Single-copy locus proteomics of early- and late-firing DNA replication origins identifies a role of Ask1/DASH complex in replication timing control

The chromatin at origins of replication is thought to influence DNA replication initiation in eukaryotic genomes. However, it remains unclear how the chromatin composition controls the firing of early-efficient (EE) or late-inefficient (LI) origins. Here, we used site-specific recombination and single-locus chromatin isolation to purify EE and LI replication origins in Saccharomyces cerevisiae. Using mass spectrometry, we define the histone modification landscape and identify the protein composition of native chromatin regions surrounding the EE and LI replication start sites. In addition to the known origin interactors, we find novel origin-associated factors, such as the kinetochore-associated Ask1/DASH complex. Strikingly, we show that Ask1 regulates the replication timing control of specific origins in yeast. Thus, our unbiased approach identifies functionally-relevant proteomes at single-copy loci and would be widely applicable to provide an in-depth quantitative characterization of histone modification and protein interaction networks of chromatin at any genomic locus of interest. Overall design: DNA sequencing based copy number analysis of origin activity in HU-arrested and synchronously released S. cerevisiae cells after targeted degradation of Ask1 or targeted recruitment of Ask1 at specific replication origins ARS305, ARS313 or ARS316.

真核基因组中，复制起点处的染色质被认为可影响DNA复制起始进程。然而目前仍不清楚染色质组成如何调控早期高效（EE）或晚期低效（LI）复制起点的激活。本研究利用位点特异性重组与单基因座染色质分离技术，纯化出酿酒酵母（Saccharomyces cerevisiae）中的EE与LI复制起点。通过质谱（mass spectrometry）技术，我们明确了EE与LI复制起始位点周围天然染色质区域的组蛋白修饰图谱，并鉴定了其蛋白质组成。除已知的起点互作蛋白外，我们还发现了新的起点相关因子，例如动粒相关的Ask1/DASH复合物。值得注意的是，我们证实Ask1可调控酿酒酵母中特定复制起点的复制时序控制。综上，我们的无偏倚方法可在单拷贝基因座上鉴定出具有功能相关性的蛋白质组，且该方法可广泛应用于对任意目标基因组位点的染色质组蛋白修饰与蛋白质互作网络进行深度定量表征。总体设计：在特定复制起点ARS305、ARS313或ARS316处实施Ask1靶向降解或靶向招募后，对经羟基脲（HU）阻滞并同步释放的酿酒酵母细胞开展基于DNA测序的拷贝数分析，以此检测复制起点活性。