非洲爪蟾起源识别复合体对于DNA复制以及MCM与染色质的结合至关重要。

The Xenopus origin recognition complex is essential for DNA replication and MCM binding to chromatin.

作者信息

Romanowski P, Madine M A, Rowles A, Blow J J, Laskey R A

机构信息

Wellcome/CRC Institute, Cambridge.

出版信息

Curr Biol. 1996 Nov 1;6(11):1416-25. doi: 10.1016/s0960-9822(96)00746-4.

DOI:10.1016/s0960-9822(96)00746-4

PMID:8939603

Abstract

BACKGROUND

The origin recognition complex (ORC) and the minichromosome maintenance (MCM) protein complex were initially discovered in yeast and shown to be essential for DNA replication. Homologues of ORC and MCM proteins exist in higher eukaryotes, including Xenopus. The Xenopus MCM proteins and the Xenopus homologues of Saccharomyces cerevisiae Orc 1p and Orc2p (XOrc1 and XOrc2) have recently been shown to be essential for DNA replication. Here, we describe the different but interdependent functions of the ORC and MCM complexes in DNA replication in Xenopus egg extracts.

RESULTS

The XOrc1 and XOrc2 proteins are present in the same multiprotein complex in Xenopus egg extracts. Immunodepletion of ORC inhibits DNA replication of Xenopus sperm nuclei. Mixing MCM-depleted and ORC-depleted extracts restores replication capacity. ORC does not co-localize with sites of DNA replication during elongation. However, at initiation the two staining patterns overlap. In contrast to MCMs, which are displaced from chromatin during S phase, XOrc1 and XOrc2 are nuclear chromatin-bound proteins throughout interphase and move to the cytoplasm in mitosis. Permeable HeLa G1- and G2-phase nuclei can replicate in ORC-depleted extract, consistent with the presence of chromatin-bound ORC in both pre-replicative and post-replicative nuclei. Interestingly, the binding of ORC to chromatin does not require the presence of MCMs; however, the binding of MCM proteins to chromatin is dependent on the presence of ORC.

CONCLUSIONS

The Xenopus ORC and the MCM protein complex perform essential, non-redundant functions in DNA replication. Xenopus ORC is bound to chromatin throughout interphase but, in contrast to S. cerevisiae ORC, it appears to be, at least partly, displaced from chromatin during mitosis. The binding of MCM proteins requires the presence of ORC. Thus, the assembly of replication-competent chromatin involves the sequential binding of ORC and MCMs to DNA.

摘要

背景

起始识别复合物（ORC）和微小染色体维持（MCM）蛋白复合物最初在酵母中被发现，并被证明对DNA复制至关重要。ORC和MCM蛋白的同源物存在于包括非洲爪蟾在内的高等真核生物中。最近研究表明，非洲爪蟾MCM蛋白以及酿酒酵母Orc 1p和Orc2p的非洲爪蟾同源物（XOrc1和XOrc2）对DNA复制至关重要。在此，我们描述了ORC和MCM复合物在非洲爪蟾卵提取物DNA复制过程中不同但相互依存的功能。

结果

XOrc1和XOrc2蛋白存在于非洲爪蟾卵提取物中的同一多蛋白复合物中。ORC的免疫耗竭会抑制非洲爪蟾精子细胞核的DNA复制。将耗尽MCM的提取物和耗尽ORC的提取物混合可恢复复制能力。在延伸过程中，ORC与DNA复制位点不共定位。然而，在起始阶段，两种染色模式重叠。与在S期从染色质上移位的MCM不同，XOrc1和XOrc2在整个间期都是与核染色质结合的蛋白，并在有丝分裂时转移到细胞质中。可渗透的HeLa G1期和G2期细胞核能够在耗尽ORC的提取物中复制，这与复制前和复制后细胞核中存在与染色质结合的ORC一致。有趣的是，ORC与染色质的结合并不需要MCM的存在；然而，MCM蛋白与染色质的结合依赖于ORC的存在。