文雅蕾，呂柯孬，徐小康，張欣，丁良，潘學(xué)峰,,3

退火解旋酶SMARCAL1在維持基因組穩(wěn)定中的作用與機(jī)制

文雅蕾1，呂柯孬2，徐小康1，張欣1，丁良1，潘學(xué)峰1,2,3

1. 河北大學(xué)醫(yī)學(xué)院藥理室，保定 071000 2. 北京理工大學(xué)生命學(xué)院，北京 100081 3. 河北大學(xué)化學(xué)與環(huán)境學(xué)院，保定 071000

SMARCAL1是屬于SWI/SNF (SWItch/Sucrose Non-Fermentable)相關(guān)、基質(zhì)相關(guān)和激動(dòng)蛋白依賴(lài)的染色質(zhì)調(diào)節(jié)因子家族成員ATP驅(qū)動(dòng)的DNA退火解旋酶。SMARCAL1在體外和體內(nèi)能催化單鏈結(jié)合蛋白R(shí)PA結(jié)合的DNA單鏈與其互補(bǔ)鏈退火成雙鏈DNA。人基因的突變與Schimke免疫骨性發(fā)育不良(Schimke immuno-osseous dysplasia, SIOD)所能表現(xiàn)出的臨床癥狀呈高度相關(guān)。本文對(duì)SMARCAL1在DNA損傷部位DNA復(fù)制叉的重塑、在DNA雙鏈斷裂(double-stranded DNA, dsDNA)處參與經(jīng)典的非同源末端連接(non-homologous end joining, NHEJ)修復(fù)，以及在人染色體端粒完整性維護(hù)等方面的作用與機(jī)制進(jìn)行了梳理，對(duì)基因突變類(lèi)型與SIOD癥狀之間的對(duì)應(yīng)關(guān)系進(jìn)行了更新，并對(duì)SMARCAL1在三核苷酸重復(fù)序列擴(kuò)增關(guān)聯(lián)的神經(jīng)–肌肉退行性病變過(guò)程中的可能作用進(jìn)行了分析和討論，旨在更好地理解該退火解旋酶在維持基因組穩(wěn)定中的作用和機(jī)制。

SMARCAL1；RPA；DNA復(fù)制叉；SIOD；三核苷酸重復(fù)序列擴(kuò)增

基因組不穩(wěn)定常見(jiàn)于人類(lèi)遺傳疾病、癌癥及神經(jīng)–肌肉退行性疾病的病理過(guò)程[1]。常見(jiàn)的基因組不穩(wěn)定主要包括基因的點(diǎn)突變、插入/缺失突變、DNA鏈斷裂(單鏈斷裂和雙鏈斷裂)、DNA鏈交聯(lián)、基因組的倍性改變等類(lèi)型[1,2]。引發(fā)基因組不穩(wěn)定的原因可依據(jù)DNA是否受損分為兩類(lèi)：一類(lèi)源于細(xì)胞內(nèi)源性或細(xì)胞外源性因素造成DNA損傷；一類(lèi)則起因于基因組內(nèi)特定DNA序列在DNA復(fù)制、轉(zhuǎn)錄或重組過(guò)程中發(fā)生了錯(cuò)誤折疊[1,2]。據(jù)統(tǒng)計(jì)，無(wú)論是原核細(xì)胞還是真核細(xì)胞，均需要130種以上的蛋白組份參與基因組穩(wěn)定維護(hù)[1]。其中，退火解旋酶SMARCAL1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1)，因廣泛參與DNA損傷部位DNA復(fù)制叉的重塑、DNA雙鏈斷裂部位的非同源末端連接(non-homologous end joining, NHEJ)修復(fù)、以及染色體端粒的維護(hù)而受到高度重視[3~7]。

SMARCAL1普遍存在于真核生物細(xì)胞中，屬于一類(lèi)依賴(lài)ATP的染色質(zhì)“重塑因子”SNF2 (sucrose non-fermenting 2, SNF2)家族的一員[4]。SNF2家族的染色質(zhì)重塑因子需要利用ATP水解供能，廣泛參與細(xì)胞周期調(diào)控、基因轉(zhuǎn)錄、DNA重組、DNA損傷修復(fù)和DNA甲基化修飾等過(guò)程[5]。除SMARCAL1之外，通過(guò)影響DNA復(fù)制過(guò)程維持基因組穩(wěn)定性的ZRANB3和HLTF也是SNF2家族的重要成員[6]。這些蛋白均含有一個(gè)由7個(gè)保守基序(motif)組成的類(lèi)似“解旋酶”(與常見(jiàn)的DNA和RNA解旋酶類(lèi)似)的ATPase結(jié)構(gòu)域[7]。盡管SMARCAL1具有DNA“退火解旋酶”(annealing helicase)活性，但目前并未被歸類(lèi)于已有的6個(gè)DNA解旋酶超家族(DNA helicase superfamily)中[8]。

人基因定位于2q34-q36區(qū)段，含17個(gè)外顯子，編碼一個(gè)由954個(gè)氨基酸殘基組成的蛋白質(zhì)[7]。在人()和小鼠()的所有組織中均見(jiàn)基因表達(dá)，如在人免疫系統(tǒng)中，在單核細(xì)胞、B淋巴細(xì)胞、CD4+T細(xì)胞、CD8+T細(xì)胞、NK細(xì)胞中的表達(dá)量分別為1.259‰、1.584‰、1.259‰、1.995‰和0.100‰；在內(nèi)分泌系統(tǒng)的胰腺細(xì)胞、前列腺細(xì)胞中表達(dá)量約為0.100‰和0.016‰；在睪丸細(xì)胞中表達(dá)量則為0.158‰[9~16]。基因突變與Schimke免疫骨性發(fā)育不良(Schimke immuno-osseous dysplasia, SIOD)密切相關(guān)[17,18]。SIOD是一種累及多系統(tǒng)、進(jìn)行性加重的罕見(jiàn)常染色體遺傳病，主要表現(xiàn)為T(mén)細(xì)胞免疫缺陷、局灶節(jié)段性腎小球硬化、腦發(fā)育受損、腎功能衰竭和骨骼發(fā)育不良造成的生長(zhǎng)遲緩等[8,12,19~21]。除此之外，部分SIOD患者還表現(xiàn)有甲狀腺功能減退、骨髓衰竭、頭發(fā)稀薄、角膜混濁、動(dòng)脈粥樣硬化、中風(fēng)和偏頭痛等[8,12,21]。

本文將對(duì)SMARCAL1在DNA損傷部位借退火解旋酶活性重塑DNA復(fù)制叉，在DNA雙鏈斷裂部位參與非同源末端連接(NHEJ)修復(fù)，以及在端粒完整性維護(hù)過(guò)程等方面的作用與機(jī)制進(jìn)行梳理。同時(shí)，對(duì)基因突變型與SIOD癥狀的相關(guān)性的最新進(jìn)展進(jìn)行了更新，對(duì)其在累及多達(dá)40余種人神經(jīng)–肌肉退行性疾病中的三核苷酸重復(fù)序列擴(kuò)增性不穩(wěn)定和脆性不穩(wěn)定發(fā)生過(guò)程中的可能作用進(jìn)行了分析。

1 SMARCAL1的結(jié)構(gòu)與功能

SMARCAL1含有復(fù)制蛋白A(replication protein A , RPA)結(jié)合基序、退火解旋酶活性結(jié)構(gòu)域、ATP酶結(jié)構(gòu)域等(圖1)[22]。其中，與RPA作用的基序位于N-末端(28個(gè)高度保守的氨基酸殘基序列)；與“退火解旋活性”有關(guān)的結(jié)構(gòu)域位于239~307和331~ 4002區(qū)間的“HARP”結(jié)構(gòu)域(每個(gè)“HARP”各含60個(gè)氨基酸殘基)[23]。C-末端是解旋酶結(jié)構(gòu)域，具有ATP酶催化活性(由115個(gè)氨基酸殘基組成的“RecA樣”結(jié)構(gòu)域，DEXDc和HELICc)和SWI/SNF“核小體重塑蛋白”結(jié)構(gòu)域(圖1)[24]。

SMARCAL1的ATP依賴(lài)DNA退火解旋酶含有2個(gè)HARP結(jié)構(gòu)域組成的ATPase[25~27]。當(dāng)DNA出現(xiàn)損傷時(shí)，與單鏈DNA結(jié)合的單鏈DNA結(jié)合蛋白R(shí)PA32識(shí)別位于SMARCAL1的N端RPA作用結(jié)構(gòu)域(圖1)，并招募SMARCAL1至dsDNA-ssDNA的單鏈DNA一側(cè)，這一反應(yīng)可見(jiàn)于S期細(xì)胞周期關(guān)卡通路激活、停滯的復(fù)制叉重塑、端粒DNA完整性維護(hù)以及利用NHEJ機(jī)制修復(fù)DNA雙鏈斷裂損傷等過(guò)程[3,24,25,28~31]。

1.1 SMARCAL1與DNA復(fù)制叉的維護(hù)

在人體細(xì)胞中，缺失常影響細(xì)胞對(duì)DNA復(fù)制逆境(replication stress)應(yīng)答能力[4]。缺陷的細(xì)胞內(nèi)可見(jiàn)DNA雙鏈斷裂，且對(duì)多種影響DNA復(fù)制的物質(zhì)敏感，表明SMARCAL1與DNA復(fù)制忠實(shí)性的監(jiān)管有關(guān)[3,24,28]。研究發(fā)現(xiàn)，SMARCAL1可通過(guò)與RPA作用參與停滯的DNA復(fù)制叉的“重塑”[29]，否則停滯的DNA復(fù)制叉有可能衍生為DNA雙鏈斷裂從而使DNA突變和染色體重排的風(fēng)險(xiǎn)增高[32]。真核生物的RPA是一個(gè)由RPA70、RPA32、RPA14亞基組成的異源三聚體，可借4個(gè)DNA結(jié)合結(jié)構(gòu)域(DNA-binding domain, DBD)與單鏈DNA結(jié)合[33]。RPA異三聚體參與DNA損傷部位修復(fù)蛋白的募集和組裝，同時(shí)規(guī)避單鏈DNA錯(cuò)誤折疊出非B型DNA構(gòu)象[33,34]。SMARCAL1借其N(xiāo)-末端與RPA32的N端結(jié)合，從而被快速高效地定位到復(fù)制叉的單鏈DNA部位[3,35~37]。

活性氧自由基、紫外輻射及一些化學(xué)物質(zhì)(如放線菌素等)會(huì)損傷DNA模板，影響細(xì)胞周期S時(shí)相的DNA復(fù)制[3]。為此，細(xì)胞需針對(duì)相應(yīng)的DNA損傷類(lèi)型做出應(yīng)答，包括利用ATR對(duì)單鏈空缺(single stranded gap, SSG)損傷應(yīng)答，利用ATM、DNA-PK等激酶對(duì)DNA DSBs應(yīng)答等[1]。這些激酶對(duì)SMARCAL1蛋白的磷酸化修飾有助于有序啟動(dòng)DNA損傷修復(fù)[38]。比如，SMARCAL1第652位絲氨酸(S652)殘基被ATR磷酸化修飾，可抑制SMARCAL1的ATP酶活性，從而降低其DNA復(fù)制叉重塑能力，避免DNA復(fù)制叉崩解[39]。

體外研究表明，SMARCAL1的退火解旋酶活性的發(fā)揮需要與DNA結(jié)合，并由ATP水解供能[22]。在這個(gè)過(guò)程中，SMARCAL1借RPA結(jié)合蛋白與單鏈DNA結(jié)合，并利用ATP水解提供的能量催化彼此互補(bǔ)的單鏈DNA重新形成雙鏈DNA，并同時(shí)解除RPA與單鏈DNA的結(jié)合(圖2，A和B)[24]。現(xiàn)已發(fā)現(xiàn)，上述反應(yīng)可以發(fā)生在DNA復(fù)制叉部位，幫助DNA復(fù)制叉內(nèi)的單鏈DNA發(fā)生“分支遷移”和“分支重塑”[4,22,40]，使停滯的DNA復(fù)制叉“重塑”出“雞爪”狀四臂交叉中間體結(jié)構(gòu)[41,42](圖2C)。圖2C中的“雞爪”結(jié)構(gòu)可以穩(wěn)定停滯的DNA復(fù)制叉，又能使帶傷的單鏈DNA重新“退回”雙鏈DNA狀態(tài)，可以贏得修復(fù)時(shí)間，或利用“模板轉(zhuǎn)換”(template switching)使新生DNA鏈的3 ?生長(zhǎng)端“繞過(guò)”帶傷DNA模板后重啟DNA復(fù)制[6,27,29,33,43](圖2，B和C)。

圖1 退火解旋酶SMARCAL1的功能結(jié)構(gòu)域分布

SMARCAL1在其N(xiāo)端區(qū)域包含與RPA相互作用基序和2個(gè)退火活性所需的HARP結(jié)構(gòu)域；在其C末端含有解旋酶結(jié)構(gòu)域，為DEXDc和HELICc基序[23,25]。

圖2 SMARCAL1修復(fù)受損DNA復(fù)制叉的機(jī)制

A：DNA鏈復(fù)制遇阻，復(fù)制型DNA聚合酶和所“偶聯(lián)”的DNA解旋酶脫離，導(dǎo)致前導(dǎo)鏈模板上產(chǎn)生ssDNA空缺；B：在停滯的復(fù)制叉上，RPA與單鏈DNA結(jié)合后行成RPA-ssDNA，并招募SMARCAL1，啟動(dòng)復(fù)制叉回轉(zhuǎn)；C：SMARCAL1在復(fù)制叉回轉(zhuǎn)后以3種可能的方式催化復(fù)制叉的修復(fù)，包括持續(xù)分支遷移產(chǎn)生“雞爪”狀的Holliday結(jié)構(gòu)；與相鄰的重新建立的DNA復(fù)制叉發(fā)生“融合”獲得拯救；以及用FANCP-MUS81處理后產(chǎn)生對(duì)應(yīng)于新生前導(dǎo)鏈的ssDNA鏈，然后生成單末端DNA雙鏈斷裂(DSB)。RPA協(xié)助SMARCAL1用互補(bǔ)的模板鏈與新生的ssDNA前導(dǎo)鏈“退火”，重構(gòu)出可正常復(fù)制的DNA復(fù)制叉[25,39,40]。

需要特別指出的是SMARCAL1催化的上述DNA復(fù)制叉重塑在體外也可以由大腸桿菌的DNA解旋酶RecG催化[29]。大腸桿菌RecG是一個(gè)3?→5?DNA解旋酶，具有催化Holliday中間體(同源重組過(guò)程中出現(xiàn)的交叉結(jié)構(gòu))發(fā)生分支遷移能力[29]。在催化DNA復(fù)制叉回轉(zhuǎn)時(shí)，RecG類(lèi)似SMARCAL1，需要借DNA單鏈結(jié)合蛋白與DNA復(fù)制叉結(jié)合。在這個(gè)過(guò)程中DNA單鏈結(jié)合蛋白還可以進(jìn)一步穩(wěn)定RecG與DNA復(fù)制叉的結(jié)合，有利于RecG催化DNA復(fù)制叉回轉(zhuǎn)(圖2)[44]。因此，雖然SMARCAL1在結(jié)構(gòu)上與大腸桿菌RNA聚合酶結(jié)合蛋白HepA相似(被稱(chēng)為HepA相關(guān)蛋白，HARP)，但在催化DNA底物結(jié)構(gòu)重塑時(shí)則更類(lèi)似于大腸桿菌的DNA解旋酶RecG[9~11]。

1.2 SMARCAL1在維持端粒穩(wěn)定過(guò)程中的作用

人染色體端粒DNA一般由“TTAGGG”六聚體重復(fù)序列組成，可形成包括G-四鏈體、D(t)環(huán)等在內(nèi)的非B型DNA結(jié)構(gòu)。盡管這些DNA結(jié)構(gòu)為端粒穩(wěn)定所必需，但如果它們出現(xiàn)在DNA復(fù)制過(guò)程中則會(huì)造成DNA復(fù)制叉停滯，使端粒變短[45~47]。維持端粒穩(wěn)定是保證細(xì)胞增殖能力的關(guān)鍵。腫瘤細(xì)胞的端粒長(zhǎng)度的維持機(jī)制有兩種，一是重新激活端粒酶，一是利用同源重組“替補(bǔ)”機(jī)制維持(統(tǒng)稱(chēng)為端粒延長(zhǎng)替代機(jī)制，alterative lengthening of telomere, ALT)[48]。ALT細(xì)胞(缺乏端粒酶而需要ALT維持端粒長(zhǎng)度的細(xì)胞)中可見(jiàn)由端粒DNA的額外染色體形成的C-環(huán)(C-circles)，可用于ALT活性的標(biāo)記[43]。

DNA損傷修復(fù)系統(tǒng)對(duì)端粒酶復(fù)制穩(wěn)定端粒發(fā)揮著重要的調(diào)控作用[49]。Cox等[49]發(fā)現(xiàn)SMARCAL1參與端粒的ALT維持機(jī)制。在ALT細(xì)胞內(nèi)，SMARCAL1與端粒DNA結(jié)合，協(xié)助停滯的DNA復(fù)制叉重啟，應(yīng)對(duì)端粒DNA復(fù)制應(yīng)激[49]。而缺失SMARCAL1的ALT細(xì)胞則會(huì)出現(xiàn)端粒DNA復(fù)制困難，表現(xiàn)為DNA復(fù)制叉持續(xù)停滯，并最終形成DSBs，表現(xiàn)出高強(qiáng)度染色體融合(圖3)[49]。與此同時(shí)，SMARCAL1缺失會(huì)影響端粒的長(zhǎng)度異質(zhì)性，推測(cè)與ALT細(xì)胞中SMARCAL1水平過(guò)低催生了更多的C-環(huán)有關(guān)[49]。

此外，Poole等[50]發(fā)現(xiàn)SMARCAL1在端粒處發(fā)揮作用時(shí)無(wú)需RPA協(xié)助，因?yàn)槎肆２课恢貜?fù)DNA序列更傾向于形成G-四鏈體等非B型DNA二級(jí)結(jié)構(gòu)，而G-四鏈體本身就可有效招募SMARCAL1。但是，當(dāng)SMARCAL1與DNA復(fù)制叉內(nèi)的前導(dǎo)鏈模板結(jié)合時(shí)則需要RPA激活[33,50]。

1.3 SMARCAL1與DNA雙鏈斷裂修復(fù)

真核細(xì)胞內(nèi)與DNA DSBs修復(fù)有關(guān)的機(jī)制有兩類(lèi)，一類(lèi)是依賴(lài)DNA同源性的同源重組修復(fù)(homo-logous recombination, HR)，一類(lèi)則是不嚴(yán)格依賴(lài)DNA同源性(無(wú)需模板)的非同源末端連接(NHEJ)[51]。HR常用于細(xì)胞周期的S、G2等時(shí)相，而NHEJ則可用于整個(gè)細(xì)胞周期，特別當(dāng)細(xì)胞處于G0/G1和S期早期，由于缺乏“同源染色體”，NHEJ對(duì)DSBs修復(fù)起著關(guān)鍵的作用[52~54]。

Keka等[55]發(fā)現(xiàn)SMARCAL1參與G1期DSBs的NHEJ修復(fù)過(guò)程。SMARCAL1的退火解旋酶活性可以協(xié)助DNA末端結(jié)合蛋白Ku70/80“獲取”雙鏈DNA末端，有利于進(jìn)一步依序招募參與NHEJ修復(fù)的蛋白因子，以完成NHEJ或MMEJ (小同源末端連接，microhomology-mediated end-joining，MMEJ)在G1時(shí)相對(duì)DNA DSBs修復(fù)。在此過(guò)程中，SMA-RCAL1的ATP依賴(lài)的退火能力可能避免了RPA在DSB末端與ssDNA結(jié)合，因此促進(jìn)Ku70/Ku80/DNA- PKcs復(fù)合體對(duì)DNA雙鏈末端識(shí)別、結(jié)合和保護(hù)，為后續(xù)XRCC4和DNA連接酶IV在斷裂末端的積聚提供便利，從而提供了NHEJ修復(fù)的精確度[55~60](圖4)。

SMARCAL1在Ku70/Ku80/DNA-PKcs聚積的DNA末端協(xié)助Mre11、RAD50、Nbs1組裝成MRN復(fù)合物(Mre11-RAD50-Nbs1)[65,66]。具體過(guò)程如下：DSB信號(hào)被ATM“捕獲”，ATM將H2AX磷酸化成γ-H2AX。γ-H2AX與Nbs1作用進(jìn)一步促進(jìn)RAD50和Mre11在DSB斷端處形成MRN復(fù)合體(RAD50- Mre11-Nbs1, MRN)[67~69]。其中，RAD50二聚體上的ATP酶(Walker A和Walker B)負(fù)責(zé)與DSB兩個(gè)末端結(jié)合，避免末端錯(cuò)位或漂移。Nbs1則協(xié)助SMA-RCAL1退火可被NHEJ修復(fù)的DNA斷端，使之保持雙鏈狀態(tài)，并由RAD50的絞鏈區(qū)將兩個(gè)斷端“固定”[70]。

圖3 SMARCAL1在維持端粒穩(wěn)定中的作用

ALT細(xì)胞內(nèi)的染色體端粒易出現(xiàn)復(fù)制逆境，SMARCAL1幫助重塑端粒DNA上停滯的復(fù)制叉，確保端粒序列的完全復(fù)制[46,47]。在SMARCAL1缺陷的情況下，ALT端粒上持續(xù)停滯的復(fù)制叉會(huì)催生DNA雙鏈斷裂，出現(xiàn)端粒DNA復(fù)制障礙及形成C-環(huán)，此時(shí)，染色體易融合，造成基因組不穩(wěn)定[49,50]。

圖4 SMARCAL1在NHEJ修復(fù)DNA雙鏈斷裂中的可能作用

A：DSB形成后，RPA識(shí)別并結(jié)合DNA斷端，之后，招募SMARCAL1。SMARCAL1的退火解旋酶活性保證DSB斷口DNA呈雙鏈狀態(tài)；B：Ku70/Ku80組成的異源二聚體與DNA末端結(jié)合；C：DNA-Ku復(fù)合體招募DNA-PKcs，形成Ku70/Ku80/DNA-PKcs復(fù)合體；D：SMARCAL1促進(jìn)Ku70/Ku80/DNA-PKcs復(fù)合體穩(wěn)定結(jié)合在DSB端口，并激活DNA-PKcs。DNA-PKcs對(duì)包括自身在內(nèi)的蛋白進(jìn)行磷酸化修飾；E：DSB斷端的SMARCAL1促進(jìn)Ku70/Ku80/DNA-PKcs復(fù)合體招募DNA連接酶IV和XRCC4復(fù)合體，進(jìn)一步形成Ku70/Ku80/DNA-PKcs/XRCC4/DNA連接酶IV修復(fù)復(fù)合體；F：含有連接酶的復(fù)合體完成兩個(gè)DNA斷端的連接[55~64]。

此外，SMARCAL1也和RAD50一起參與阻止復(fù)制叉反轉(zhuǎn)，通過(guò)調(diào)節(jié)Mre11的核酸酶活性，防止Mre11過(guò)度降解新生DNA區(qū)段[71]。

1.4 SMARCAL1與基因轉(zhuǎn)錄

SMARCAL1作為SWI/SNF家族(負(fù)責(zé)催化核小體重塑)中的成員同時(shí)擁有解旋酶(helicase)和ATPase活性，因此有可能參與某些基因轉(zhuǎn)錄過(guò)程中的核小體重塑。當(dāng)前研究較多的是SMARCAL1對(duì)基因轉(zhuǎn)錄的調(diào)節(jié)。編碼一種亮氨酸拉鏈蛋白，參與人類(lèi)基因組中5%~15%的基因的轉(zhuǎn)錄，在細(xì)胞增殖、分化、生長(zhǎng)和凋亡中發(fā)揮重要作用[72~74]。SMARCAL1作為輔因子參與基因的轉(zhuǎn)錄[72~74]。Heravi等[75]發(fā)現(xiàn)SMARCAL1通過(guò)ATP依賴(lài)性方式改變DNA結(jié)構(gòu)，調(diào)節(jié)的轉(zhuǎn)錄[75]。Tapan等[76]發(fā)現(xiàn)SMARCAL1是轉(zhuǎn)錄的負(fù)調(diào)控因子。通過(guò)與激活蛋白BRG1和RNA聚合酶Ⅱ(RNAPⅡ)“爭(zhēng)奪”基因P1啟動(dòng)子上游的一段富含GC堿基的159 bp DNA區(qū)域(-B159)，當(dāng)激活蛋白BRG1和RNAPⅡ占據(jù)時(shí)，基因轉(zhuǎn)錄；當(dāng)該區(qū)域被SMARCAL1占據(jù)時(shí)，基因關(guān)閉[76]。SMARCAL1與結(jié)合后使得相應(yīng)部位的染色質(zhì)結(jié)構(gòu)更難與BRG1和RNAPⅡ結(jié)合，故可抑制的轉(zhuǎn)錄[76]。

2 SMARCAL1在三核苷酸重復(fù)序列擴(kuò)增中的潛在作用

人基因組中特定基因部位處的三核苷酸重復(fù)序列(CAG)n·(CTG)n的“動(dòng)態(tài)”擴(kuò)增與多種遺傳性脊髓–小腦共濟(jì)失調(diào)、亨廷頓疾病、阿爾茲海默綜合征等神經(jīng)–肌肉系統(tǒng)退行性病變的發(fā)生密切相關(guān)[77~79]。已有的研究結(jié)果表明，(CAG)n重復(fù)序列的擴(kuò)增與DNA剪切修復(fù)過(guò)程中產(chǎn)生的單鏈DNA錯(cuò)誤、DNA復(fù)制過(guò)程中出現(xiàn)DNA鏈斷裂、DNA復(fù)制過(guò)程中新生鏈和模板鏈間發(fā)生“滑動(dòng)”、鏈轉(zhuǎn)換(strand swit-ching)、蛋白質(zhì)與重復(fù)DNA序列結(jié)合等許多因素有關(guān)。上述過(guò)程可能起因于(CAG)n重復(fù)DNA形成的含錯(cuò)配堿基對(duì)的DNA發(fā)卡結(jié)構(gòu)，DNA發(fā)卡結(jié)構(gòu)可能會(huì)直接干擾DNA復(fù)制、修復(fù)和重組。不僅如此，在基因轉(zhuǎn)錄過(guò)程中(CAG)n三核苷酸重復(fù)序列一旦形成R環(huán)結(jié)構(gòu)(DNA雜交體與非模板鏈形成)也可影響DNA復(fù)制叉的移動(dòng)，導(dǎo)致非模板鏈DNA斷裂，甚至可被用于DNA重新復(fù)制的引物[77~79]。本實(shí)驗(yàn)室已有的研究結(jié)果表明，基因的正本基因()功能喪失的大腸桿菌細(xì)胞內(nèi)，疾病相關(guān)的(CAG)n·(CTG)n序列呈特異性擴(kuò)增(待發(fā)表)。鑒于SMARCAL1在催化DNA復(fù)制叉重塑及避免DNA復(fù)制叉崩解的過(guò)程中與RecG功能一致，推測(cè)出現(xiàn)在人類(lèi)患者細(xì)胞內(nèi)的(CAG)n·(CTG)n序列擴(kuò)增有可能與DNA復(fù)制叉重塑和DNA復(fù)制重起始失敗有關(guān)(圖5A)，或出現(xiàn)在(CAG)n重復(fù)序列內(nèi)的R環(huán)結(jié)構(gòu)影響了DNA復(fù)制，并在RecG/SMARCAL1功能異常的情況下誘生DNA鏈重排或造成該處DNA局部額外復(fù)制[77~79](圖5B)。最近，類(lèi)似圖5A的工作機(jī)制已被Kononenko等[80]的工作證實(shí)。他們利用小鼠幼紅細(xì)胞白血病細(xì)胞系驗(yàn)證了可導(dǎo)致人脆性X染色體綜合征(馬丁–貝爾綜合征)的三核苷酸重復(fù)序列(CGG)n的擴(kuò)增不穩(wěn)定與SMARCAL1所具有的功能直接有關(guān)[80]。但是，目前尚無(wú)有工作表明SMARCAL1自身是否具有類(lèi)似于RecG的RNA解旋酶活性或與其他RNA解旋酶一起參與RNA轉(zhuǎn)錄，故圖5B所描述的機(jī)制依然有待進(jìn)一步檢驗(yàn)。

為了闡釋長(zhǎng)期困擾國(guó)際醫(yī)學(xué)界的亨廷頓疾病、阿爾茲海默綜合征和多種遺傳性脊髓–小腦共濟(jì)失調(diào)綜合征等發(fā)病過(guò)程中出現(xiàn)的(CAG)n擴(kuò)增的原因，以及明確其他與三核苷酸重復(fù)序列失穩(wěn)定引發(fā)的人類(lèi)神經(jīng)–肌肉退行性病變的病理機(jī)制，當(dāng)前亟需深入分析SMARCAL1在包括(CAG)n重復(fù)序列在內(nèi)的三核苷酸重復(fù)序列穩(wěn)定維護(hù)過(guò)程中可能發(fā)揮的作用[77~79]。

3 SMARCAL1的基因突變型與SIOD的癥狀關(guān)聯(lián)

世界范圍內(nèi)SIOD的發(fā)病率約為1/300萬(wàn)~1/100萬(wàn)[8,9,81,82]。導(dǎo)致SIOD發(fā)生的基因突變多為雙等位基因功能缺失、錯(cuò)義突變、插入/缺失(inser-tion and deletion, Ins/Del)、大片段缺失以及SMAR-CAL1 mRNA拼接錯(cuò)誤(表1)[9,83]。上述基因改變常出現(xiàn)在SMARCAL1的RecA樣結(jié)構(gòu)域I中(圖1)，由于突變影響了SMARCAL1的ATP酶活性，故常見(jiàn)疾病的嚴(yán)重程度與突變體SMARCAL1所表現(xiàn)出的ATP酶活性成反比[82]。SMARCAL1等位基因缺失、無(wú)義或移碼突變常見(jiàn)于重癥患者。重癥SIOD患者的癥狀在孕期外顯，表現(xiàn)為胎兒生長(zhǎng)遲緩、甲狀腺功能減退癥、骨髓衰竭、短暫性腦缺血發(fā)作、中風(fēng)和腎衰竭等，一般死于5歲前。SMARCAL1等位基因錯(cuò)義突變患者癥狀較輕，多數(shù)發(fā)病較晚，常見(jiàn)8~13歲以后發(fā)病，數(shù)年后進(jìn)展至腎衰[84,85](表1)。

圖5 SMARCAL1及其可能的突變體基因在三核苷酸重復(fù)序列穩(wěn)定維護(hù)中的潛在作用

A：SMARCAL1參與三核苷酸重復(fù)序列部位處DNA復(fù)制叉的重塑。根據(jù)參考文獻(xiàn)[80]修改繪制；B：SMARCAL1自身或引導(dǎo)其他RNA解旋酶清除在三核苷酸重復(fù)序列轉(zhuǎn)錄過(guò)程中形成的R-環(huán)結(jié)構(gòu)[77~79]。

表1 Smarcal1基因突變型與SIOD的癥狀關(guān)聯(lián)

續(xù)表

“？”核苷酸或氨基酸突變的位置待確認(rèn)。

SIOD的共有臨床癥狀是T細(xì)胞免疫缺陷。在淋巴細(xì)胞發(fā)育過(guò)程中，編碼免疫球蛋白和T細(xì)胞受體抗原結(jié)合域的功能基因需要經(jīng)過(guò)與NHEJ類(lèi)似的V(D)J重組才能形成[98,99]。與NHEJ類(lèi)似，V(D)J重排也需產(chǎn)生DNA雙鏈斷裂，并由NHEJ機(jī)制完成斷鏈末端的連接[98,99]。而突變體SMARCAL1常影響NHEJ在V(D)J重排重組的連接效率，這可能是SIOD患者常見(jiàn)T細(xì)胞免疫缺陷的原因之一[96,100]。此外，突變體SMARCAL1有可能通過(guò)影響-基因表達(dá)進(jìn)一步影響SIOD的征候。研究發(fā)現(xiàn)成年小鼠的腎臟和腦組織中，不表達(dá)而高表達(dá)[13,101]。暗示SIOD患者表現(xiàn)出的腎功能不全和腦發(fā)育受損可能與SMARCAL1突變體不能正確調(diào)節(jié)表達(dá)有關(guān)[19]。Tapan等[76]發(fā)現(xiàn)SIOD患者的染色質(zhì)也會(huì)出現(xiàn)異常，這種情況暗示SMARCAL1可能依然具有染色質(zhì)重塑活性?？赡苡捎趖rxG和PcG的復(fù)合體誘導(dǎo)組蛋白的翻譯后修飾，影響染色體的結(jié)構(gòu)。而SMARCAL1通過(guò)結(jié)合染色質(zhì)直接影響基因表達(dá)。

4 結(jié)語(yǔ)與展望

目前，DNA退火酶SMARCAL1的功能異常與SIOD之間的關(guān)聯(lián)已經(jīng)得到了明確，但SIOD患者所能表現(xiàn)的癥狀征候差異與不同基因突變型的對(duì)應(yīng)關(guān)系的細(xì)節(jié)依然處于不斷更新?tīng)顟B(tài)。由于臨床上SIOD癥狀可累及人體多個(gè)系統(tǒng)，輕重患者之間的臨床表現(xiàn)并不盡然一致，也很難呈現(xiàn)一種癥狀的漸進(jìn)發(fā)展特征，暗示基因突變可能依附攜帶突變的組織和器官，一種可能的情形是基因突變對(duì)不同組織細(xì)胞內(nèi)的染色質(zhì)結(jié)構(gòu)、基因轉(zhuǎn)錄、DNA損傷修復(fù)依細(xì)胞類(lèi)型不同而具有差異。因此，目前亟需對(duì)有關(guān)基因表達(dá)的組織特異性機(jī)制加深了解。在鑒定出的分子機(jī)制方面，SMARCAL1的退火酶活性在復(fù)制叉重塑過(guò)程中的作用已被體外實(shí)驗(yàn)證實(shí)，但尚缺乏體內(nèi)實(shí)驗(yàn)證據(jù)的支持。不僅如此，不同突變型對(duì)其活性的影響呈現(xiàn)多樣性(表1)，如何關(guān)聯(lián)SIOD癥狀也需要進(jìn)一步積累體內(nèi)實(shí)驗(yàn)數(shù)據(jù)。近年來(lái)本實(shí)驗(yàn)室對(duì)SMA-RCAL1的原核生物功能類(lèi)似物DNA解旋酶RecG的研究發(fā)現(xiàn)，RecG缺陷突變后的大腸桿菌細(xì)胞容許與亨廷頓疾病、阿爾茲海默綜合癥以及多種遺傳型小腦–脊髓共濟(jì)失調(diào)在內(nèi)的40多種神經(jīng)-肌肉系統(tǒng)退行性疾病直接有關(guān)的三核苷酸重復(fù)序列CAG出現(xiàn)特異性擴(kuò)增，類(lèi)似的情形是否也有可能出現(xiàn)在攜帶某些突變類(lèi)型的患者體內(nèi)需要給予重視。當(dāng)前關(guān)于SMARCAL1分子機(jī)制的研究主要集中于其對(duì)DNA復(fù)制叉重塑、DNA雙鏈斷裂損傷修復(fù)及端粒完整性維持等方面。作為SWI/SNF相關(guān)、基質(zhì)相關(guān)和激動(dòng)蛋白依賴(lài)的染色質(zhì)調(diào)節(jié)因子家族成員的SMARCAL1在染色質(zhì)重塑、組蛋白修飾編碼(hist-one code)方面是否依然具有作用尚缺乏更多了解。綜上所述，深入開(kāi)展基因及其突變型的分子遺傳學(xué)和SMARCAL1蛋白的體內(nèi)分子生物學(xué)研究將不僅有助于系統(tǒng)理解SIOD發(fā)病機(jī)制，而且對(duì)破解長(zhǎng)期困擾人類(lèi)健康的40余種進(jìn)行性神經(jīng)-肌肉系統(tǒng)退行性疾病的致病原因提供借鑒和參考。

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SMARCAL1, roles and mechanisms in genome stability maintenance

Yalei Wen1, Kenao Lü2, Xiaokang Xu1, Xin Zhang1, Liang Ding1, Xuefeng Pan1,2,3

SMARCAL1 is an ATP-driven DNA annealing helicase that is similar in structure to the chromatin regulators in the subfamily A group of the SWI/SNF-related matrix-associated actin-dependent chromatin regulators. SMARCAL1 catalyzes the formation of dsDNA by annealing the single-stranded binding protein RPA coated ssDNA with its complementary strand bothand. In humans, different mutations ofgene are found to be closely related to different symptoms shown in individuals with Schimke immuno-osseous dysplasia (SIOD). This paper reviews the recent research progress of SMARCAL1 functions in remodeling DNA replication forks at damaged DNA sites, working in classical non-homologous end joining (NHEJ) repair of DNA double-stranded breaks, and in maintaining chromosomal telomere integrity. The relationships between the mutations ofgene in different SIOD symptoms, and the possible involvements of SMARCAL1 in neuromuscular degenerative diseases associated with trinucleotide repeats expansions are also updated and discussed to better understand the roles and mechanisms of the annealing helicase in genome stability maintenance.

SMARCAL1; RPA; DNA replication fork; SIOD; expansions of trinucleotide repeats

2019-07-07;

2019-09-04

北京自然科學(xué)基金項(xiàng)目(編號(hào)：5132014)和河北省醫(yī)學(xué)科學(xué)重點(diǎn)項(xiàng)目(編號(hào)：20160051)資助[Supported by the Beijing Natural Science Foundation (No. 5132014) and the Hebei Provincial Key Research Programs for Medical Science (No. 20160051)]

文雅蕾，碩士研究生，專(zhuān)業(yè)方向：分子藥理學(xué)。E-mail: 491574395@qq.com

丁良，博士，教授，研究方向：分析化學(xué)。E-mail: 345823685@qq.com

潘學(xué)峰，博士，教授，研究方向：生物化學(xué)、分子遺傳學(xué)和藥理等。E-mail: xuefengpancam@aliyun.com

10.16288/j.yczz.19-158

2019/9/27 9:18:00

URI: http://kns.cnki.net/kcms/detail/11.1913.R.20190926.1625.002.html

(責(zé)任編委: 盧大儒)