介导BLM解旋酶细胞核定位的关键氨基酸位点鉴定

BLM解旋酶是人RecQ DNA解旋酶家族重要成员之一,在机体的DNA复制、重组、损伤修复以及维护基因组稳定性等方面发挥重要作用。早期研究表明,BLM解旋酶通过自身携带的核定位信号（nuclear localization signal, NLS）进入细胞核,但是介导其细胞核定位的关键氨基酸位点尚不清楚。本研究构建了BLM解旋酶C端(aa642 1417)截短体克隆,首先通过截短表达的方法确证其NLS结构域。在此基础上,构建重组真核表达载体pEGFP NLS/BLM NES/Rev,通过观察BLM NLS碱性氨基酸位点突变对EGFP NLS/ BLM NES/Rev融合蛋白细胞核定位的影响,以此快速鉴定NLS中介导BLM解旋酶细胞核定位的关键氨基酸位点。结果表明,BLM(aa642 1417) C端截短体具有与全长BLM解旋酶相同的细胞核定位,同时确证1344RSKRRK1349是BLM解旋酶NLS结构域的活性位点,且具有与SV40 NLS相同的核输入能力。氨基酸位点突变试验结果表明,R1344A、K1346A、R1348A和K1349A点突变均减少了EGFP NLS/BLM NES/Rev和EGFP BLM(642 1417)融合蛋白的细胞核定位。因此,这4个位点是介导BLM解旋酶细胞核定位的关键氨基酸位点。此结果为后续研究BLM解旋酶细胞核定位的分子机制奠定了基础。     

SIRT3与细胞代谢及心血管疾病的相关性

蛋白去乙酰化酶在细胞生理过程中发挥着极为重要的作用。人蛋白去乙酰化酶包括HDACⅠ、HDACⅡ、HDACⅢ和HDACⅣ4个家族。其中第Ⅲ类即Sir2(Silent information regulator 2)家族包括7个成员——SIRT1~ SIRT7,每个成员都具有不同的细胞定位,并且发挥不同的生物学功能。作为主要定位于线粒体的组蛋白去乙酰化酶,SIRT3不仅调节细胞的能量代谢,并在细胞凋亡、肿瘤生长和一些疾病中发挥作用。文章综述了SIRT3在细胞代谢中的生物学功能以及其在心血管疾病中的研究进展。     

新基因水稻 OsLSD1 的克隆及拟南芥和水稻类 LSD1 基因家族的生物信息学分析

类 LSD1 (LSD1-like) 基因家族是一类特殊的 C2C2 型锌指蛋白基因,编码植物特有的转录因子 . 目前已经研究的 2 个成员拟南芥 LSD1 (lesions stimulating disease resistance 1) 和 LOL1 (LSD-One-Like 1) 基因均参与植物细胞程序化死亡 (programmed cell death, PCD) 的调控 . 从水稻 cDNA 文库中克隆到 1 个类 LSD1 基因,命名为 OsLSD1. 该基因长 988 bp ,包含一个 432 bp 的开放阅读框,推导的氨基酸序列 (143 个氨基酸 ) 含有 3 个内部保守的锌指结构域 . DNA 印迹结果表明 OsLSD1 基因在水稻基因组中为单拷贝,且在根、茎和叶中表达 . 借助于生物信息学分析技术,从拟南芥和水稻数据库中各识别出 5 个和 7 个 ( 包括 OsLSD1) 类 LSD1 基因 . 分析了这些类 LSD1 基因的结构,蛋白质结构域组成 . 系统进化分析表明,无论基于编码区的核苷酸或氨基酸序列都可以将这些类 LSD1 基因分为 2 类 . 虽然不存在拟南芥或水稻特有的类 LSD1 蛋白,但有些结构域是水稻所特有的,也有些基因是来源于复制事件 .     

新基因水稻OsLSD1的克隆及拟南芥和水稻类 LSD1基因家族的生物信息学分析

类LSD1 (LSD1-like)基因家族是一类特殊的C2C2型锌指蛋白基因,编码植物特有的转录因子.目前已经研究的2个成员拟南芥LSD1(1esions stimulating disease resistance 1)和LOL1(LSD-One-Like 1)基因均参与植物细胞程序化死亡(programmed cell death,PCD)的调控.从水稻cDNA文库中克隆到1个类LSD1基因,命名为OsLSD1.该基因长988 bp,包含一个432bp的开放阅读框,推导的氨基酸序列(143个氨基酸)含有3个内部保守的锌指结构域.DNA印迹结果表明OsLSD1基因在水稻基因组中为单拷贝,且在根、茎和叶中表达.借助于生物信息学分析技术,从拟南芥和水稻数据库中各识别出5个和7个(包括OsLSD1)类LSD1基因.分析了这些类LSD1基因的结构,蛋白质结构域组成.系统进化分析表明,无论基于编码区的核苷酸或氨基酸序列都可以将这些类LSD1基因分为2类.虽然不存在拟南芥或水稻特有的类LSD1蛋白,但有些结构域是水稻所特有的,也有些基因是来源于复制事件.     

Oct-4/Sox-2 协同调控下游基因表达的分子机制

Oct-4属POU家族蛋白,是一类在动物早期胚胎发育过程中起重要作用的转录因子,参与维持细胞的全能性及未分化状态。Oct-4蛋白的主要结构特征为具有POU家族特有的保守结构域（POUS）和POU同源异型结构域（POUHD）,这两个结构域可与DNA上特定区域形成双向结合,进而对基因转录进行调控。Sox-2是另一种转录因子,其HMG结构域可结合在DNA的特定序列上,并可通过与Oct-4的POUs结构域之间的蛋白质．蛋白质相互作用形成POU／HMG／DNA三元复合体以调控下游靶基因的表达。文章就POU家族成员Oct-4和HMG-box家族成员Sox-2在动物早期胚胎发育中调控部分下游基因表达的分子机制进行了概述。     

蕨类植物问荆DEAD-box RNA解旋酶基因的克隆与分析

DEAD-box RNA解旋酶参与RNA多方面的代谢,在植物生长发育和逆境反应中起重要作用。本研究从蕨类植物问荆（Equisetum arvense）中克隆到一条DEAD-box RNA解旋酶c DNA全长序列,命名为EaRH1,并在Gen Bank注册登记（KJ734026）。序列分析显示：该c DNA全长3 230 bp,包含一个从487 bp到2 799 bp编码770个氨基酸的开放读码框,其对应的蛋白序列包含9个保守模块结构。EaRH1与其它物种DEAD-box RNA解旋酶蛋白序列比对结果显示：模块Ⅰa、Ⅱ和Ⅲ序列几乎完全相同,模块Q、Ⅰ和Ⅳ序列存在一些差异。EaRH1与江南卷柏（Selaginella moellendorffii）基因组一条假定序列相似度高达69%,其中相似度最高的区域集中在包含9个保守模块的结构域。系统进化树分析显示：EaRH1与拟南芥（Arabidopsis thaliana）DEAD-box RNA解旋酶At3g22320在氨基酸序列上有相对较高的同源性。序列结构比较和进化分析可推测出EaRH1可能参与植物体生长发育、miRNA生物合成、与RNA结合蛋白的相互作用和非生物胁迫应答。本文的研究为探索问荆DEAD-box RNA解旋酶的进一步功能提供参考。     

Y-box结合蛋白功能及对肿瘤发生的影响

Y-box结合蛋白家族成员是一类高度保守的顺式作用元件, 广泛存在于原核及真核生物细胞中。它是一种多功能蛋白, 与转录调节、翻译调控、mRNA选择性剪接、DNA的修复、细胞增殖和再生等有关。Y-box结合蛋白的氨基酸序列包含3个结构域: 氨基酸N末端, 亲水结构域C末端, 冷休克结构域(cold shock domain CSD), 保守的冷休克结构域决定了Y-box结合蛋白的大部分功能。最近研究发现, 定位于细胞核中的YB-1蛋白在局部晚期非小细胞肺癌的预防上可作为新的靶位点, YB-1蛋白还可通过对抑癌基因p53启动子抑制起负调控作用, 此外, YB-1蛋白在PI3K/Akt信号通路中也起到重要的作用, 这些研究都为肿瘤的治疗提供了新的线索和启示。文章就Y-box结合蛋白功能及其对肿瘤发生的影响等方面进行概述。     

真核生物中锌指蛋白的结构与功能

真核生物中的许多蛋白质分子包含锌指结构区,这类蛋白称为锌指蛋白.锌指蛋白因其包含特殊的指状结构,在对DNA、蛋白质和RNA的识别和结合中起重要作用.许多锌指蛋白的锌指结构域包含能与DNA特异结合的区域,并与某些效应结构域（如KRAB、SCAN、BTB/POZ、SNAG、SANT和PLAG等）相连,这类锌指蛋白常作为转录因子起作用,可调控靶基因的转录.一些锌指蛋白包含蛋白质识别结构域（如LIM锌指、MYND锌指、PHD锌指和RING锌指等）,它们能够特异地介导蛋白质之间的相互作用,因此被称作蛋白适配器.此外,某些锌指蛋白还可以结合RNA,起转录后调控作用.本文就锌指蛋白与DNA、RNA以及蛋白质分子间的相互作用作一综述.     

酿酒酵母DNA复制的精确时空控制

DNA复制是最基本的生命活动之一。DNA复制本身的错误及其过程控制的异常是细胞内基因组不稳定的主要来源,会导致细胞生长异常、衰老、癌变乃至死亡。为了保证基因组DNA能够精确且完整的复制,DNA复制受到严格的调控。在G1期,DNA复制解旋酶的核心组分Mcm2-7复合体被招募到复制起点,获得复制许可资格。进入S期后,在两个周期性蛋白激酶及多个支架蛋白的作用下,复制解旋酶的激活因子Cdc45和GINS复合体被招募至Mcm2-7,形成解旋酶全酶Cdc45-Mcm2-7-GINS (CMG)复合体。随后,众多复制相关蛋白在精准的时空控制下被招募至CMG平台并组装成复制机器,起始DNA双向复制。当相向而行的两个复制叉相遇,复制机器会从DNA链上解离下来,从而完成DNA复制。关于DNA复制过程的研究在近十年来取得了跨越式的突破。本文以酿酒酵母为例,围绕所有真核生物中都高度保守的DNA复制控制开关——CMG解旋酶,对真核生物DNA复制的最新进展进行综述。     

SUN结构域家族蛋白研究进展

SUN(Sad-1,UNC-84)结构域家族蛋白是一种广泛分布于酵母、线虫等真核生物的膜蛋白,主要定位于细胞核膜以及内质网。由于在酵母中发现Sad-1突变后的表型与线虫中UNC-84突变的表型一致,并且两者C-端有近一半的同源相似性,因此得名SUN结构域,其家族成员也都具有SUN结构域。根据SUN结构域所在家族成员中蛋白质一级序列位置的不同,分为经典及非经典家族蛋白,经典的家族蛋白一般通过与KASH(Klarsicht、ANC-1、Syne homology)蛋白相互作用行使功能。越来越多的研究结果表明,SUN蛋白可能参与核膜锚定、核膜重塑、细胞迁移和DNA损伤修复等过程,其形成的复合体与人类进行性肌营养不良等疾病的发生发展也有紧密联系。该文就SUN家族各成员蛋白的结构特性以及功能特点的研究进展进行简要综述。     

Two steps forward,one step back: Determining XPD helicase mechanism by single-molecule fluorescence and high-resolution optical tweezers

XPD-like helicases constitute a prominent DNA helicase family critical for many aspects of genome maintenance. These enzymes share a unique structural feature, an auxiliary domain stabilized by an iron-sulphur (FeS) cluster, and a 5′–3′ polarity of DNA translocation and duplex unwinding. Biochemical analyses alongside two single-molecule approaches, total internal reflection fluorescence microscopy and high-resolution optical tweezers, have shown how the unique structural features of XPD helicase and its specific patterns of substrate interactions tune the helicase for its specific cellular function and shape its molecular mechanism. The FeS domain forms a duplex separation wedge and contributes to an extended DNA binding site. Interactions within this site position the helicase in an orientation to unwind the duplex, control the helicase rate, and verify the integrity of the translocating strand. Consistent with its cellular role, processivity of XPD is limited and is defined by an idiosyncratic stepping kinetics. DNA duplex separation occurs in single base pair steps punctuated by frequent backward steps and conformational rearrangements of the protein–DNA complex. As such, the helicase in isolation mainly stabilizes spontaneous base pair opening and exhibits a limited ability to unwind stable DNA duplexes. The presence of a cognate ssDNA binding protein converts XPD into a vigorous helicase by destabilizing the upstream dsDNA as well as by trapping the unwound strands. Remarkably, the two proteins can co-exist on the same DNA strand without competing for binding. The current model of the XPD unwinding mechanism will be discussed along with possible modifications to this mechanism by the helicase interacting partners and unique features of such bio-medically important XPD-like helicases as FANCJ (BACH1), RTEL1 and CHLR1 (DDX11).     

FANCJ helicase defective in Fanconia anemia and breast cancer unwinds G-quadruplex DNA to defend genomic stability

FANCJ mutations are associated with breast cancer and genetically linked to the bone marrow disease Fanconi anemia (FA). The genomic instability of FA-J mutant cells suggests that FANCJ helicase functions in the replicational stress response. A putative helicase with sequence similarity to FANCJ in Caenorhabditis elegans (DOG-1) and mouse (RTEL) is required for poly(G) tract maintenance, suggesting its involvement in the resolution of alternate DNA structures that impede replication. Under physiological conditions, guanine-rich sequences spontaneously assemble into four-stranded structures (G quadruplexes [G4]) that influence genomic stability. FANCJ unwound G4 DNA substrates in an ATPase-dependent manner. FANCJ G4 unwinding is specific since another superfamily 2 helicase, RECQ1, failed to unwind all G4 substrates tested under conditions in which the helicase unwound duplex DNA. Replication protein A stimulated FANCJ G4 unwinding, whereas the mismatch repair complex MSH2/MSH6 inhibited this activity. FANCJ-depleted cells treated with the G4-interactive compound telomestatin displayed impaired proliferation and elevated levels of apoptosis and DNA damage compared to small interfering RNA control cells, suggesting that G4 DNA is a physiological substrate of FANCJ. Although the FA pathway has been classically described in terms of interstrand cross-link (ICL) repair, the cellular defects associated with FANCJ mutation extend beyond the reduced ability to repair ICLs and involve other types of DNA structural roadblocks to replication.     

Warsaw Breakage Syndrome, a Cohesinopathy Associated with Mutations in the XPD Helicase Family Member DDX11/ChlR1

The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.     

Insight into the Roles of Helicase Motif Ia by Characterizing Fanconi Anemia Group J Protein (FANCJ) Patient Mutations

Helicases are molecular motors that couple the energy of ATP hydrolysis to the unwinding and remodeling of structured DNA or RNA, which is coordinated by conserved helicase motifs. FANCJ is a DNA helicase that is genetically linked to Fanconi anemia, breast cancer, and ovarian cancer. Here, we characterized two Fanconi anemia patient mutations, R251C and Q255H, that are localized in helicase motif Ia. Our genetic complementation analysis revealed that both the R251C and Q255H alleles failed to rescue cisplatin sensitivity of a FANCJ null cell line as detected by cell survival or γ-H2AX foci formation. Furthermore, our biochemical assays demonstrated that both purified recombinant proteins abolished DNA helicase activity and failed to disrupt the DNA-protein complex. Intriguingly, R251C impaired DNA binding ability to single-strand DNA and double-strand DNA, whereas Q255H retained higher binding activity to these DNA substrates compared with wild-type FANCJ protein. Consequently, R251C abolished its DNA-dependent ATP hydrolysis activity, whereas Q255H retained normal ATPase activity. Physically, R251C had reduced ATP binding ability, whereas Q255H had normal ATP binding ability and could translocate on single-strand DNA. Although both proteins were recruited to damage sites in our laser-activated confocal assays, they lost their DNA repair function, which explains why they exerted a domain negative effect when expressed in a wild-type background. Taken together, our work not only reveals the structural function of helicase motif Ia but also provides the molecular pathology of FANCJ in related diseases.     

Novel Function of the Fanconi Anemia Group J or RECQ1 Helicase to Disrupt Protein-DNA Complexes in a Replication Protein A-stimulated Manner

Understanding how cellular machinery deals with chromosomal genome complexity is an important question because protein bound to DNA may affect various cellular processes of nucleic acid metabolism. DNA helicases are at the forefront of such processes, yet there is only limited knowledge how they remodel protein-DNA complexes and how these mechanisms are regulated. We have determined that representative human RecQ and Fe-S cluster DNA helicases are potently blocked by a protein-DNA interaction. The Fanconi anemia group J (FANCJ) helicase partners with the single-stranded DNA-binding protein replication protein A (RPA) to displace BamHI-E111A bound to duplex DNA in a specific manner. Protein displacement was dependent on the ATPase-driven function of the helicase and unique properties of RPA. Further biochemical studies demonstrated that the shelterin proteins TRF1 and TRF2, which preferentially bind the telomeric repeat found at chromosome ends, effectively block FANCJ from unwinding the forked duplex telomeric substrate. RPA, but not the Escherichia coli single-stranded DNA-binding protein or shelterin factor Pot1, stimulated FANCJ ejection of TRF1 from the telomeric DNA substrate. FANCJ was also able to displace TRF2 from the telomeric substrate in an RPA-dependent manner. The stimulation of helicase-catalyzed protein displacement is also observed with the DNA helicase RECQ1, suggesting a conserved functional interaction of RPA-interacting helicases. These findings suggest that partnerships between RPA and interacting human DNA helicases may greatly enhance their ability to dislodge proteins bound to duplex DNA, an activity that is likely to be highly relevant to their biological roles in DNA metabolism.