Sulfolobus islandicus plasmids pRN1 and pRN2 share distant but common evolutionary ancestry

The complete sequence of the plasmid pRN2 from the thermoacidophile Sulfolobus islandicus has been determined. The plasmid was found to be circular and 6959 bp in length. S. islandicus harbors another endogenous plasmid, pRN1, and comparison of pRN1 and pRN2 revealed that these two plasmids are essentially homologous, although very distantly related. pRN1 and pRN2 share several stretches of highly conserved noncoding DNA and three common open reading frames. Two of these reading frames are likely related to replication, one encoding a large protein with a helicase domain similar to viral helicases, and the other a copy number control protein, CopG. Received: November 19, 1997 / Accepted: March 10, 1998     

Effect of DNA binding protein Sshl2 from hyperthermophilic archaeon Sulfolobus shibatae on DNA supercoiling

An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.     

Effect of DNA binding protein Sshl2 from hyperthermophilic archaeon Sulfolobus shibatae on DNA supercoiling

An 11.5-ku DNA binding protein, designated as Sshl2, was purified from the hyperthermophilic archaeon Sulfolobus shibatae by column chromatography in SP Sepharose, DNA cellulose and phosphocellulose. Sshl2 accounts for about 4 % of the total cellular protein. The protein is capable of binding to both negatively supercoiled and relaxed DNAs. Nick closure analysis revealed that Sshl2 constrains negative supercoils upon binding to DNA. While the ability of the protein to constrain supercoils is weak at 22℃ , it is enhanced substantially at temperatures higher than 37℃ . Both the cellular content and supercoil-constraining ability of Sshl2 suggest that the protein may play an important role in the organization and stabilization of the chromosome of S. shibatae.     

Folding and association of an extremely stable dimeric protein from Sulfolobus islandicus

ORF56 is a plasmid-encoded protein from Sulfolobus islandicus, which probably controls the copy number of the pRN1 plasmid by binding to its own promotor. The protein showed an extremely high stability in denaturant, heat, and pH-induced unfolding transitions, which can be well described by a two-state reaction between native dimers and unfolded monomers. The homodimeric character of native ORF56 was confirmed by analytical ultracentrifugation. Far-UV circular dichroism and fluorescence spectroscopy gave superimposable denaturant-induced unfolding transitions and the midpoints of both heat as well as denaturant-induced unfolding depend on the protein concentration supporting the two-state model. This model was confirmed by GdmSCN-induced unfolding monitored by heteronuclear 2D NMR spectroscopy. Chemical denaturation was accomplished by GdmCl and GdmSCN, revealing a Gibbs free energy of stabilization of -85.1 kJ/mol at 25 degrees C. Thermal unfolding was possible only above 1 M GdmCl, which shifted the melting temperature (t(m)) below the boiling point of water. Linear extrapolation of t(m) to 0 M GdmCl yielded a t(m) of 107.5 degrees C (5 microM monomer concentration). Additionally, ORF56 remains natively structured over a remarkable pH range from pH 2 to pH 12. Folding kinetics were followed by far-UV CD and fluorescence after either stopped-flow or manual mixing. All kinetic traces showed only a single phase and the two probes revealed coincident folding rates (k(f), k(u)), indicating the absence of intermediates. Apparent first-order refolding rates depend linearly on the protein concentration, whereas the unfolding rates do not. Both lnk(f) and lnk(u) depend linearly on the GdmCl concentration. Together, folding and association of homodimeric ORF56 are concurrent events. In the absence of denaturant ORF56 refolds fast (7.0 x 10(7)M(-1)s(-1)) and unfolds extremely slowly (5.7 year(-1)). Therefore, high stability is coupled to a slow unfolding rate, which is often observed for proteins of extremophilic organisms.     

超嗜热古菌Sulfolobus tokodaii RadA蛋白的克隆表达及 其辐射可诱导性

RecA/Rad51/RadA家族蛋白是细胞内重要的重组修复蛋白,在功能上非常保守.研究发现在细菌、真核生物、甲烷古菌和嗜盐古菌细胞内RecA/Rad51/RadA均可以受紫外线辐射诱导转录.而对极端嗜热古菌中的RadA辐射可诱导性仍存在争议.通过体外表达极端嗜热古菌Sulfolobus tokodaii的RadA蛋白,制备抗体,利用免疫学方法并结合RT-PCR分析,对嗜热古菌S.tokodaii中RadA的辐射诱导进行了研究.经过100J/m2和200J/m2 UV辐照处理,radA基因的转录分别上调了2倍和3倍,同时RadA蛋白的表达分别上升了1.5倍和1倍.实验结果表明S.tokodaii中RadA可以被紫外线辐射诱导表达,证实了极端嗜热古菌S.tokodaii细胞中存在DNA损伤诱导反应的观点.     

Surface resistance to SSVs and SIRVs in pilin deletions of Sulfolobus islandicus

Characterizing the molecular interactions of viruses in natural microbial populations offers insights into virus–host dynamics in complex ecosystems. We identify the resistance of Sulfolobus islandicus to Sulfolobus spindle-shaped virus (SSV9) conferred by chromosomal deletions of pilin genes, pilA1 and pilA2 that are individually able to complement resistance. Mutants with deletions of both pilA1 and pilA2 or the prepilin peptidase, PibD, show the reduction in the number of pilins observed in TEM and reduced surface adherence but still adsorb SSV9. The proteinaceous outer S-layer proteins, SlaA and SlaB, are not required for adsorption nor infection demonstrating that the S-layer is not the primary receptor for SSV9 surface binding. Strains lacking both pilins are resistant to a broad panel of SSVs as well as a panel of unrelated S. islandicus rod-shaped viruses (SIRVs). Unlike SSV9, we show that pilA1 or pilA2 is required for SIRV8 adsorption. In sequenced Sulfolobus strains from around the globe, one copy of each pilA1 and pilA2 is maintained and show codon-level diversification, demonstrating their importance in nature. By characterizing the molecular interactions at the initiation of infection between S. islandicus and two different types of viruses we hope to increase the understanding of virus–host interactions in the archaeal domain.     

Repair Responses to DNA Damage: Enzymatic Pathways in E coli and Human Cells

Bacteria and eukaryotic cells employ a variety of enzymatic pathways to remove damage from DNA or to lessen its impact upon cellular functions. Most of these processes were discovered in Escherichia coli and have been most extensively analyzed in this organism because suitable mutants have been isolated and characterized. Analogous pathways have been inferred to exist in mammalian cells from the presence of enzyme activities similar to those known to be involved in repair in bacteria, from the analysis of events in cells treated with DNA damaging agents, and from the analysis of the few naturally occurring mutant cell types. Excision repair of pyrimidine dimers produced by UV in E coli is initiated by an incision event catalyzed by a complex composed of uvrA, uvrB, and uvrC gene products. Multiple exonuclease and polymerase activities are available for the subsequent excision and resynthesis steps. In addition to the constitutive pathway, which produces short patches of 20–30 nucleotides, an inducible excision repair process exists that produces much longer patches. This long patch pathway is controlled by the recA-lexA regulatory circuit and also requires the recF gene. It is apparently not responsible for UV-induced mutagenesis. However, the ability to perform inducible long patch repair correlates with enhanced bacterial survival and with a major component of the Weigle reactivation of bacteriophage with double-strand DNA genomes. Mammalian cells possess an excision repair pathway similar to the constitutive pathway in E coli. Although not as well understood, the incision event is at least as complex, and repair resynthesis produces patches of about the same size as the constitutive short patches. In mammalian cells, no patches comparable in size to those produced by the inducible pathway of E coli are observed. Repair in mammalian cells may be more complicated than in bacteria because of the structure of chromatin, which can affect both the distribution of DNA damage and its accessibility to repair enzymes. A coordinated alteration and reassembly of chromatin at sites of repair may be required. We have observed that the sensitivity of digestion by staphylococcal nuclease (SN) of newly synthesized repair patches resulting from excision of furocoumarin adducts changes with time in the same way as that of patches resulting from excision of pyrimidine dimers. Since furocoumarin adducts are formed only in the SN-sensitive linker DNA between nucleosome cores, this suggests that after repair resynthesis is completed, the nucleosome cores in the region of the repair event do not return exactly to their original positions. We have also studied excision repair of UV and chemical damage in the highly repeated 172 base pair α DNA sequence in African green monkey cells. In UV irradiated cells, the rate and extent of repair resynthesis in this sequence is similar to that in bulk DNA. However, in cells containing furocoumarin adducts, repair resynthesis in α DNA is only about 30% of that in bulk DNA. Since the frequency of adducts does not seem to be reduced in α DNA, it appears that certain adducts in this unique DNA may be less accessible to repair. Endonuclease V of bacteriophage T4 incises DNA at pyrimidine dimers by cleaving first the glycosylic bond between deoxyribose and the 5′ pyrimidine of the dimer and then the phosphodiester bond between the two pyrimidines. We have cloned the gene (denV) that codes for this enzyme and have demonstrated its expression in uvrA recA and uvrB recA cells of E coli. Because T4 endonuclease V can alleviate the excision repair deficiency of xeroderma pigmentosum when added to permeabilized cells or to isolated nuclei after UV irradiation, the cloned denV gene may ultimately be of value for analyzing DNA repair pathways in cultured human cells.     

Genomics and genetics of Sulfolobus islandicus LAL14/1, a model hyperthermophilic archaeon

The 2 465 177 bp genome of Sulfolobus islandicus LAL14/1, host of the model rudivirus SIRV2, was sequenced. Exhaustive comparative genomic analysis of S. islandicus LAL14/1 and the nine other completely sequenced S. islandicus strains isolated from Iceland, Russia and USA revealed a highly syntenic common core genome of approximately 2 Mb and a long hyperplastic region containing most of the strain-specific genes. In LAL14/1, the latter region is enriched in insertion sequences, CRISPR (clustered regularly interspaced short palindromic repeats), glycosyl transferase genes, toxin–antitoxin genes and MITE (miniature inverted-repeat transposable elements). The tRNA genes of LAL14/1 are preferential targets for the integration of mobile elements but clusters of atypical genes (CAG) are also integrated elsewhere in the genome. LAL14/1 carries five CRISPR loci with 10 per cent of spacers matching perfectly or imperfectly the genomes of archaeal viruses and plasmids found in the Icelandic hot springs. Strikingly, the CRISPR_2 region of LAL14/1 carries an unusually long 1.9 kb spacer interspersed between two repeat regions and displays a high similarity to pING1-like conjugative plasmids. Finally, we have developed a genetic system for S. islandicus LAL14/1 and created ΔpyrEF and ΔCRISPR_1 mutants using double cross-over and pop-in/pop-out approaches, respectively. Thus, LAL14/1 is a promising model to study virus–host interactions and the CRISPR/Cas defence mechanism in Archaea.     

端粒结合蛋白在端粒复制与损伤修复中的作用

端粒位于真核细胞线性染色体末端,正常的端粒长度与结构对于细胞基因组稳定的维持有重要作用.端粒DNA序列的高度重复性使其容易形成一些特殊的二级结构,相比染色体其他位置更难复制.结合在端粒上的Shelterin蛋白复合体由六个端粒结合蛋白组成,该复合体可以通过抑制端粒处异常DNA损伤修复途径的激活维持端粒的稳定.此外,近几...     

极端嗜热古菌———芝田硫化叶菌DNA 连接酶的生化性质

极端嗜热古菌———芝田硫化叶菌 DNA 连接酶 (Ssh 连接酶 ) 的最适辅因子为 ATP ,在 dATP 存在时,该酶也能表现出较弱的连接活性 . ATP 或 dATP 都能够使该酶发生腺苷化,腺苷化的 Ssh 连接酶能够将腺苷基团转移至含切刻的 DNA 上 . 电泳迁移率改变实验表明, Ssh 连接酶能够结合双链 DNA ,且与含切刻及不含切刻的 DNA 结合的亲和力相同,但不结合单链 DNA. 酵母双杂交实验显示,硫磺矿硫化叶菌 ( 与芝田硫化叶菌亲缘关系很近 ) 的 DNA 连接酶,与该菌所含的 3 个增殖细胞核抗原 (PCNA) 同源蛋白中的一个 (PCNA-1) 有相互作用,而与另外 2 个同源蛋白 (PCNA-like 和 PCNA-2) 则无相互作用 . 在古菌中高度保守的 Sac10b 蛋白家族成员 Ssh10b 能够激活 Ssh 连接酶的活性,而硫化叶菌中的主要染色体蛋白——— 7 ku DNA 结合蛋白 (Ssh7) 则对该酶活性没有影响 .     

DNA损伤反应性蛋白参与中心体调控

中心体是动物细胞有丝分裂期微管组织中心,对于细胞有丝分裂期形成纺锤体、正常分裂及染色体精确分离至关重要. 中心体失调控常造成遗传物质错误分配,最终诱发肿瘤形成.因此,对中心体结构及数量的精密调控将对细胞命运起着决定 作用.目前发现,中心体至少包含100多种调节蛋白,这些蛋白在细胞内的功能各异.最近很多研究显示,多种DNA损伤修复及 应答通路的激酶或磷酸酶定位于中心体,并且参与中心体调控.本文将对中心体结构、中心体复制、中心体分离、中心体扩 增、DNA损伤与中心体异常及DNA损伤反应性蛋白在中心体调控中的功能作一综述.     

Sirtuins家族在DNA损伤修复中的作用

Sirtuins家族蛋白是一类依赖NAD的去乙酰化酶,属于第Ш类去乙酰化酶(HDACs),哺乳动物Sirtuins家族成员共有7个(SIRT1-7),其主要具有去乙酰化酶的活性,可以使多种蛋白发生去乙酰化,进而参与DNA的损伤修复、基因的转录调控、细胞凋亡、代谢及衰老等诸多生物进程。本文主要对Sirtuins家族在DNA损伤修复中的作用及其相关机制进行阐述。     

Domain organization and biochemical features of Sulfolobus solfataricus DNA polymerase

DNA polymerase from Sulfolobus solfataricus, strain MT4 (Sso DNA pol), was one of the first archaeal DNA polymerases to be isolated and characterized. Its encoding gene was cloned and sequenced, indicating that Sso DNA pol belongs to family B of DNA polymerases. By limited proteolysis experiments carried out on the recombinant homogeneous protein, we were able to demonstrate that the enzyme has a modular organization of its associated catalytic functions (DNA polymerase and 3′-5′ exonuclease). Indeed, the synthetic function was ascribed to the enzyme C-terminal portion, whereas the N-terminal half was found to be responsible for the exonucleolytic activity. In addition, partial proteolysis studies were utilized to map conformational changes on DNA binding by comparing the cleavage map in the absence or presence of nucleic acid ligands. This analysis allowed us to identify two segments of the Sso DNA pol amino acid chain affected by structural modifications following nucleic acid binding: region 1 and region 2, in the middle and at the C-terminal end of the protein chain, respectively. Site-directed mutagenesis studies will be performed to better investigate the role of these two protein segments in DNA substrate interaction. Received: January 22, 1998 / Accepted: February 16, 1998     

PHF1在肿瘤DNA损伤修复中的作用

轻微的DNA损伤可启动损伤修复途径,严重的DNA损伤则会启动细胞休眠或凋亡途径。PHF1是PcG蛋白家族中的重要组分,参与复杂的生物学过程,包括DNA损伤修复、细胞休眠或凋亡、组蛋白翻译后修饰和染色体重排。本文主要对PHF1的结构、参与的信号通路、翻译后修饰及生物学功能做小结和展望,为PHF1进一步研究提供理论基础。     

Characterization of the plant homolog of Nijmegen breakage syndrome 1: Involvement in DNA repair and recombination

The Nbs1 gene is known to code for a protein involved in the hereditary cancer-prone disease, Nijmegen breakage syndrome. This gene is conserved in animals and fungi, but no plant homolog is known. The work reported here describes a homolog of Nbs1 isolated from higher plants. The Nbs1 proteins from both Arabidopsis thaliana and Oryza sativa are smaller in size than animal or yeast Nbs1, but both contain the conserved Nbs1 domains such as the FHA/BRCT domain, the Mre11-binding domain, and the Atm-interacting domain in orientations similar to what is seen in animal Nbs1. The OsNbs1 protein interacted not only with plant Mre11, but also with animal Mre11. In plants, OsNbs1 mRNA expression was found to be higher in the shoot apex and young flower, and AtNbs1 expression increased when plants were exposed to 100 Gy of X-rays. These results suggest that plant Nbs1 could participate in a Rad50/Mre11/Nbs1 complex, and could be essential for the regulation of DNA recombination and DNA damage responses.     

Studies on DNA Damage and Repair in the Mammalian Brain

Methods for studying breaks in DNA strands and their repair, originally developed for prokaryotes and cultured cell lines, have been applied to preparations from rat brain. The relative sensitivities of these methods, which include alkaline sucrose density gradient sedimentation, nucleoid sedimentation, and ADP-ribosyltransferase assay, are compared.     

DNA损伤修复过程表观遗传学改变

多种化学、物理及生物因素可诱发细胞DNA损伤,损伤后DNA损伤位点被相关损伤感受器识别,激活相应的修复通路进行DNA修复。越来越多的证据表明DNA甲基化状态、蛋白翻译后修饰、染色质重塑、miRNA等修饰方式参与了DNA的损伤修复。文章通过不同损伤修复通路中这些修饰的特点,阐述表观遗传学改变在DNA损伤修复发展过程中的作用机制。     

蛋白激酶CK2与DNA断裂修复

蛋白激酶CK2（酪蛋白激酶Ⅱ）是真核细胞中普遍存在的一种信使非依赖的丝氨酸／苏氨酸蛋白激酶,它底物众多,功能广泛。DNA断裂修复是一个涉及很多种酶和蛋白的过程,CK2在其中起着很重要的作用。