RECQL4在维持基因组稳定中的作用
2014-05-04唐超智杨钧棠王文晟
唐超智++杨钧棠++王文晟
摘要:RECQL4属于人类RECQ解旋酶家族,其变异已引起至少3种基因组不稳定性疾病。除了同家族其他成员一样参与DNA损伤修复外,RECQL4独特的结构还使其参与了复制的起始。最新研究结果表明,RECQL4可维持端粒和线粒体DNA的稳定,提示RECQL4的作用可能更为广泛。对RECQL4维持基因组稳定的不同途径及其机制进行了归纳,以期为人们探索RECQL4相关性疾病的发病机理和治疗方案提供思路。
关键词:RECQL4;复制;修复;端粒;线粒体
中图分类号:Q71 文献标识码:A 文章编号:0439-8114(2014)04-0745-05
Role of RECQL4 in Maintaining the Stability of Genome
TANG Chao-zhi,YANG Jun-tang,WANG Wen-sheng
(College of Life Sciences, Henan Normal University, Xinxiang 453007, Henan,China)
Abstract: RECQL4 belonging to RECQ helicase family is responsible for more then three human diseases of genomic disorder. Being involved in DNA damage repair like other family members, the special structure of RECQL4 makes it involve in initiating of DNA replication. It was found that RECQL4 helps maintain the stability of telomere and mitochondrial DNA, revealing that RECQL4 may have broad functions. Different ways that RECQL4 maintains the stability of the genome were summarized to provide advices on exploring the mechanisms and therapies of RECQL4 related diseases.
Key words: RECQL4; replication; repair; telomere; mitochondrion
大量的研究表明,人类疾病往往与基因组的稳定性密切相关[1-3],而基因组的稳定涉及到DNA复制、修复、端粒维持及线粒体DNA稳定等多个环节[4-7],因此,在这些环节中起关键性作用的分子成为当今医学分子生物学研究的热点。在这些分子中,RECQ解旋酶家族成员因以多种方式参与了维持基因组的稳定而备受关注[8-12]。
人类RECQ解旋酶家族由5个成员组成,即RECQL1、RECQL2(BLM)、RECQL3(WRN)、RECQL4和RECQL5。其中BLM、WRN和RECQL4的变异被证明与一系列基因组异常的疾病有关,而RECQL1和RECQL5目前并没有相应的发现。另外,在导致疾病的3个解旋酶中,BLM和WRN的变异均分别导致一种疾病,而RECQL4被发现与至少3种基因组紊乱疾病如RTS(Rothmund-Thomson syndrome)、RAPA(Rapadilino syndrome)和BGS(Baller-Gerold syndrome)等均有关的蛋白,且这些疾病的症状不尽相同[13-16],因此,揭示RECQL4参与维持基因组稳定的机制可能更具有代表意义。
结合之前对RECQ解旋酶家族成员功能的了解[17,18]及最新的研究动态,本文对RECQL4在维持基因组稳定中发挥的作用进行了综述,并探讨了进一步研究的方向。
1 RECQL4在DNA复制中的作用
最初认为RECQL4可能参与DNA复制的是Sangrithi小组,该小组受非洲爪蟾(Xenopus laevis)xRTS蛋白(RECQL4同源产物)N末端与出芽酵母中参与复制叉形成的SLD2因子存在同源结构的启发,发现xRTS促进DNA聚合酶α结合到染色体上进而参与了爪蟾卵母细胞DNA复制的起始[19,20]。后来,从事哺乳类研究的Hoki等[21]观察到RECQL4缺陷小鼠会出现发育阻滞和皮肤畸形生长,证实RECQL4与DNA复制和细胞分裂关系密切。
真核细胞DNA复制的G1期,复制前复合体(Pre-ORC)的各组分会被逐步装载到复制起始位点,之后在G1/S期转换阶段, CDC45、MCM2-7、GINS在磷酸激酶CDK和DDK的作用下组装成CMG复合体,进而启动DNA复制[22-25]。2009年Im等[25]利用双分子荧光互补技术证明了CMG复合体结合到染色质的能力在RECQL4删除后减弱。同时,Xu等[26]发现了MCM10在G1/S和S期通过磷酸化RECQL4的N末端的前200个氨基酸来调节其与MCM2-7间的相互作用。该两项研究很好地解释了RECQL4在复制起始中的具体作用。
此外,Xu等[26]也认为RECQL4在完成复制起始后会与TIM/TIPIN互作,进一步促进复制的延伸。之后,Abe等[27]发现在RECQL4删除的DT40细胞中表达RECQL4的N末端496个氨基酸可使细胞恢复生长能力;Ohlenschlager等[28]通过酵母双杂交和GST沉淀技术证实了RECQL4通过其N末端54个氨基酸与TopBP1互作,而后者被多次证实参与了DNA复制的起始[29,30]。以上研究均表明RECQL4参与了DNA复制,对基因组的稳定遗传十分重要。
2 RECQL4在DNA损伤修复中的作用
众所周知,机体在受到外部刺激(如紫外线、过氧化物)时,会造成DNA损伤,而细胞在进化过程中产生了一系列的修复方式对DNA损伤进行修复。对RTS患者(RECQL4变异)细胞的研究发现,其对多种 DNA 损伤试剂均表现出脆弱的抵抗能力[31-33],这暗示RECQL4可能在DNA损伤修复中发挥着重要作用。
2005年Petkovic等[34]在使用不同的双链断裂试剂处理细胞后,通过免疫荧光染色发现RECQL4会与RAD51互作,定位在双链断裂处,而后者已被证实在DNA双链断裂修复中发挥重要的作用[35]。次年,Werner等[36]发现在人成纤维细胞里,RECQL4主要分布于细胞质中,但用H2O2处理过后,RECQL4会转运到细胞核内以应对氧化剂引发的DNA损伤。同时,Woo等[37]的试验使用T7噬菌体展示技术发现RECQL4通过C末端与DNA修复酶PARP1(Poly ADP-ribose polymerase-1)互作,参与由γ射线造成的DNA损伤应答。之后,Fan等[38]发现细胞在清除紫外线引发的变异时,RECQL4能够直接与XPA(Xeroderma pigmentosum group A)互作,参与核酸切除修复。接着,Schurman等[39]发现RECQL4通过与APE1(Apurinic endonuclease 1)的N端相互作用激活其外切酶活性,同时提高FEN1(Fflap endonuclease 1)的切割能力,进而参与到细胞碱基切除修复的过程中。2012年Kohzaki等[40]又发现细胞在缺少RECQL4整个C末端的情况下,受γ射线的刺激会导致DNA复制的提前终止和复制叉的停滞, Singh等[41]发现了RECQL4能够促进BLM在DNA损伤处的解旋酶活性。上述证据均表明,RECQL4在DNA修复中起着不可或缺的作用。
3 RECQL4在端粒维持中的作用
端粒是位于染色体末端的核苷酸序列[42],其功能是保护染色体在复制时不会缩短,因此端粒也是维持基因组稳定性的重要因素之一[43,44]。Ghosh等[6]最近研究发现,RECQL4可能参与了端粒的维持。
Ghosh等[6]观察到,RTS病人细胞端粒末端存在易断位点的水平较高,且RECQL4沉默细胞的端粒处会发生更多的姐妹染色体交换、积累更多的易断位点、更容易出现双链断裂。该研究小组也发现,在骨肉瘤细胞U2OS的RECQL4删除后,DNA损伤应答蛋白53BP1会定位于端粒处并被激活。体外酶活检测试验发现,分别在加入10 nmol/L和20 nmol/L的TRF2(Telomere repeat-binding factors 2)后,RECQL4解旋端粒D-loop结构的能力可分别被提高1.75倍和2.00倍,而TRF2单独则不能解旋D-loop结构。同样的方法,该研究小组又发现RECQL4可以提高WRN解旋D-loop的活性,而解旋D-loop结构是端粒复制和修复的前提。该研究小组还应用免疫共沉淀的方法进一步证明RECQL4分别与TRF2和WRN存在直接相互作用。
此外,Bodelon等[45]也通过SNP分析发现,地中海人群的黑色素瘤症状与RECQL4有关,而端粒的缩短被证明与黑素瘤有关[46],这也佐证了RECQL4在端粒维持中发挥着重要作用。
4 RECQL4在线粒体DNA维持中的作用
线粒体DNA作为人类基因组的重要部分,其变异会导致线粒体的功能紊乱。而线粒体作为细胞内ATP的主要来源, DNA的复制和修复等需要ATP参与。因此,与线粒体DNA稳定性相关的分子也成为近年来基因组不稳定性疾病研究的热点[47]。
2009年Jiang等[48]通过蛋白质组学的分析发现RECQL4在细胞受到辐射损伤后定位于线粒体中。2012年Chi等[7]发现在RECQL4的C末端有两个细胞质定位序列PNES2和PNES3,并且用免疫荧光定位的方法证明了RECQL4存在于线粒体中。同年Croteau等[49]使用不同的RECQL4抗体,通过免疫荧光发现RECQL4存在于U2OS细胞和HeLa细胞的线粒体中,为了进一步印证这一试验结果,该研究小组又分离出人SH-SY5Y细胞的线粒体,然后用RECQL4抗体免疫染色,并获得了阳性结果,这充分说明了RECQL4会定位于线粒体中。
为了研究RECQL4在线粒体中的功能,Croteau等[49]使用RECQL4敲除的细胞作为研究对象,发现RECQL4缺陷细胞从缺氧环境转入有氧环境后,对氧气的需求量明显降低,同时RTS患者的细胞也表现出线粒体功能的紊乱。Chi等发现HEK293细胞内过量表达RECQL4可以提高线粒体DNA的拷贝数,相反,使用shRNA抑制RECQL4在U2OS细胞内(该细胞RECQL4内源性表达量相对较高)的表达则会导致线粒体DNA拷贝数下降,此外也发现U2OS细胞受到过氧化损伤后,其线粒体中RECQL4的量是对照组的5.4倍[7],而RECQL4已被证明当核DNA损伤时会向核内大量转运参与核DNA的修复[36]。De等[50]发现,在正常细胞中,RECQL4与P53互作,协助后者进入线粒体,而后者被证实参与了维持线粒体DNA准确复制和修复的过程[51-53]。以上试验结果均表明,RECQL4可能对线粒体DNA的稳定具有重要作用。
5 结语与展望
综上所述,RECQL4不仅参与了核DNA的复制和修复,还可能参与了端粒的维持和线粒体DNA的稳定,在整个基因组稳定性方面发挥着十分重要的作用。恰恰也正因为RECQL4的功能如此复杂,其成为当前基因组稳定性维持及与此相关的人类遗传病、早衰和癌症等研究领域的热门话题[54-56]。
然而,在DNA复制中SLD2结构域和解旋酶结构域是怎样协调起来的还需要进一步阐释[57,58]。而最新的研究发现,RECQL4是RECQ解旋酶家族中惟一一个被发现可定位于线粒体的成员,并极有可能参与了线粒体DNA的复制与损伤修复,那么其在核DNA复制与线粒体DNA复制中发挥作用的机理又是否相同,另外,之前的研究发现RECQL5-/-BLM-/-细胞比BLM-/-细胞有着更高的SCE(Sister chromatid exchange)[17,18],表明解旋酶家族蛋白之间在某些功能上可能有协同性,而RECQL4与家族其他蛋白之间关系的研究鲜有报道。这一系列问题都将是未来有关RECQL4研究的重要内容,相信这些问题的阐明不仅有助于解释基因组不稳定性疾病的发病原因,也可能使人们对RTS并发的早衰和癌症等难以攻克的医学课题有了新的认识,并会找到较好的治疗手段。
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