into proteins and subsequent ligation of biotin to Aha-containing proteins through the [3+2] cycloaddition reaction between the azide group of Aha and an alkyne linked to biotin. To measure turnover rates, we treat cells briefly with Aha, couple biotin to nucleosomes containing newly incorporated histones, affinity purify with strepavidin, wash stringently to remove non-histone proteins and H2A/H2B dimers, and analyze the affinity-purified DNA using tiling microarrays. We call this strategy 'CATCH-IT' for Covalent Attachment of Tags to Capture Histones and Identify Turnover. Keywords: Chromatin affinity-purification on microarray All experiments were done using strepavidin pulldown DNA cohybridized with total input DNA to the same array. Two channels per array, Cy5 and Cy3, were used in each experiment.

本研究通过将甲硫氨酸（methionine，Met）的替代衍生物叠氮高丙氨酸（azidohomoalaine，Aha）共翻译掺入蛋白质，并借助Aha的叠氮基团与连接生物素的炔基之间的[3+2]环加成反应，将生物素共价连接至含Aha的蛋白质，以此直接测定核小体周转动力学。为测定核小体周转速率，我们使用Aha短暂处理细胞，将生物素与包含新掺入组蛋白的核小体进行偶联，通过链霉亲和素（streptavidin）完成亲和纯化，随后严格洗涤以去除非组蛋白及H2A/H2B二聚体，并采用平铺式微阵列（tiling microarray）分析经亲和纯化得到的DNA。我们将该实验策略命名为'CATCH-IT'，即'共价标记组蛋白并识别周转的标签连接法（Covalent Attachment of Tags to Capture Histones and Identify Turnover）'的缩写。关键词：基于微阵列的染色质亲和纯化（Chromatin affinity-purification on microarray）。所有实验均采用链霉亲和素下拉得到的DNA与总输入DNA共同杂交至同一微阵列。每次实验均使用阵列的两个荧光通道：Cy5与Cy3。