The Origin Recognition Complex interacts with a subset of metabolic genes tightly linked to origins of replication.

The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell, such as in the orc2-1 mutant, only sites that bind ORC tightly, such as HMR-E, remain fully occupied by ORC, while lower affinity sites, including most origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC “affinity” genome-wide by performing an ORC ChIP-on-chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1-resistant ORC-interacting chromosomal sites (ORF-ORC sites) that did not function as replication origins or silencers. Instead, ORF-ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC-silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF-ORC genes were enriched in proximity to origins of replication, and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection between ORC, replication origins and cellular metabolism. Two arrays are used, one for WT orc2 and one for mutant orc2-1. On each array Cy3 was the IP and Cy5 was the input (total sheared genomic DNA). The arrays were custom designed to tile the S. cerevisiae genome (as of 8/16/2006) with a 51 nt probe every 32 bp. The probes alternated strands as they tiled the genome. Microarray synthesis, fluorescent-labeling of ChIP DNA, hybridization of labeled DNA to the microarray, and scanning of the microarray after hybridization were all performed by NimbleGen at a NimbleGen facility.

起始识别复合物（origin recognition complex, ORC）可将染色体位点标记为复制起始位点，且对复制起始过程至关重要。在酵母中，ORC还可结合被称为沉默子的DNA元件，其主要功能是招募沉默信息调节因子（silent information regulator, SIR）蛋白以建立转录沉默。事实上，沉默子作为染色体复制起始位点的功能极差。针对HMR-E的多项遗传学、分子生物学及生物化学研究提出了如下模型：当细胞内ORC水平受限时（如orc2-1突变体中），只有紧密结合ORC的位点（如HMR-E）仍能被ORC完全占据，而亲和力较低的位点（包括大多数复制起始位点）则会失去ORC的结合。鉴于HMR-E具有独特的非复制功能，我们推测其他紧密结合ORC的位点可能揭示ORC在染色体上的新功能。因此，我们通过在ORC2及orc2-1菌株中开展ORC染色质免疫沉淀芯片（ChIP-on-chip）实验，在全基因组范围内全面测定了ORC的"亲和力"。本文报道了一类全新的、对orc2-1具有抗性的ORC结合染色体位点（ORF-ORC sites），这类位点既不具备复制起始位点功能，也不发挥沉默子作用。与之相反，ORF-ORC位点由转录水平极高的代谢基因的蛋白编码区域构成。与ORC-沉默子作用范式不同的是，转录激活会促进ORC与这些基因的结合。值得注意的是，ORF-ORC基因富集于复制起始位点附近，且在多个案例中，这些复制起始位点会对其进行转录调控。综上，本研究结果揭示了ORC、复制起始位点与细胞代谢之间此前未被发现的关联。本实验使用了两张芯片，一张针对野生型ORC2菌株，另一张针对orc2-1突变体菌株。每张芯片中，Cy3标记免疫沉淀组分，Cy5标记输入组分（即总剪切基因组DNA）。该定制芯片以每隔32 bp设置一条51 nt探针的密度，覆盖酿酒酵母（Saccharomyces cerevisiae, S. cerevisiae）全基因组（截至2006年8月16日）。探针在基因组上平铺时会交替对应正负两条链。芯片合成、ChIP DNA的荧光标记、标记DNA与芯片的杂交以及杂交后芯片的扫描，均由NimbleGen公司在其实验基地完成。