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1.
Proteolytic truncation of microtubule associated human (h) Tau protein by caspase-3 at the carboxy (C) terminus has been linked to the pathogenesis of Alzheimer’s Disease (AD). This cleavage likely occurs between Asp421↓Ser422 leading to the formation of 421-mer truncated Tau protein which has been found to be present as aggregate in high level after phosphorylation in mortal AD brain tissue compared to normal. At least 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed in hTau and a selected number of them have been implicated in hTau aggregation following latter’s proteolytic truncation. Interestingly, it is further noted that Ser422 residue present in the P1′ position of hTau caspase-3 cleavage region is a potential phosphorylation site. So we became interested to examine in vitro the effect of phospho-Ser422 residue on hTau cleavage by caspase-3 which is a crucial upstream event associated with hTau self-assembly leading to AD pathogenesis. The goal of this project is to study in vitro the caspase-3 cleavage site of hTau protein and to examine the kinetics of this cleavage following Ser422 phosphorylation and treatment with caspase-3 inhibitors. This is achieved by designing peptides from the sequence of hTau protein containing the proposed caspase-3 cleavage region. Peptides were designed from 441-mer major human Tau protein sequence that encompasses the proposed caspase-3 cleavage site [Asp421↓Ser422]. Corresponding phospho-, dextro-Ser422 and dextro-Asp421 analogs were also designed. Peptides were synthesized by solid phase chemistry, purified and fully characterized by mass spectrometry. These were then incubated with recombinant caspase-3 enzyme under identical condition for digestion and analyzed for cleavage by mass spectrometry and RP-HPLC chromatograms. Our results indicated that while the control peptide is efficiently cleaved by caspase-3 at Asp421↓Ser422 site producing the expected N- and C-terminal fragment peptides, the corresponding phospho-Ser422 peptide remained completely resistant to the cleavage. Substitution of Asp421 by its dextro isoform also blocks peptide cleavage by caspase-3. However substitution of Ser422 by its dextro isoform in the peptide did not affect the cleavage significantly. The above results were further confirmed by caspase-3 digestion experiment in the presence of varying amounts of caspase-3 inhibitor (Ac-DQVD-aldehyde) which was found to block this cleavage in a highly effective manner. Our results highlighted the crucial significance of Ser422 phosphorylation and suggest that the kinase associated with this Ser-phosphorylation may protect Tau from aggregation. Thus specific promoters/activators of this kinase may find useful therapeutic benefits in arresting Tau truncation by caspase-3 and the progression of AD. In addition our data demonstrated that Tau-peptides where Ser422 or Asp421 are substituted by their respective dextro isomers, exhibit different cleavage kinetics by caspase-3 and this may have important implications in therapeutic intervention of Tau aggregation and associated AD.  相似文献   

2.
Two chloroplast tRNAAsp species from barley were purified by chromatography on benzoylated DEAE-cellulose and sequenced. They differ in the modification at position 34, where queuosine (Q) is present in one of the species. The same chromatographic procedure yielded only one tRNAGlu species, corroborating the assumption that the same tRNAGlu species participates in both protein and chlorophyll biosynthesis. The level of tRNAGlu remains unchanged after light treatment of etiolated seedlings, whereas the amount of tRNAAsp decreases to about 50% relative to the level of dark-grown plants.  相似文献   

3.
Glutamyl-queuosine-tRNAAsp synthetase (Glu-Q-RS) is a paralog of glutamyl-tRNA synthetase (GluRS) and is found in more than forty species of proteobacteria, cyanobacteria, and actinobacteria. Glu-Q-RS shows striking structural similarity with N-terminal catalytic domain of GluRS (NGluRS) but it lacks the C-terminal anticodon binding domain (CGluRS). In spite of structural similarities, Glu-Q-RS and NGluRS differ in their functional properties. Glu-Q-RS glutamylates the Q34 nucleotide of the anticodon of tRNAAsp whereas NGluRS constitutes the catalytic domain of GluRS catalyzing the transfer of Glu on the acceptor end of tRNAGlu. Since NGluRS is able to catalyze aminoacylation of only tRNAGlu the glutamylation capacity of tRNAAsp by Glu-Q-RS is surprising. To understand the substrate specificity of Glu-Q-RS we undertook a systemic approach by investigating the biophysical and biochemical properties of the NGluRS (1–301), CGluRS (314–471) and Glu-Q-RS-CGluRS, (1–298 of Glu-Q-RS fused to 314–471 from GluRS). Circular dichroism, fluorescence spectroscopy and differential scanning calorimetry analyses revealed absence of N-terminal domain (1–298 of Glu-Q-RS) and C-terminal domain (314–471 from GluRS) communication in chimera, in contrast to the native full length GluRS. The chimeric Glu-Q-RS is still able to aminoacylate tRNAAsp but has also the capacity to bind tRNAGlu. However the chimeric protein is unable to aminoacylate tRNAGlu probably as a consequence of the lack of domain–domain communication.  相似文献   

4.
Polyamine (putrescine, spermidine and spermine) and agmatine uptake by the human organic cation transporter 2 (hOCT2) was studied using HEK293 cells transfected with pCMV6-XL4/hOCT2. The Km values for putrescine and spermidine were 7.50 and 6.76 mM, and the Vmax values were 4.71 and 2.34 nmol/min/mg protein, respectively. Spermine uptake by hOCT2 was not observed at pH 7.4, although it inhibited both putrescine and spermidine uptake. Agmatine was also taken up by hOCT2, with Km value: 3.27 mM and a Vmax value of 3.14 nmol/min/mg protein. Amino acid residues involved in putrescine, agmatine and spermidine uptake by hOCT2 were Asp427, Glu448, Glu456, Asp475, and Glu516. In addition, Glu524 and Glu530 were involved in putrescine and spermidine uptake activity, and Glu528 and Glu540 were weakly involved in putrescine uptake activity. Furthermore, Asp551 was also involved in the recognition of spermidine. These results indicate that the recognition sites for putrescine, agmatine and spermidine on hOCT2 strongly overlap, consistent with the observation that the three amines are transported with similar affinity and velocity. A model of spermidine binding to hOCT2 was constructed based on the functional amino acid residues.  相似文献   

5.
We expressed full-length Na+-Ca2+ exchangers (NCXs) with mutations in two Ca2+-binding domains (CBD1 and CBD2) to determine the roles of the CBDs in Ca2+-dependent regulation of NCX. CBD1 has four Ca2+-binding sites, and mutation of residues Asp421 and Glu451, which primarily coordinate Ca2+ at sites 1 and 2, had little effect on regulation of NCX by Ca2+. In contrast, mutations at residues Glu385, Asp446, Asp447, and Asp500, which coordinate Ca2+ at sites 3 and 4 of CBD1, resulted in a drastic decrease in the apparent affinity of peak exchange current for regulatory Ca2+. Another mutant, M7, with 7 key residues of CBD1 replaced, showed a further decrease in apparent Ca2+ affinity but retained regulation, confirming a contribution of CBD2 to Ca2+ regulation. Addition of the mutation K585E (located in CBD2) into the M7 background induced a marked increase in Ca2+ affinity for both steady-state and peak currents. Also, we have shown previously that the CBD2 mutations E516L and E683V have no Ca2+-dependent regulation. We now demonstrate that introduction of a positive charge at these locations rescues Ca2+-dependent regulation. Finally, our data demonstrate that deletion of the unstructured loops between β-strands F and G of both CBDs does not alter the regulation of the exchanger by Ca2+, indicating that these segments are not important in regulation. Thus, CBD1 and CBD2 have distinct roles in Ca2+-dependent regulation of NCX. CBD1 determines the affinity of NCX for regulatory Ca2+, although CBD2 is also necessary for Ca2+-dependent regulation.  相似文献   

6.
This study investigates how calcium modulates the properties of dual positional specific maize lipoxygenase-1, including its interaction with substrate, association with subcellular membrane and alteration of product distribution. Bioinformatic analyses identified Asp38, Glu127 and Glu201 as putative calcium binding residues and Leu37 as a flanking hydrophobic residue also potentially involved in calcium-mediated binding of the enzyme to subcellular membranes. Asp38 and Leu37 were shown to be important but not essential for calcium-mediated association of maize lipoxygenase-1 to subcellular membranes in vitro. Kinetic studies demonstrate that catalytic efficiency (Vmax/Km) shows a bell-shaped dependence on log of the molar ratio of substrate to unbound calcium. Calcium also modulates product distribution of the maize lipoxygenase-1 reaction, favoring 13-positional specificity and increasing the relative amount of (E,Z)-isomeric products. The results suggest that calcium regulates the maize lipoxygenase-1 reaction by binding to substrate, and by promoting binding of substrate to enzyme and association of maize lipoxygenase-1 to subcellular membranes.  相似文献   

7.
Necula M  Chirita CN  Kuret J 《Biochemistry》2005,44(30):10227-10237
Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Small molecules capable of both inhibiting aggregation and promoting filament disaggregation have been discovered, but knowledge of their mechanism of action and potential for testing in biological models is fragmentary. To clarify these issues, the interaction between a small-molecule inhibitor of tau fibrillization, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), and full-length four-repeat tau protein was characterized in vitro using transmission electron microscopy and fluorescence spectroscopy. Analysis of reaction time courses performed in the presence of anionic fibrillization inducers revealed that increasing concentrations of N744 decreased the total filament length without modulating lag time, indicating that filament extension but not nucleation was affected by inhibitor under the conditions that were investigated. Critical concentration measurements confirmed that N744 shifted equilibria at filament ends away from the fibrillized state, resulting in endwise filament disaggregation when it was added to synthetic filaments. Both increasing bulk tau concentrations and filament stabilizing modifications such as pseudophosphorylation and glycation antagonized N744 activity. The results illustrate the importance of mechanism for the design and interpretation of pharmacological studies in biological models of tau aggregation.  相似文献   

8.
9.
IF (intermediate filament) proteins can be cleaved by caspases to generate proapoptotic fragments as shown for desmin. These fragments can also cause filament aggregation. The hypothesis is that disease-causing mutations in IF proteins and their subsequent characteristic histopathological aggregates could involve caspases. GFAP (glial fibrillary acidic protein), a closely related IF protein expressed mainly in astrocytes, is also a putative caspase substrate. Mutations in GFAP cause AxD (Alexander disease). The overexpression of wild-type or mutant GFAP promotes cytoplasmic aggregate formation, with caspase activation and GFAP proteolysis. In this study, we report that GFAP is cleaved specifically by caspase 6 at VELD225 in its L12 linker domain in vitro. Caspase cleavage of GFAP at Asp225 produces two major cleavage products. While the C-GFAP (C-terminal GFAP) is unable to assemble into filaments, the N-GFAP (N-terminal GFAP) forms filamentous structures that are variable in width and prone to aggregation. The effect of N-GFAP is dominant, thus affecting normal filament assembly in a way that promotes filament aggregation. Transient transfection of N-GFAP into a human astrocytoma cell line induces the formation of cytoplasmic aggregates, which also disrupt the endogenous GFAP networks. In addition, we generated a neo-epitope antibody that recognizes caspase-cleaved but not the intact GFAP. Using this antibody, we demonstrate the presence of the caspase-generated GFAP fragment in transfected cells expressing a disease-causing mutant GFAP and in two mouse models of AxD. These findings suggest that caspase-mediated GFAP proteolysis may be a common event in the context of both the GFAP mutation and excess.  相似文献   

10.
ACA8 is a type 2B Ca2+-ATPase having a regulatory N terminus whose auto-inhibitory action can be suppressed by binding of calmodulin (CaM) or of acidic phospholipids. ACA8 N terminus is able to interact with a region of the small cytoplasmic loop connecting transmembrane domains 2 and 3. To determine the role of this interaction in auto-inhibition we analyzed single point mutants produced by mutagenesis of ACA8 Glu252 to Asn345 sequence. Mutation to Ala of any of six tested acidic residues (Glu252, Asp273, Asp291, Asp303, Glu302, or Asp332) renders an enzyme that is less dependent on CaM for activity. These results highlight the relevance in ACA8 auto-inhibition of a negative charge of the surface area of the small cytoplasmic loop. The most deregulated of these mutants is D291A ACA8, which is less activated by controlled proteolysis or by acidic phospholipids; the D291A mutant has an apparent affinity for CaM higher than wild-type ACA8. Moreover, its phenotype is stronger than that of D291N ACA8, suggesting a more direct involvement of this residue in the mechanism of auto-inhibition. Among the other produced mutants (I284A, N286A, P289A, P322A, V344A, and N345A), only P322A ACA8 is less dependent on CaM for activity than the wild type. The results reported in this study provide the first evidence that the small cytoplasmic loop of a type 2B Ca2+-ATPase plays a role in the attainment of the auto-inhibited state.  相似文献   

11.
The Vc-NhaD is an Na+/H+ antiporter from Vibrio cholerae belonging to a new family of bacterial Na+/H+ antiporters, the NhaD family. In the present work we mutagenized five conserved Asp and Glu residues and one conserved Thr residue to Ala in order to identify amino acids that are critical for the antiport activity. All mutations fall into two distinct groups: (i) four variants, Glu100Ala, Glu251Ala, Glu342Ala, and Asp393Ala, did not abolish antiport activity but shifted the pH optimum to more alkaline pH, and (ii) variants Asp344Ala, Asp344Asn, and Thr345Ala caused a complete loss of both Na+/H+ and Li+/H+ antiport activity whereas the Asp344Glu variant exhibited reduced Na+/H+ and Li+/H+ antiport activity. This is the first mutational analysis of the antiporter of NhaD type and the first demonstration of Thr residue being indispensable for Na+/H+ antiport. We discuss the possible role of Asp344 and Thr345 in the functioning of Vc-NhaD.  相似文献   

12.
Escherichia coli encodes YadB, a protein displaying 34% identity with the catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-binding domain. We show that YadB is a tRNA modifying enzyme that evidently glutamylates the queuosine residue, a modified nucleoside at the wobble position of the tRNAAsp QUC anticodon. This conclusion is supported by a variety of biochemical data and by the inability of the enzyme to glutamylate tRNAAsp isolated from an E.coli tRNA-guanosine transglycosylase minus strain deprived of the capacity to exchange guanosine 34 with queuosine. Structural mimicry between the tRNAAsp anticodon stem and the tRNAGlu amino acid acceptor stem in prokaryotes encoding YadB proteins indicates that the function of these tRNA modifying enzymes, which we rename glutamyl-Q tRNAAsp synthetases, is conserved among prokaryotes.  相似文献   

13.
A spermidine excretion protein in Escherichia coli was looked for among 33 putative drug exporters thus far identified. Cell toxicity and inhibition of growth due to overaccumulation of spermidine were examined in an E. coli strain deficient in spermidine acetyltransferase, an enzyme that metabolizes spermidine. Toxicity and inhibition of cell growth by spermidine were recovered in cells transformed with pUCmdtJI or pMWmdtJI, encoding MdtJ and MdtI, which belong to the small multidrug resistance family of drug exporters. Both mdtJ and mdtI are necessary for recovery from the toxicity of overaccumulated spermidine. It was also found that the level of mdtJI mRNA was increased by spermidine. The spermidine content in cells cultured in the presence of 2 mM spermidine was decreased, and excretion of spermidine from cells was enhanced by MdtJI, indicating that the MdtJI complex can catalyze excretion of spermidine from cells. It was found that Tyr4, Trp5, Glu15, Tyr45, Tyr61, and Glu82 in MdtJ and Glu5, Glu19, Asp60, Trp68, and Trp81 in MdtI are involved in the excretion activity of MdtJI.  相似文献   

14.
Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's disease and other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentration of free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify how negative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudophosphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tau aggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensity by stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative charge into the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.  相似文献   

15.
Glutamyl-queuosine-tRNAAsp synthetase (Glu-Q-RS) and glutamyl-tRNA synthetase (GluRS), differ widely by their function although they share close structural resemblance within their catalytic core of GluRS. In particular both Escherichia coli GluRS and Glu-Q-RS contain a single zinc-binding site in their putative tRNA acceptor stem-binding domain. It has been shown that the zinc is crucial for correct positioning of the tRNAGlu acceptor-end in the active site of E. coli GluRS. To address the role of zinc ion in Glu-Q-RS, the C101S/C103S Glu-Q-RS variant is constructed. Energy dispersive X-ray fluorescence show that the zinc ion still remained coordinated but the variant became structurally labile and acquired aggregation capacity. The extent of aggregation of the protein is significantly decreased in presence of the small substrates and more particularly by adenosine triphosphate. Addition of zinc increased significantly the solubility of the variant. The aminoacylation assay reveals a decrease in activity of the variant even after addition of zinc as compared to the wild-type, although the secondary structure of the protein is not altered as shown by the Fourier transform infrared spectroscopy study.  相似文献   

16.
A novel endodextranase from Paenibacillus sp. (Paenibacillus sp. dextranase; PsDex) was found to mainly produce isomaltotetraose and small amounts of cycloisomaltooligosaccharides (CIs) with a degree of polymerization of 7–14 from dextran. The 1,696-amino acid sequence belonging to the glycosyl hydrolase family 66 (GH-66) has a long insertion (632 residues; Thr451–Val1082), a portion of which shares identity (35% at Ala39–Ser1304 of PsDex) with Pro32–Ala755 of CI glucanotransferase (CITase), a GH-66 enzyme that catalyzes the formation of CIs from dextran. This homologous sequence (Val837–Met932 for PsDex and Tyr404–Tyr492 for CITase), similar to carbohydrate-binding module 35, was not found in other endodextranases (Dexs) devoid of CITase activity. These results support the classification of GH-66 enzymes into three types: (i) Dex showing only dextranolytic activity, (ii) Dex catalyzing hydrolysis with low cyclization activity, and (iii) CITase showing CI-forming activity with low dextranolytic activity. The fact that a C-terminal truncated enzyme (having Ala39–Ser1304) has 50% wild-type PsDex activity indicates that the C-terminal 392 residues are not involved in hydrolysis. GH-66 enzymes possess four conserved acidic residues (Asp189, Asp340, Glu412, and Asp1254 of PsDex) of catalytic candidates. Their amide mutants decreased activity (1/1, 500 to 1/40, 000 times), and D1254N had 36% activity. A chemical rescue approach was applied to D189A, D340G, and E412Q using α-isomaltotetraosyl fluoride with NaN3. D340G or E412Q formed a β- or α-isomaltotetraosyl azide, respectively, strongly indicating Asp340 and Glu412 as a nucleophile and acid/base catalyst, respectively. Interestingly, D189A synthesized small sized dextran from α-isomaltotetraosyl fluoride in the presence of NaN3.  相似文献   

17.
Summary An amino-terminal extension of endothelin-l by the lys-Arg dipeptide in the prosequence (KR-ET-1) greatly increased the ratio of native-type to non-native-type disulfide isomer (96/4 versus 71/29) during the oxidative folding reaction. This improvement was completely abolished by substituting Asn for Asp at position 8 (D8N-KR-ET-1), whereas most of it was maintained with similar carboxamide analogues replaced at Glu10 or Asp18. Structure analyses by circular dichroism spectroscopy revealed that (i) in the carboxylate state, the α-helical content of the native-type isomer of KR-ET-l is higher than that of the native-type isomer of ET-1, while such a variation is not observed in the corresponding non-native-type isomer of KR-ET-l; and (ii) the enhanced α-helicity resulting from the Lys-Arg extension is largely diminished in D8N-KR-ET-l. From these results and our previous findings that the helical structure in KR-ET-l is stabilized by a particular salt bridge between the extended Arg−1 basic moiety and either the Asp8 or Glu10 acidic side chain in Et-1 [Aumelas, A. et al., Biochemistry, 34 (1995) 4546], we conclude that the formation of a specific salt bridge between the side chains of Arg−1 and Asp8 in KR-ET-1 is critical for the predominant generation of the native-type disulfide isomer, probably because it stabilizes the helical structure of parental ET-1.  相似文献   

18.
Using four clones isolated from Echinococcus multilocularis cDNA library with alveolar echinococcosis (AE) patient sera, various antigens were expressed as ThioHis tag-fused protein. Recombinant EmII/3 antigen was produced as the five fragments divided into the N-terminal (#5 and #5s), the central (#6 and #6s) and the C-terminal domain (#7). Immunoblot analysis revealed that the #7 showed significant reactivity whereas those of #5 and #5s were relatively low. The #6 and #6s also showed lower reactivity than that of #7, although the two minor bands of #6 reacted with every serum. These results suggested that an immunodominant region of EmII/3 locate within the C-terminal one third. The #8s recombinant antigen, Ser23–Glu176 of actin filament fragmenting protein (AFFP), apparently reacted with the AE patient sera, while the #1 antigen synthesized as a full-length antigen B1 did not show such high reactivity. Thus, #7 and #8s antigens showed significant potential for use in immunodetection of AE. In addition, the specific antibodies against #7 and #8s reacted with specific antigens in crude extract of E. multilocularis cyst, indicating that these antigens retained antigenicity common to native EmII/3 and AFFP, respectively.  相似文献   

19.
Glutamyl-queuosine tRNAAsp synthetase (Glu-Q-RS) from Escherichia coli is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS) that catalyzes glutamylation of queuosine in the wobble position of tRNAAsp. Despite important structural similarities, Glu-Q-RS and GluRS diverge strongly by their functional properties. The only feature common to both enzymes consists in the activation of Glu to form Glu-AMP, the intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes differ by the mechanism of selection of the cognate amino acid and by the mechanism of its activation. Whereas GluRS selects l-Glu and activates it only in the presence of the cognate tRNAGlu, Glu-Q-RS forms Glu-AMP in the absence of tRNA. Moreover, while GluRS transfers the activated Glu to the 3′ accepting end of the cognate tRNAGlu, Glu-Q-RS transfers the activated Glu to Q34 located in the anticodon loop of the noncognate tRNAAsp. In order to gain insight into the structural elements leading to distinct mechanisms of amino acid activation, we solved the three-dimensional structure of Glu-Q-RS complexed to Glu and compared it to the structure of the GluRS·Glu complex. Comparison of the catalytic site of Glu-Q-RS with that of GluRS, combined with binding experiments of amino acids, shows that a restricted number of residues determine distinct catalytic properties of amino acid recognition and activation by the two enzymes. Furthermore, to explore the structural basis of the distinct aminoacylation properties of the two enzymes and to understand why Glu-Q-RS glutamylates only tRNAAsp among the tRNAs possessing queuosine in position 34, we performed a tRNA mutational analysis to search for the elements of tRNAAsp that determine recognition by Glu-Q-RS. The analyses made on tRNAAsp and tRNAAsn show that the presence of a C in position 38 is crucial for glutamylation of Q34. The results are discussed in the context of the evolution and adaptation of the tRNA glutamylation system.  相似文献   

20.
PrimPol is a human primase/polymerase specialized in downstream repriming of stalled forks during both nuclear and mitochondrial DNA replication. Like most primases and polymerases, PrimPol requires divalent metal cations, as Mg2+ or Mn2+, used as cofactors for catalysis. However, little is known about the consequences of using these two metal cofactors in combination, which would be the most physiological scenario during PrimPol-mediated reactions, and the individual contribution of the putative carboxylate residues (Asp114, Glu116 and Asp280) acting as metal ligands. By site-directed mutagenesis in human PrimPol, we confirmed the catalytic relevance of these three carboxylates, and identified Glu116 as a relevant enhancer of distinctive PrimPol reactions, which are highly dependent on Mn2+. Herein, we evidenced that PrimPol Glu116 contributes to error-prone tolerance of 8oxodG more markedly when both Mg2+ and Mn2+ ions are present. Moreover, Glu116 was important for TLS events mediated by primer/template realignments, and crucial to achieving an optimal primase activity, processes in which Mn2+ is largely preferred. EMSA analysis of PrimPol:ssDNA:dNTP pre-ternary complex indicated a critical role of each metal ligand, and a significant impairment when Glu116 was changed to a more conventional aspartate. These data suggest that PrimPol active site requires a specific motif A (DxE) to favor the use of Mn2+ ions in order to achieve optimal incoming nucleotide stabilization, especially required during primer synthesis.  相似文献   

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