MCM2-7復(fù)合體參與DNA復(fù)制機(jī)制及調(diào)控特點(diǎn)

王明剛，毛德文，張榮臻，龍富立，王秀峰

廣西中醫(yī)藥大學(xué)第一附屬醫(yī)院肝病一區(qū)，廣西 南寧 530023

綜述

MCM2-7復(fù)合體參與DNA復(fù)制機(jī)制及調(diào)控特點(diǎn)

王明剛，毛德文，張榮臻，龍富立，王秀峰

廣西中醫(yī)藥大學(xué)第一附屬醫(yī)院肝病一區(qū)，廣西 南寧 530023

脫氧核糖核酸(deoxyribonucheic acid, DNA)無(wú)差錯(cuò)復(fù)制是基因組信息成功遺傳到子代的主要保障，對(duì)于生命的遺傳信息傳遞和延續(xù)意義重大。微小染色體維持蛋白(minichromosome maintenance proteins, MCM)家族是廣泛存在的高保守蛋白，MCM2-7六聚體是DNA復(fù)制所必須的。MCMs基因轉(zhuǎn)錄與啟動(dòng)子區(qū)E2F結(jié)構(gòu)域密切相關(guān)，在基因表達(dá)層面可受到miRNA-34s和miRNA-1269的影響。在MCM2-7生物學(xué)功能過程中可受到Cdt1、cyclinE、p27Kip1、Rb、ATM、ATR蛋白的調(diào)控和影響。

DNA復(fù)制；微小染色體維持蛋白；調(diào)控；微小核糖核酸

脫氧核糖核酸(deoxyribonucleic acid,DNA)是由兩條脫氧多核苷酸鏈反向平行盤繞所形成的雙螺旋結(jié)構(gòu)，內(nèi)部由許多脫氧核苷酸按一定堿基順序以3′,5′-磷酸二酯鍵相連構(gòu)成[1]。DNA是生命遺傳信息的主要存儲(chǔ)形式。DNA復(fù)制是單鏈模板DNA復(fù)制成兩個(gè)互補(bǔ)DNA鏈的生物學(xué)過程。對(duì)于細(xì)胞分裂，DNA復(fù)制是必須的，且多發(fā)生在細(xì)胞周期的S期，一個(gè)細(xì)胞周期DNA復(fù)制僅發(fā)生一次。DNA無(wú)差錯(cuò)復(fù)制是基因組信息成功遺傳到子代的主要保障，對(duì)于生命的遺傳信息傳遞和延續(xù)意義重大[2]。

微小染色體維持蛋白(minichromosome maintenance proteins, MCM)家族是高度保守的DNA解旋蛋白復(fù)合體，其保守性可追溯到上古時(shí)代單細(xì)胞形成[3]。在細(xì)胞內(nèi)MCM2、MCM3、MCM4、MCM5、MCM6、MCM7形成六聚體才具有解螺旋酶活性，MCMs蛋白功能正常是DNA復(fù)制所必須的[4]。新近研究發(fā)現(xiàn)，MCMs蛋白家族除了經(jīng)典的MCM2-7外，MCM1、MCM8、MCM9、MCM10相繼被發(fā)現(xiàn)和證實(shí)[5-7]。本文總結(jié)了近年來(lái)經(jīng)典MCM2-7蛋白復(fù)合體參與DNA復(fù)制機(jī)制及該蛋白復(fù)合體被調(diào)控的特點(diǎn)。

1 真核細(xì)胞DNA復(fù)制進(jìn)程

DNA復(fù)制是真核細(xì)胞有絲分裂S期的重要生物學(xué)事件，其主要分為DNA復(fù)制起始和延伸兩個(gè)部分[8]。DNA 復(fù)制源識(shí)別復(fù)合物 (origin recognition complex, ORC)首先識(shí)別并結(jié)合到復(fù)制起始位點(diǎn)上，作為預(yù)復(fù)制復(fù)合物組裝平臺(tái)(pre-replication complex, pre-RC)[9]。隨后招募Cdc6、Cdt1和MCM2-7蛋白，完成pre-RC組裝[10]。真核細(xì)胞有絲分裂進(jìn)入S期后，CDK和DDK蛋白將依次磷酸化激活pre-RC蛋白，活化的pre-RC進(jìn)一步招募Cdc45 和 GINS形成CMG復(fù)合物(Cdc45-MCM-GINS complex)，CMG復(fù)合物具有解螺旋活性可激活起始復(fù)制源[11]。后續(xù)RPA、RFC、PCNA及 DNA 聚合酶依次結(jié)合到被激活的復(fù)制源上開始DNA復(fù)制進(jìn)程[12]。

2 MCMs蛋白結(jié)構(gòu)的特點(diǎn)及功能學(xué)基礎(chǔ)

MCMs蛋白首先在釀酒酵母中被發(fā)現(xiàn)[13]，結(jié)構(gòu)研究表明MCM蛋白是AAA+ATPase家族的一個(gè)亞群，中央結(jié)構(gòu)域是由約200個(gè)氨基酸組成的高保守結(jié)構(gòu)區(qū)，MCMs蛋白形成六聚體是功能活性的基礎(chǔ)[14]。此外，MCM4、MCM6和MCM7在蛋白N端鑲嵌了一個(gè)鋅指結(jié)構(gòu)域，該結(jié)構(gòu)域?qū)CM形成六聚體起關(guān)鍵作用；MCM2和MCM3中具有核定位序列；MCM2和MCM4的N端具有額外的細(xì)胞周期蛋白依賴激酶(cyclin dependent kinase, CDK)結(jié)合序列[15]。在MCM2-7六聚體中，MCM4、MCM6和MCM7形成核心，其他MCMs蛋白依次結(jié)合，依賴復(fù)合體核定位序列進(jìn)入細(xì)胞核和CDK結(jié)合序列招募CDK蛋白[16]。

3 MCM2-7參與的DNA復(fù)制過程

MCM2-7(MCM2、MCM3、MCM4、MCM5、MCM6、MCM7)蛋白六聚體是較早被發(fā)現(xiàn)的MCM蛋白功能復(fù)合體，復(fù)合體中MCM蛋白的特殊結(jié)構(gòu)是實(shí)現(xiàn)核-膜穿梭和磷酸化激活的基礎(chǔ)。晶體研究顯示MCM2-7復(fù)合體呈指環(huán)狀,中間有足夠容納dsDNA通過的孔道[17]。DNA復(fù)制過程中dsDNA從中間孔道通過，解螺旋后的ssDNA從側(cè)邊孔道延出[18]。在真核細(xì)胞有絲分裂G期MCM2-7復(fù)合體與染色體結(jié)合，完成預(yù)復(fù)制復(fù)合物組裝，進(jìn)入S期后，MCM2-7復(fù)合體被激酶活化，誘發(fā)下游聚合酶-引物合成酶裝配，同時(shí)和DNA聚合酶結(jié)合，形成功能性復(fù)制叉，開始DNA解螺旋與復(fù)制[19]。MCM2-7必須在G期結(jié)合到起始點(diǎn)上，缺少M(fèi)CM2-7的結(jié)合，DNA復(fù)制將不能進(jìn)行[20]。

4 MCM2-7的轉(zhuǎn)錄調(diào)控

MCM2-7基因的轉(zhuǎn)錄有明顯的周期性規(guī)律，具體表現(xiàn)在基因轉(zhuǎn)錄mRNA表達(dá)的高峰在細(xì)胞周期的G1晚期[21]，進(jìn)入S期后mRNA表達(dá)將受到抑制[22]。在哺乳動(dòng)物中，MCMs基因轉(zhuǎn)錄與啟動(dòng)子區(qū)E2F結(jié)構(gòu)域密切相關(guān)，細(xì)胞轉(zhuǎn)錄因子E2F結(jié)合到啟動(dòng)子特定區(qū)域是MCMs基因轉(zhuǎn)錄所必須的[23]。在正常情況下E2F與Rb蛋白結(jié)合，抑制E2F與啟動(dòng)子結(jié)合。當(dāng)細(xì)胞進(jìn)入分裂周期以后，cyclin D結(jié)合CDK4與CDK6磷酸化Rb蛋白，Rb蛋白磷酸化后，E2F從復(fù)合體中解離，下游轉(zhuǎn)錄大量蛋白，包括MCMs蛋白家族，推進(jìn)細(xì)胞周期變化[24]。在細(xì)胞周期的G1晚期，Rb蛋白被磷酸化，E2F從復(fù)合體中大量解離，結(jié)合到啟動(dòng)子區(qū)E2F結(jié)構(gòu)域上，轉(zhuǎn)錄靶基因(見圖1)。

圖1 E2F參與的MCM基因轉(zhuǎn)錄 Fig 1 E2F transcription involved in MCM gene

5 MCM2-7功能過程的調(diào)控

MCM2-7生物學(xué)功能的調(diào)控可分為間接調(diào)控和直接調(diào)控。在預(yù)復(fù)制復(fù)合物組裝過程中，Geminin蛋白可與Cdt1緊密結(jié)合，阻斷Cdt1與Cdc6結(jié)合及下游招募MCM2-7[25]。CDKs可磷酸化Cdc6，促進(jìn)Cdc6降解，MCM2-7復(fù)合體將不能與染色體結(jié)合[26]。在哺乳動(dòng)物中，cyclin E蛋白是MCM2-7復(fù)合體與染色體結(jié)合所必須的[27]。另外，MCM2-7復(fù)合體可受到一些因子的直接調(diào)控，p27Kip1可與MCM7相互作用，誘導(dǎo)細(xì)胞周期阻滯[28]。負(fù)性調(diào)控基因產(chǎn)物Rb蛋白可與MCM7相互作用，影響細(xì)胞周期[29]。細(xì)胞周期點(diǎn)檢驗(yàn)蛋白ATM和ATR可介導(dǎo)MCM2和MCM3蛋白磷酸化，影響MCM2-7復(fù)合體的ATP酶活性和解鏈酶活性[30]。

在正常細(xì)胞周期下Cdc6 結(jié)合Cdt1招募MCM2-7，完成pre-RC組裝。Geminin蛋白可與Cdt1緊密結(jié)合，阻斷Cdt1與Cdc6結(jié)合及下游招募MCM2-7。CDKs可磷酸化Cdc6，致使Cdc6降解，阻斷MCM2-7與染色體結(jié)合(見圖2)。

圖2 MCM2-7在結(jié)合到染色體前受到的調(diào)控Fig 2 Regulation of MCM2-7 binding to chromosomes

6 miRNA參與的MCM2-7表達(dá)調(diào)控

miRNA是21-25 nt的單鏈小分子RNA，是非編碼RNA的重要組成部分，miRNA具有高度的保守性、時(shí)序性和組織特異性[31]。成熟的單鏈miRNA與蛋白質(zhì)復(fù)合物miRNP結(jié)合，引導(dǎo)復(fù)合物通過部分互補(bǔ)結(jié)合靶基因mRNA 3′UTR，從而抑制靶基因翻譯，或通過直接切割互補(bǔ)mRNA，使目標(biāo)mRNA失效[32]。目前關(guān)于miRNA與MCMs表達(dá)調(diào)控及交互影響的相關(guān)研究報(bào)道尚少。已證實(shí)miRNA-1269可靶向調(diào)控MCM2基因表達(dá)，影響前列腺癌細(xì)胞增殖[33]。基因組關(guān)聯(lián)分析發(fā)現(xiàn)p53表達(dá)依賴的miRNA-34家族與MCM基因3′UTR有互補(bǔ)序列[34]，后在細(xì)胞模型中證實(shí)miRNA-34家族可靶向調(diào)控MCM2-7基因表達(dá)，影響DNA復(fù)制進(jìn)程[35]。

MCMs蛋白家族在細(xì)胞中廣泛存在，其高度的保守性預(yù)示著其功能的重要性。MCM2-7復(fù)合體是維持基因組穩(wěn)定和參與DNA復(fù)制所必須的。MCMs表達(dá)水平與細(xì)胞增殖狀態(tài)及再生能力呈正相關(guān)。MCMs生物學(xué)過程了MCMs基因轉(zhuǎn)錄、MCM2-7復(fù)合體形成、核膜穿梭、與起始位點(diǎn)結(jié)合、DNA解螺旋及最后與染色體解離等眾多環(huán)節(jié)。目前對(duì)各環(huán)節(jié)的激活原因及調(diào)控過程了解并不十分透徹，MCMs的功能和機(jī)制研究仍有進(jìn)一步深入研究的必要，探索非編碼RNA與MCMs的交互調(diào)控機(jī)制將打開更廣闊的研究視野。

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(責(zé)任編輯：馬 軍)

MCM2-7 complex participates in the mechanism and control of DNA replication

WANG Minggang, MAO Dewen, ZHANG Rongzhen, LONG Fuli, WANG Xiufeng

Department of Hepatology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530023, China

Deoxyribonucleic acid (DNA) replication error-freely is the main security of genomic information successfully inherited to offspring. There is great significance for the life of the genetic information transfer and continuation. The minichromosome maintenance proteins (MCM) family is highly conserved protein. The MCM2-7 hexamer is essential for DNA replication. MCMs gene transcription is closely related to the promoter region E2F domain, at the gene expression level, it can be affected by miRNA-34s and miRNA-1269. In biological function MCM2-7 can be subjected by Cdt1, cyclinE, p27Kip1, Rb, ATM, ATR proteins.

DNA replication; MCMs; Regulate; MicroRNA

廣西高等學(xué)校高水平創(chuàng)新團(tuán)隊(duì)及卓越學(xué)者項(xiàng)目；國(guó)家中醫(yī)藥管理局“慢性重型肝炎解毒化瘀”重點(diǎn)研究室；國(guó)家自然科學(xué)基金課題(81460718)；廣西中醫(yī)藥大學(xué)第一附屬醫(yī)院青年基金(QN14010)

王明剛，碩士，研究方向：肝衰竭肝細(xì)胞再生機(jī)制及中醫(yī)藥的調(diào)控研究。E-mail:wmgyx2012@163.com

毛德文，博士，研究方向：肝衰竭的中醫(yī)藥療效機(jī)制研究。E-mail:mdwboshi2005@163.com

10.3969/j.issn.1006-5709.2017.07.024

Q75

A

1006-5709(2017)07-0809-03

2016-08-03