Tada S, Chong J P, Mahbubani H M, Blow J J
CRC Chromosome Replication Research Group Department of Biochemistry University of Dundee Dundee DD1 5EH Scotland UK.
Curr Biol. 1999 Feb 25;9(4):211-4. doi: 10.1016/s0960-9822(99)80092-x.
Replication licensing factor (RLF) is an essential initiation factor that can prevent re-replication of DNA in a single cell cycle [1] [2]. It is required for the initiation of DNA replication, binds to chromatin early in the cell cycle, is removed from chromatin as DNA replicates and is unable to re-bind replicated chromatin until the following mitosis. Chromatography of RLF from Xenopus extracts has shown that it consists of two components termed RLF-B and RLF-M [3]. The RLF-M component consists of complexes of all six Xenopus minichromosome maintenance (MCM/P1) proteins (XMcm2-7), which bind to chromatin in late mitosis and are removed as replication occurs [3] [4] [5] [6] [7]. The identity of RLF-B is currently unknown. At least two factors must be present on chromatin before licensing can occur: the Xenopus origin recognition complex (XORC) [8] [9] and Xenopus Cdc6 (XCdc6) [10]. XORC saturates Xenopus sperm chromatin at approximately one copy per replication origin whereas XCdc6 binds to chromatin only if XORC is bound first [9] [10] [11]. Although XORC has been shown to be a distinct activity from RLF-B [9], the relationship between XCdc6 and RLF-B is currently unclear. Here, we show that active XCdc6 is loaded onto chromatin in extracts with defective RLF, and that both RLF-M and RLF-B are still required for the licensing of XCdc6-containing chromatin. Furthermore, RLF-B can be separated from XCdc6 by immunoprecipitation and standard chromatography. These experiments demonstrate that RLF-B is both functionally and physically distinct from XCdc6, and that XCdc6 is loaded onto chromatin before RLF-B function is executed.
复制许可因子（RLF）是一种必需的起始因子，可防止DNA在单个细胞周期中重新复制[1][2]。它是DNA复制起始所必需的，在细胞周期早期与染色质结合，随着DNA复制从染色质上移除，并且在接下来的有丝分裂之前无法重新结合已复制的染色质。从非洲爪蟾提取物中对RLF进行色谱分析表明，它由两个组分组成，称为RLF-B和RLF-M[3]。RLF-M组分由所有六种非洲爪蟾微型染色体维持（MCM/P1）蛋白（XMcm2 - 7）的复合物组成，这些蛋白在有丝分裂后期与染色质结合，并在复制发生时被移除[3][4][5][6][7]。RLF-B的身份目前尚不清楚。在许可发生之前，染色质上必须至少存在两个因子：非洲爪蟾起源识别复合物（XORC）[8][9]和非洲爪蟾Cdc6（XCdc6）[10]。XORC以每个复制起点约一个拷贝的量饱和非洲爪蟾精子染色质，而XCdc6仅在XORC首先结合时才与染色质结合[9][10][11]。尽管XORC已被证明是一种与RLF-B不同的活性物质[9]，但目前XCdc6与RLF-B之间的关系尚不清楚。在这里，我们表明活性XCdc6在RLF有缺陷的提取物中被加载到染色质上，并且含XCdc6的染色质的许可仍需要RLF-M和RLF-B两者。此外，RLF-B可以通过免疫沉淀和标准色谱法与XCdc6分离。这些实验表明，RLF-B在功能和物理上均与XCdc6不同，并且在RLF-B功能执行之前，XCdc6被加载到染色质上。
