泉古菌Sulfolobus tokodaii RadA同系物stRad55B与RadA的共表达及其对RadA重组活性的抑制

ST0838（定义为stRad55B）是超嗜热古菌（Sulfolobus tokodaii）编码的4个RadA的同系物（或Rad55同源蛋白）之一．研究发现,它能够被紫外线（UV）辐射损伤诱导,可能参与了细胞内的DNA损伤修复过程,然而利用常规方法,该蛋白不能体外可溶性地表达．通过和RadA共表达,得到了具有热稳定性的可溶stRad55B蛋白,并对其活性进行了初步检测．stRad55B优先结合单链DNA,并且具有不依赖DNA的ATP酶活性．另外,DNA链交换实验发现stRad55B能够明显抑制RadA催化的链重组活性,表现出一个重组修复系统抑制蛋白的特征．实验结果为进一步研究古菌中RadA同系蛋白的功能以及相互作用机制,揭示古菌DNA同源重组修复机理提供了依据．     

极端嗜盐古菌蛋白类抗生素——嗜盐菌素

古菌(Archaea)是一类与细菌及真核生物显著不同的生命的第三种形式[1],大多生活在极端或特殊环境,主要包括产甲烷古菌(Methanogenic Achaea)、极端嗜盐古菌(Extremely Halophilic Archaea)和极端嗜热古菌(Extremely Thermophilic Archaea)等三大类.极端古菌是极端环境微生物的重要成员,也是极端环境微生物资源开发的重要领域.其中,嗜盐古菌可产生一类蛋白类抗生素,称为嗜盐菌素(halocin).     

极端嗜热古菌的热休克蛋白

随着生物工程产业对于耐高温酶和菌体的需求, 极端嗜热古菌热休克蛋白(heat shock proteins, HSPs)的研究更受重视, 其热休克蛋白体系非常简洁, 不含HSP100s和HSP90s, 就是HSP70(DnaK)、HSP40、(DnaJ)和GrpE等嗜温古菌可能含有的在极端嗜热古菌中几乎不含有, 即仅包括HSP60, sHSP, prefoldin和AAA+蛋白四大类, 因此对其结构、功能和作用机制的研究在理论和实践上都特别有意义。系统地介绍了这四大类组分的结构、功能和作用机制和协同作用的研究进展, 论述了极端嗜热古菌热休克蛋白的系列研究难点和困惑, 展望了进一步的研究方向和重点。     

极端嗜热古菌DNA修复核酸内切酶的研究进展

极端嗜热古菌由于生活在高温环境,其基因组DNA面临着严重的挑战,因此,它们如何维持其基因组稳定是本研究领域最为关注的科学问题之一。极端嗜热古菌具有与常温微生物相似的自发突变频率,暗示着它们比常温微生物具有更加有效的DNA修复体系进行修复高温所造成的基因组DNA损伤。目前,极端嗜热古菌DNA修复的分子机制尚不清楚。核酸内切酶在DNA修复途径中发挥着重要的作用。基因组序列显示极端嗜热古菌编码多种DNA修复核酸内切酶,但是其研究尚处于初期阶段。本文综述了极端嗜热古菌DNA修复核酸内切酶Nuc S、Endo V、Endo Q、XPF和Hjc的研究进展,并对今后的研究提出了展望。     

极端嗜盐古菌蛋白类抗生素——嗜盐菌素

古菌 (Ａｒｃｈａｅａ)是一类与细菌及真核生物显著不同的生命的第三种形式[1] ,大多生活在极端或特殊环境 ,主要包括产甲烷古菌 (ＭｅｔｈａｎｏｇｅｎｉｃＡｃｈａｅａ)、极端嗜盐古菌 (ＥｘｔｒｅｍｅｌｙＨａｌｏｐｈｉｌｉｃＡｒｃｈａｅａ)和极端嗜热古菌 (ＥｘｔｒｅｍｅｌｙＴｈｅｒｍｏｐｈｉｌｉｃＡｒｃｈａｅａ)等三大类。极端古菌是极端环境微生物的重要成员 ,也是极端环境微生物资源开发的重要领域。其中 ,嗜盐古菌可产生一类蛋白类抗生素 ,称为嗜盐菌素 (ｈａｌｏｃｉｎ)。与细菌素相似[2 ] ,嗜盐菌素是由质粒编码、核糖体合…     

极端嗜热古菌——芝田硫化叶菌DNA结合蛋白Ssh12对DNA超螺旋的影响

通过SPSepharose,DNA纤维素和磷酸纤维素等柱层析 ,从极端嗜热古菌———芝田硫化叶菌 (Sulfolobusshibatae)中纯化得到分子量为 11.5ku的DNA结合蛋白Ssh12 .Ssh12约占细胞总蛋白的 4% .该蛋白既能与负超螺旋DNA也能与松弛DNA结合 .利用含单切刻环状DNA进行的切刻闭合分析表明 ,Ssh12在与DNA结合时能够固定负超螺旋 .这种能力在室温 ( 2 2℃ )下很弱 ,而在 3 7℃以上则大大增强 .Ssh12的细胞内含量和固定负超螺旋的能力提示 ,该蛋白对于芝田硫化叶菌染色体DNA的组织以及热稳定性起着重要作用 .     

极端嗜热古菌尿嘧啶DNA糖苷酶的研究进展

高温会加快碱基脱氨基反应形成损伤碱基的速率,进一步对脱氨基的碱基进行复制会导致突变。因此,极端嗜热古菌基因组的稳定性面临着其生存高温环境的挑战。胞嘧啶脱氨基形成尿嘧啶,是常见的脱碱基类型,复制DNA中尿嘧啶会造成GC→AT的突变。尿嘧啶DNA糖苷酶(Uracil DNA glycosylase,UDG)是修复DNA中尿嘧啶的关键酶。基于识别底物的特异性,UDG分为6个家族,广泛分布在细菌、古菌、真核生物以及一些病毒中。基因组序列显示,极端嗜热古菌至少编码一种UDG。目前,对于细菌和真核生物的UDG已进行了大量的研究,但是关于极端嗜热古菌UDG的研究相对较少,尚处于初期阶段。本文综述了极端嗜热古菌UDG的研究进展,并对今后的研究提出了展望。     

嗜酸嗜热古菌Sulfolobus acidocaldarius编码尿嘧啶DNA糖苷酶表达，纯化与酶学特征

摘要:【目的】嗜高温微生物面临dC脱氨基生成dU损伤的巨大压力,鉴定嗜酸嗜热古菌S.acidocaldarius来源的尿嘧啶DNA糖苷酶(UDG)切除dU损伤的酶学活性。【方法】重组表达来源于S.acidocaldarius的IV和V型UDG,经亲和纯化得到电泳纯重组蛋白。然后利用人工合成的dU(deoxyuracil)修饰寡核苷酸片段作为底物,体外鉴定两种重组UDG 的酶学特性。【结果】来源于S.acidocaldarius的IV和V型重组UDG具有相似的酶学特性。IV型UDG催化效率更高,比活性是V型重组UDG的750倍左右。作为来自嗜热微生物 的蛋白,S.acidocaldarius的IV和V型UDG的最适反应温度为65－75℃。【结论】IV型UDG比V型UDG水解dU碱基和脱氧核糖之间糖苷键的能力更强。     

Ssh10b2与其同源蛋白Ssh10b在细胞含量及固定DNA超螺旋的能力上存在明显差异

极端嗜热古菌———芝田硫化叶菌(Sulfolobus shibatae)基因组含一对亲缘关系较远的同源基因,ssh10b和ssh10b2。这对同源基因编码的蛋白(Ssh10b和Ssh10b2)属于古菌Sac10b DNA结合蛋白家族。关于Ssh10b以及与其极为相似的硫矿硫化叶菌(S.solfataricus)Sso10b、嗜酸热硫化叶菌(S.acidocaldarius)Sac10b蛋白已有较多研究,推测这些蛋白可能在染色体组织和包装、DNA重组、基因表达调控等方面起作用。克隆并在大肠杆菌中表达了ssh10b2基因,纯化了重组Ssh10b2蛋白。免疫印迹定量分析表明,ssh10b2在芝田硫化叶菌中有表达,但其细胞含量仅相当于Ssh10b的约十分之一。重组Ssh10b2对双链DNA的亲和力低于Ssh10b。此外,Ssh10b2和Ssh10b在与双链DNA结合时表现出相似的凝胶阻滞模式。有意思的是,Ssh10b2固定DNA负超螺旋的能力明显低于Ssh10b。这些结果提示,Ssh10b和Ssh10b2可能具有不同的生理作用。     

Co-expression with RadA and the characterization of stRad55B, a RadA paralog from the hyperthermophilic crenarchaea Sulfolobus tokodaii

ST0838 (designed stRad55B) is one of the four RadA paralogs (or Rad55 homologues) in the genome of the hyperthermophilic crenarchaeon Sulfolobus tokodaii. The gene is induced by UV irradiation, suggesting that it is involved in DNA recombinational repair in this organism. However, this protein could not be expressed normally in vitro. In this study, thermostable and soluble stRad55B was obtained by co-expression with S. tokodaii RadA (stRadA) in E. coli, and the enzymatic properties were examined. It was found that stRad55B bound ssDNA preferentially and had a very weak ATPase activity that was not stimulated by DNA. The recombinant protein inhibited the strand exchange activity promoted by stRadA, indicating that stRad55B might be an inhibitor to the homologous recombination in this archaeon. The results will be helpful for further functional and interaction analysis of RadA paralogs and for the understanding of the mechanism of recombinational repair in archaea.     

Structural and Functional Characterisation of a Conserved Archaeal RadA Paralog with Antirecombinase Activity

DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.     

RadA protein from Archaeoglobus fulgidus forms rings, nucleoprotein filaments and catalyses homologous recombination

Proteins that catalyse homologous recombination have been identified in all living organisms and are essential for the repair of damaged DNA as well as for the generation of genetic diversity. In bacteria homologous recombination is performed by the RecA protein, whereas in the eukarya a related protein called Rad51 is required to catalyse recombination and repair. More recently, archaeal homologues of RecA/Rad51 (RadA) have been identified and isolated. In this work we have cloned and purified the RadA protein from the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus and characterised its in vitro activities. We show that (i) RadA protein forms ring structures in solution and binds single- but not double-stranded DNA to form nucleoprotein filaments, (ii) RadA is a single-stranded DNA-dependent ATPase at elevated temperatures, and (iii) RadA catalyses efficient D-loop formation and strand exchange at temperatures of 60–70°C. Finally, we have used electron microscopy to visualise RadA-mediated joint molecules, the intermediates of homologous recombination. Intriguingly, RadA shares properties of both the bacterial RecA and eukaryotic Rad51 recombinases.     

Interactions of RadB, a DNA repair protein in archaea, with DNA and ATP

The RecA family of recombinases (RecA, Rad51, RadA and UvsX) catalyse strand-exchange between homologous DNA molecules by utilising conserved DNA-binding modules and a common core ATPase domain. RadB was identified in archaea as a Rad51-like protein on the basis of conserved ATPase sequences. However, RadB does not catalyse strand exchange and does not turn over ATP efficiently. RadB does bind DNA, and here we report a triplet of residues (Lys-His-Arg) that is highly conserved at the RadB C terminus, and is crucial for DNA binding. This is consistent with the motif forming a "basic patch" of highly conserved residues identified in an atomic structure of RadB from Thermococcus kodakaraensis. As the triplet motif is conserved at the C terminus of XRCC2 also, a mammalian Rad51-paralogue, we present a phylogenetic analysis that clarifies the relationship between RadB, Rad51-paralogues and recombinases. We investigate interactions between RadB and ATP using genetics and biochemistry; ATP binding by RadB is needed to promote survival of Haloferax volcanii after UV irradiation, and ATP, but not other NTPs, induces pronounced conformational change in RadB. This is the first genetic analysis of radB, and establishes its importance for maintaining genome stability in archaea. ATP-induced conformational change in RadB may explain previous reports that RadB controls Holliday junction resolution by Hjc, depending on the presence or the absence of ATP.     

Comparison of bacteriophage T4 UvsX and human Rad51 filaments suggests that RecA-like polymers may have evolved independently

The UvsX protein from bacteriophage T4 is a member of the RecA/Rad51/RadA family of recombinases active in homologous genetic recombination. Like RecA, Rad51 and RadA, UvsX forms helical filaments on DNA. We have used electron microscopy and a novel method for image analysis of helical filaments to show that UvsX-DNA filaments exist in two different conformations: an ADP state and an ATP state. As with RecA protein, these two states have a large difference in pitch. Remarkably, even though UvsX is only weakly homologous to RecA, both UvsX filament states are more similar to the RecA crystal structure than are RecA-DNA filaments. We use this similarity to fit the RecA crystal structure into the UvsX filament, and show that two of the three previously described blocks of similarity between UvsX and RecA are involved in the subunit-subunit interface in both the UvsX filament and the RecA crystal filament. Conversely, we show that human Rad51-DNA filaments have a different subunit-subunit interface than is present in the RecA crystal, and this interface involves two blocks of sequence similarity between Rad51 and RecA that do not overlap with those found between UvsX and RecA. This suggests that helical filaments in the RecA/Rad51/RadA family may have arisen from convergent evolution, with a conserved core structure that has assembled into multimeric filaments in a number of different ways.     

Domain analysis of an archaeal RadA protein for the strand exchange activity

Archaeal RadA, like eukaryotic Rad51 and bacterial RecA, promotes strand exchange between DNA strands with homologous sequences in vitro and is believed to participate in the homologous recombination in cells. The amino acid sequences of the archaeal RadA proteins are more similar to the eukaryotic Rad51s rather than the bacterial RecAs, and the N-terminal region containing domain I is conserved among the RadA and Rad51 proteins but is absent from RecA. To understand the structure-function relationship of RadA, we divided the RadA protein from Pyrococcus furiosus into two parts, the N-terminal one-third (RadA-n) and the residual C-terminal two-thirds (RadA-c), the latter of which contains the central core domain (domain II) of the RecA/Rad51 family proteins. RadA-c had the DNA-dependent ATPase activity and the strand exchange activity by itself, although much weaker (10%) than that of the intact RadA. These activities of RadA-c were restored to 60% of those of RadA by addition of RadA-n, indicating that the proper active structure of RadA was reconstituted in vitro. These results suggest that the basic activities of the RecA/Rad51 family proteins for homologous recombination are derived from domain II, and the N-terminal region may help to enhance the catalytic efficiencies.     

The Rad51/RadA N-terminal domain activates nucleoprotein filament ATPase activity

Proteins in the RecA/RadA/Rad51 family form helical filaments on DNA that function in homologous recombination. While these proteins all have the same highly conserved ATP binding core, the RadA/Rad51 proteins have an N-terminal domain that shows no homology with the C-terminal domain found in RecA. Both the Rad51 N-terminal and RecA C-terminal domains have been shown to bind DNA, but no role for these domains has been established. We show that RadA filaments can be trapped in either an inactive or active conformation with respect to the ATPase and that activation involves a large rotation of the subunit aided by the N-terminal domain. The G103E mutation within the yeast Rad51 N-terminal domain inactivates the filament by failing to make proper contacts between the N-terminal domain and the core. These results show that the N-terminal domains play a regulatory role in filament activation and highlight the modular architecture of the recombination proteins.     

A RecA / RAD51 homologue from a hyperthermophilic archaeon retains the major RecA domain only

 A gene encoding a RecA/RAD51 homologue from a hyperthermophilic archaeon, Pyrococcus sp. KOD1 (Pk), was cloned, sequenced and expressed in Escherichia coli. The deduced 210-amino acid sequence was compared to homologues from bacteria (RecA), eukaryotes (RAD51, DMC1) and archaea (RadA). The entire protein from Pk (Pk-REC) basically corresponds to the essential central domain of its counterparts and lacks the two smaller RecA subdomains at the N- and C-termini. The sequence comparison suggests that Pk-REC represents a common prototype of RecA, RAD51, DMC1 and RadA, with higher enzymatic activity. Recombinant Pk-REC was fully active and complemented the ultraviolet light sensitivity of an E. coli recA mutant strain. Received: 11 June 1996 / Accepted: August 31 1996     

Archaeal RecA homologues: different response to DNA-damaging agents in mesophilic and thermophilic Archaea

Two archaeal proteins, RadA and RadB, share similarity with the RecA/Rad51 family of recombinases, with RadA being the functional homologue. We have studied and compared the RadA and RadB proteins of mesophilic and thermophilic Archaea. In growing cells, RadA levels are similar in mesophilic Methanococcus species and the hyperthermophile Methanococcus jannaschii. Treatment of cells with mutagenic agents (methylmethane sulfonate or UV light) increased the expression of RadA (as evidenced by higher levels of both mRNA and protein) in all organisms tested, but the increase was greater in the mesophiles than in the thermophiles M. jannaschii and Sulfolobus solfataricus. Recombinantly expressed RadA proteins from the mesophile M. voltae and the thermophile M. jannaschii were similar in their ATPase- and DNA-binding activities. All the data are consistent with proposals that RadA plays the same role as eukaryotic Rad51. Surprisingly, the data also suggested that the thermophiles do not need more RadA protein or activity than the mesophiles. On the other hand, RadB is not coregulated with RadA, and its role remains unclear. Neither RadA nor RadB from a mesophile or from a thermophile rescued the UV-sensitive phenotype of an Escherichia coli recA- host.