Differential correction of lagging-strand replication errors made by DNA polymerases {alpha} and {delta}

Mismatch repair (MMR) of replication errors requires DNA ends that can direct repair to the newly synthesized strand containing the error. For all but those organisms that use adenine methylation to generate nicks, the source of these ends in vivo is unknown. One possibility is that MMR may have a "special relation to the replication complex" [Wagner R, Jr., Meselson M (1976) Proc Natl Acad Sci USA 73:4135-4139], perhaps one that allows 5' or 3' DNA ends associated with replication to act as strand discrimination signals. Here we examine this hypothesis, based on the logic that errors made by yeast DNA polymerase α (Pol α), which initiates Okazaki fragments during lagging-strand replication, will always be closer to a 5' end than will be more internal errors generated by DNA polymerase δ (Pol δ), which takes over for Pol α to complete lagging-strand replication. When we compared MMR efficiency for errors made by variant forms of these two polymerases, Msh2-dependent repair efficiencies for mismatches made by Pol α were consistently higher than for those same mismatches when made by Pol δ. Thus, one special relationship between MMR and replication is that MMR is more efficient for the least accurate of the major replicative polymerases, exonuclease-deficient Pol α. This observation is consistent with the close proximity and possible use of 5' ends of Okazaki fragments for strand discrimination, which could increase the probability of Msh2-dependent MMR by 5' excision, by a Msh2-dependent strand displacement mechanism, or both.     

Structural and functional analysis of the interaction between the nucleoporin Nup98 and the mRNA export factor Rae1

The export of mRNAs is a multistep process, involving the packaging of mRNAs into messenger ribonucleoprotein particles (mRNPs), their transport through nuclear pore complexes, and mRNP remodeling events prior to translation. Ribonucleic acid export 1 (Rae1) and Nup98 are evolutionarily conserved mRNA export factors that are targeted by the vesicular stomatitis virus matrix protein to inhibit host cell nuclear export. Here, we present the crystal structure of human Rae1 in complex with the Gle2-binding sequence (GLEBS) of Nup98 at 1.65 Å resolution. Rae1 forms a seven-bladed β-propeller with several extensive surface loops. The Nup98 GLEBS motif forms an ≈50-Å-long hairpin that binds with its C-terminal arm to an essentially invariant hydrophobic surface that extends over the entire top face of the Rae1 β-propeller. The C-terminal arm of the GLEBS hairpin is necessary and sufficient for Rae1 binding, and we identify a tandem glutamate element in this arm as critical for complex formation. The Rae1•Nup98^GLEBS surface features an additional conserved patch with a positive electrostatic potential, and we demonstrate that the complex possesses single-stranded RNA-binding capability. Together, these data suggest that the Rae1•Nup98 complex directly binds to the mRNP at several stages of the mRNA export pathway.     

Salicylate,an aspirin metabolite,specifically inhibits the current mediated by glycine receptors containing α1-subunits

Background and purpose:

Aspirin or its metabolite sodium salicylate is widely prescribed and has many side effects. Previous studies suggest that targeting neuronal receptors/ion channels is one of the pathways by which salicylate causes side effects in the nervous system. The present study aimed to investigate the functional action of salicylate on glycine receptors at a molecular level.

Experimental approach:

Whole-cell patch-clamp and site-directed mutagenesis were deployed to examine the effects of salicylate on the currents mediated by native glycine receptors in cultured neurones of rat inferior colliculus and by glycine receptors expressed in HEK293T cells.

Key results:

Salicylate effectively inhibited the maximal current mediated by native glycine receptors without altering the EC₅₀ and the Hill coefficient, demonstrating a non-competitive action of salicylate. Only when applied simultaneously with glycine and extracellularly, could salicylate produce this antagonism. In HEK293T cells transfected with either α1-, α2-, α3-, α1β-, α2β- or α3β-glycine receptors, salicylate only inhibited the current mediated by those receptors that contained the α1-subunit. A single site mutation of I240V in the α1-subunit abolished inhibition by salicylate.

Conclusions and implications:

Salicylate is a non-competitive antagonist specifically on glycine receptors containing α1-subunits. This action critically involves the isoleucine-240 in the first transmembrane segment of the α1-subunit. Our findings may increase our understanding of the receptors involved in the side effects of salicylate on the central nervous system, such as seizures and tinnitus.     

A new T677C mutation of the aspartoacylase gene encodes for a protein with no enzymatic activity

ObjectiveTo verify the effect of and to date the unknown T677C mutation of the human N-acetylaspartoacylase (hASPA) gene on the function of the mutated enzyme.Design and methodsWild type and I226T-mutated proteins were expressed and purified from a transformed Escherichia coli colony. Enzymatic activities were measured in the presence of varying substrate concentrations.ResultsWhilst kinetic parameters of wild type hASPA were in line with data in literature, I226T-mutated hASPA showed no enzymatic activity.ConclusionData indicated that this new mutation might be responsible in homozygosis for the phenotype corresponding to Canavan disease.     

Identification of ginsenoside interaction sites in 5-HT3A receptors

We previously demonstrated that 20(S)-ginsenoside Rg(3) (Rg(3)), one of the active components of Panax ginseng, non-competitively inhibits 5-HT(3A) receptor channel activity on extracellular side of the cell. Here, we sought to elucidate the molecular mechanisms underlying Rg(3)-induced 5-HT(3A) receptor regulation. We used the two-microelectrode voltage-clamp technique to investigate the effect of Rg(3) on 5-HT-mediated ion currents (I(5-HT)) in Xenopus oocytes expressing wild-type or 5-HT(3A) receptors harboring mutations in the gating pore region of transmembrane domain 2 (TM2). In oocytes expressing wild-type 5-HT(3A) receptors, Rg(3) dose-dependently inhibited peak I(5-HT) with an IC(50) of 27.6+/-4.3microM. Mutations V291A, F292A, and I295A in TM2 greatly attenuated or abolished the Rg(3)-induced inhibition of peak I(5-HT). Mutation V291A but not F292A and I295A induced constitutively active ion currents with decrease of current decay rate. Rg(3) accelerated the rate of current decay with dose-dependent manner in the presence of 5-HT. Rg(3) and TMB-8, an open channel blocker, dose-dependently inhibited constitutively active ion currents. The IC(50) values of constitutively active ion currents in V291A mutant receptor were 72.4+/-23.1 and 6.5+/-0.7microM for Rg(3) and TMB-8, respectively. Diltiazem did not prevent Rg(3)-induced inhibition of constitutively active ion currents in occlusion experiments. These results indicate that Rg(3) inhibits 5-HT(3A) receptor channel activity through interactions with residues V291, F292, and I295 in the channel gating region of TM2 and further demonstrate that Rg(3) regulates 5-HT(3A) receptor channel activity in the open state at different site(s) from those of TMB-8 and diltiazem.     

登革2型病毒全长cDNA克隆定点诱变的OL-PCR方法

目的 对带有登革2型病毒（DEN-2）全长cDNA的质粒pDVWS501上E62、E203位点进行定点诱变。方法 设计4对点诱变引物,运用OL-PCR（overlapPCR)法,扩增出分别在E62或E203位带有点突变的2条DNA片段,克隆至T载体,获T-TB62、T-TB203两个克隆,将T-TB62用ClaⅠ和SphⅠ分别酶切,T-TB203用SphⅠ NheⅠ酶切后,用T4连接酶分别连接至pDVWS501,获重组质粒TB62和TB203。对TB62和TB203进行序列测定。结果 成功得到分别在E62、E203位带有点突变的TB62、TB203克隆。结论 OL-PCR法是具有较高突变效率的定点诱变方法。     

VLDL-R中配体结合重复序列的结合特性及结构分析

目的 研究VLDL-R中8个配体结合重复序列(1igand-bindingrepeats，LBR)在配体结合中的作用并探讨结合位点的结构。方法采用基因缺失诱变方法，构建不同LBR缺失的VLDL-R重组体。将其分别导人无LDL-R功能性表达的1dl-A7细胞。配体结合实验观察转染细胞与荧光标记的VLDL、β-VLDL的结合能力，并采用同源建模的方法预测受体N-端328个氨基酸的配体结合域的空间结构。结果 配体结合实验显示LBR1和LBR2对受体结合VLDL、pVLDL最为重要；LBR3对结合VLDL也有重要作用，但对结合pVLDL无明显影响。模型显示受体N-端328个氨基酸的配体结合域呈现弧形口袋样结构，前3个LBR结构紧凑，呈棒状，负电荷相对集中，与功能特性吻合，LBR5与LBR6之间的连接区赋予口袋结构一定的伸缩性，适应配体大小的变化。结论 受体N-端的前3个LBR含有与配体结合的位点。该区域特定的空间结构与理化特性是结合功能的重要基础。     

基因变构IL-2重组克隆的构建

①目的 构建基因变构IL-2（88ArgIL-2)的重组克隆。②方法 将正常人的扁桃体细胞经PHA刺激后，获得cDNA文库，并以此为模板，经套式PCR扩增得IL-2的基因编码序列。测序确认正确后，设计含有突变位点的引物，经pCR定点诱变技术获得变构IL-2克隆并进行序列确定。③结果 IL-2基因定点诱变成功，并获得了基因变构IL-2重组克隆。④结论 IL-2重组基因变构克隆的构建为进一步在原核和真核细胞中表达以及研究制备低毒、高效的新型基因变构IL-2奠定了基础。     

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca²⁺-dependent regulation. Force then depended on the negative logarithm of Ca²⁺ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa₅₀, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca²⁺, but restoration of Ca²⁺ dependence required incubation with both TnI and TnC. Relaxation at low Ca²⁺ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca²⁺ sensitivity, i.e. the pCa₅₀, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca²⁺ than those reconstituted with cTnC and cTnI (difference in pCa₅₀ approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G¹⁰⁴-K-F-K-R-P-P-L-R-R-V-R¹¹⁵-). The four mutants produced by substitution of T for P¹¹⁰, G for P¹¹⁰, G for L¹¹¹, and G for K¹⁰⁵ were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S₁ ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca²⁺ dependence could be restored when ^gly110sTnI, ^thr110sTnI or ^gly111sTnI was used for reconstitution. The mutant ^gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca²⁺ concentrations and it caused an increase in Ca²⁺ sensitivity. Received: 5 October 1995/Received after revision and accepted: 1 December 1995     

酞酸酯对果蝇生存天数影响及其遗传毒性

目的探讨邻苯二甲酸二丁酯(di-n-butyl phthalate，DBP)和邻苯二甲酸二辛酯(di-n-octyl phthalate，DOP)对果蝇生存时间的影响和遗传毒性。方法用等质量混合的DBP和DOP对果蝇经口联合染毒。采用果蝇寿命试验和果蝇伴性隐性致死试验观察DBP、DOP对果蝇生存时间的影响对生殖细胞的致突变作用。结果从0．2％浓度组开始，果蝇平均寿命与对照组相比差异有统计学意义，600，1800mg／L浓度组果蝇突变率与对照组相比差异有统计学意义。结论DBP、DOP干扰了果蝇正常的生长发育和新陈代谢，并加速其衰老；DBP、DOP对果蝇生殖细胞有致突变作用。