Kelly R E, DeRose M L, Draper B W, Wahl G M
Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Mol Cell Biol. 1995 Aug;15(8):4136-48. doi: 10.1128/MCB.15.8.4136.
Most DNA replication origins in eukaryotes localize to nontranscribed regions, and there are no reports of origins within constitutively expressed genes. This observation has led to the proposal that there may be an incompatibility between origin function and location within a ubiquitously expressed gene. The biochemical and functional evidence presented here demonstrates that an origin of bidirectional replication (OBR) resides within the constitutively expressed housekeeping gene CAD, which encodes the first three reactions of de novo uridine biosynthesis (carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase). The OBR was localized to a 5-kb region near the center of the Syrian hamster CAD transcriptional unit. DNA replication initiates within this region in the single-copy CAD gene in Syrian baby hamster kidney cells and in the large chromosomal amplicons that were generated after selection with N-phosphonacetyl-L-aspartate, a specific inhibitor of CAD. DNA synthesis also initiates within this OBR in autonomously replicating extrachromosomal amplicons (CAD episomes) located in an N-phosphonacetyl-L-aspartate-resistant clone (5P20) of CHOK1 cells. CAD episomes consist entirely of a multimer of Syrian hamster CAD cosmid sequences (cCAD1). These data limit the functional unit of replication initiation and timing control to the 42 kb of Syrian hamster sequences contained in cCAD1. In addition, the data indicate that the origin recognition machinery is conserved across species, since the same OBR region functions in both Syrian and Chinese hamster cells. Importantly, while cCAD1 exhibits characteristics of a complete replicon, we have not detected autonomous replication directly following transfection. Since the CAD episome was generated after excision of chromosomally integrated transfected cCAD1 sequences, we propose that prior localization within a chromosome may be necessary to "license" some biochemically defined OBRs to render them functional.
真核生物中的大多数DNA复制起点定位于非转录区域，目前尚无关于组成型表达基因内存在复制起点的报道。这一观察结果促使人们提出，复制起点功能与在普遍表达基因中的位置之间可能存在不相容性。本文提供的生化和功能证据表明，双向复制起点（OBR）位于组成型表达的管家基因CAD内，该基因编码从头合成尿苷的前三个反应（氨甲酰磷酸合成酶、天冬氨酸氨甲酰转移酶和二氢乳清酸酶）。OBR定位于叙利亚仓鼠CAD转录单元中心附近的一个5kb区域。在叙利亚仓鼠肾细胞中的单拷贝CAD基因以及在用CAD特异性抑制剂N-膦酰乙酰-L-天冬氨酸选择后产生的大染色体扩增子中，DNA复制在该区域内起始。DNA合成也在位于CHOK1细胞的N-膦酰乙酰-L-天冬氨酸抗性克隆（5P20）中的自主复制的染色体外扩增子（CAD附加体）的这个OBR内起始。CAD附加体完全由叙利亚仓鼠CAD黏粒序列（cCAD1）的多聚体组成。这些数据将复制起始和时间控制的功能单位限制在cCAD1中包含的42kb叙利亚仓鼠序列内。此外，数据表明，由于相同的OBR区域在叙利亚仓鼠和中国仓鼠细胞中均起作用，复制起点识别机制在物种间是保守的。重要的是，虽然cCAD1表现出完整复制子的特征，但我们在转染后未直接检测到自主复制。由于CAD附加体是在切除染色体整合的转染cCAD1序列后产生的，我们提出，先前在染色体内的定位可能是使一些生化定义的OBR“获得许可”从而使其发挥功能所必需的。
