第4章DNA复制研究.pptVIP

E3-1: 体外 In vitro 实验系统 纯化的 DNA : 完成复制所需的所有的蛋白质与其它因子 DNA replication Put DNA and protein together and ask replication question 小的动物病毒分子：猿猴病毒 SV40 (simian virus 40, 5 kb) are good mammalian models for elongation (replication fork) but not for initiation. 2. 酵母Yeast (Saccharomyces cerevisiae): 1.4 X 107 bp in 16 chromosomes, 400 replicons, much simpler than mammalian system and can serve as a model system 3. 无细胞提取物Cell-free extract ：非洲爪蟾卵细胞提取物或者小麦胚芽提取物. E3-2: 多复制子的起始（Initiation of multiple replicons） DNA replication Timing Order 20-50 复制子族 在细胞周期 S期同步进行 早S-phase: 常染色质的复制 （euchromatin replication） 晚 S-phase: 异染色质复制 （heterochromatin replication） 最后：着丝粒与端粒复制 （Centromeric and telomeric DNA replication ） 2.准许因子（Licensing factor） : Only initiate once per cell cycle 起始要求，用后失活（required for initiation and inactivated after use） 有丝分裂核膜裂解时才能进入胞核（Can only enter into nucleus when the nuclear envelope dissolves at mitosis） Licensing factor Initiation Initiation: origin 自主复制序列ARSs: autonomously replicating sequences Individual yeast replication origins (ARS) have been cloned into prokaryotic plasmids which allow these plasmids to replicate in yeast (a eukaryote). Minimal sequence: 11 bp [A/T]TTTAT[A/G]TTT[A/T] (TATA box) 起始点识别复合体ORC (origin recognition complex) binds to ARS, upon activation by CDKs, ORC will open the DNA for replication. 准许因子（Licensing factor） E3-3: 复制叉与延伸 Replication fork elongation DNA replication unwinding enzymes Replication fork Unwinding DNA from nucleosomes: 50 bp/sec, need helicases and replication protein A (RP-A) New nucleosomes are assembled to DNA from a mixture of old and newly synthesized histones after the fork passes. Elongation: 三类 DNA聚合酶 参与. DNA pol a: 引发酶的活性合成RNA，接着延伸DNA，但很快被其它两个酶取代。 DNA pol d: 前导链leading strand取代DNA pol a. 合成长的DNA DNA pol e: 后随链lagging strand取代DNA pol a. 合成长的DNA.合成的冈崎片段（ Okazaki fragments）非常短 (135 bp in SV40), 反应了来自每个核小体的DNA的解旋的数量. E3-4: 核基质Nuclear Matrix DNA replicati