【文章內(nèi)容簡介】 酸長的 RNA， 作為DNA 合成的引物；這段引物是由引物酶合成的。 是在特定環(huán)境下發(fā)揮作用的 RNA聚合酶，僅用于合成 DNA復(fù)制所需的一小段 RNA。 引物酶以單鏈 DNA為模板，利用核糖核苷酸 直接從頭合成RNA引物 （ 610nt)。 減少致死突變。 DNA合成中最初幾個(gè)核苷酸正確性遠(yuǎn)比其后的低。引物 RNA 隨后被降解，從而減少了復(fù)制錯(cuò)誤。 引物的意義： 引物酶催化引物 RNA的合成，但它不同于 RNA聚合酶 。 引物酶只在復(fù)制起點(diǎn)處合成 RNA引物以引發(fā) DNA復(fù)制，而 RNA聚合酶則是啟動(dòng) DNA轉(zhuǎn)錄合成 RNA ，從而將遺傳信息由 DNA傳遞到 RNA。 ，分子量為60KD,DnaG基因編碼。 ① DnaA protein molecules binds to the four 9 bp repeats in the origin， then recognizes and successively denatures the DNA in the region of the three 13 bp repeats, which are rich in A=T pairs This process requires ATP and the bacterial histonelike protein HU. HU prevents nonspecific initiation at sites other than oriC The DnaA protein then mediates the separation of the strands of the DNA duplex by acting on three ATrich tandem repeats located at the 539。end of the sequence defining oriC Formation of this 45bp “open plex” by DnaA protein is ATPdependent. ① ② ② DnaB protein (a hexamer of 50kD subunits) binds to the “open” oriC. DnaB protein is delivered to oriC by DnaC protein assisted by DnaT protein. The addition of DnaB protein pletes assembly of the prepriming plex（引發(fā)前體） . ATP hydrolysis drives the formation of this plex . DnaB protein has helicase activity and it further unwinds the DNA in the prepriming plex in both directions． ③ SSB tetramers coat single stranded regions as they arise. ② ③ ④ 引物酶（ ＤｎａＧ ）結(jié)合到引發(fā)前體上組裝成了引發(fā)體（ primosome ）。 ⑤ 在 SSB和拓?fù)洚悩?gòu)酶的參與下，引發(fā)體可在單鏈ＤＮＡ上移動(dòng)，在ＤｎａＢ的幫助下，識別復(fù)制的起始起點(diǎn)位置。 ④ ⑤ ⑥ 引發(fā)體在復(fù)制叉先合成先導(dǎo)鏈的 RNA引物，再合成后滯鏈的引物．（ Primase association with DnaB is transient。 once a primer has been synthesized, primase dissociates until another round of primer synthesis is necessary.) ⑦ 在復(fù)制叉 DNA polymerase III 組裝成 非對稱的二聚體 ，它催化第一個(gè)ｄＮＴＰ按堿基互補(bǔ)配對原則加在ＲＮＡ引物的３ ‘ －ＯＨ端，而進(jìn)入ＤＮＡ鏈的延伸階段 . ⑥ ⑥ DNA鏈的延伸需要的蛋白質(zhì) : ? DNA聚合酶 ? 滑動(dòng)夾（ Sliding DNA clamp） ? RNA酶 (RNase H 等 ) 在復(fù)制完成后切除 RNA引物。 ? DNA連接酶 (DNA ligase) 通過生成 3’5’磷酸二酯鍵連接兩條 DNA鏈。 2. DNA鏈的延伸 DNA polymerase use a single active site to catalyze DNA synthesis DNA polymerase bound to a primer:template junction palm Processivity (持續(xù)合成能力 ) ? Processivity is a characteristic of enzymes that operate on polymeric substrates. ? For DNA polymerase, the degree of processivity is defined as the average number of nucleotides added each time the enzyme binds a primer:template junction (a few~50,000). Structure of a sliding DNA clamp Sliding DNA clamps encircle the newly replicated DNA produced by an associated DNA polymerase. Sliding clamps dramatically increase DNA polymerase processivity (持續(xù)合成能力 ) The position of the DNA Pol III holoenzyme Three enzymes: ? Two copies of the DNA Pol III core enzyme ? One copy of the γplex The “trombone” model for coordinating replication by two DNA polymerase at the E. coli replication fork 岡崎片段與半不連續(xù)復(fù)制 岡崎片段 (Okazaki fragment)一般長約 10002022個(gè)堿基，原核比真核長。 前導(dǎo)鏈 Leading strand 后隨鏈 Lagging strand 然后， RNaseH降解 RNA引物， DNA聚合酶 I補(bǔ)齊缺口，再由 DNA連接酶連接兩個(gè)岡崎片段。 Steps in the synthesis of the lagging strand Removal of RNA primers from newly synthesized DNA RNAse H removes all of the RNA primer except the ribonucleotide directly linked to the DNA end. An exonuclease removes the final ribonucleotide. DNA polymerase fills the gap, leaving a a break in the backbone between the 3’OH and 5’ phosphate of the repaired strand. DNA ligase repaires this “nick”. 當(dāng)復(fù)制叉前移，遇到 20bp重復(fù)性終止子序列(Ter)時(shí)， TerTus復(fù)合物能阻擋復(fù)制叉的繼續(xù)前移，等到相反方向的復(fù)制叉到達(dá)后在 DNA拓