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1.
A completely transformation-defective point mutant of polyomavirus middle T antigen which retains full associated phosphatidylinositol kinase activity. 总被引:18,自引:8,他引:10
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By using a random mutagenesis procedure combined with a recombinant retrovirus vector, mutants of polyomavirus middle T antigen (MTAg) were generated. Three new MTAg mutants with various degrees of transformation competence were more thoroughly characterized. All of the mutants produced a stable MTAg, as assessed by metabolic labeling or immunoblotting, and each mutant possessed wild-type levels of associated tyrosine kinase activity and associated phosphatidylinositol-3 (PI-3) kinase activity. One of these mutants, with a substitution of leucine for proline at amino acid 248 of MTAg (248m) was completely transformation defective, as measured in a focus-forming assay. Furthermore, the pattern of phosphorylation of 248m in vivo was identical to that of wild-type MTAg, and the kinetics of association of MTAg with an 85-kilodalton protein, the putative PI kinase, was not altered. Similarly, the pattern of PI derivatives obtained in an in vitro kinase assay was not altered by the substitution at amino acid 248. Since the single base pair mutation at amino acid 248 resulted in an MTAg that was completely transformation defective despite possessing wild-type levels of kinase activities, this suggests that neither tyrosine kinase nor PI-3 kinase activity nor the combination of both are sufficient for transformation by MTAg. 相似文献
2.
3.
The NPEY sequence is not necessary for endocytosis and processing of insulin-receptor complexes. 总被引:2,自引:0,他引:2
P Berhanu R H Ibrahim-Schneck C Anderson W M Wood 《Molecular endocrinology (Baltimore, Md.)》1991,5(12):1827-1835
The tetrameric amino acid sequence AsnProXTyr (NPXY), where X represents any amino acid, is conserved in the intracytoplasmic domains of several membrane proteins and has been postulated to play a role in receptor-mediated endocytosis. The human insulin receptor (hIR) contains a single copy of the sequence AsnProGluTyr (NPEY) in its intracytoplasmic domain. To determine if this putative consensus sequence is necessary for endocytic functions of hIR, we constructed a mutant receptor, hIR delta NPEY, that lacks NPEY sequence, stably expressed this mutant receptor in Chinese hamster ovary cells, and then studied its endocytic functions. When compared to wild type hIR similarly expressed in Chinese hamster ovary cells, the hIR delta NPEY mutant exhibited: 1) normal subunit organization and insulin binding affinity; 2) essentially normal internalization of covalent photoaffinity labeled insulin-receptor complexes; and 3) normal internalization of receptor-bound [125I]insulin as well as normal degradation and release of the internalized insulin. Therefore, we conclude that the NPEY sequence in the juxtamembrane domain of hIR is not necessary for its endocytic function. 相似文献
4.
NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor 总被引:85,自引:0,他引:85
Rapid internalization of the cell surface low density lipoprotein (LDL) receptor requires the first 22 amino acids of the cytoplasmic domain (residues 790-811), which must include an aromatic residue at position 807. In the human LDL receptor, this position is part of a tetrameric sequence, NPVY. A similar tetramer, NPXY (where X stands for any amino acid), is conserved in LDL receptors from six species (including Xenopus laevis) and in two members of the LDL receptor gene family, human LDL receptor-related protein and rat GP330. To determine whether the NPXY sequence is necessary for coated pit-mediated internalization, we used oligonucleotide-directed mutagenesis to substitute alanines for individual amino acids in the cytoplasmic tail of the human LDL receptor. Substitution of alanine for Asn804, Pro805, or Tyr807 (but not Val806) markedly reduced internalization. Only one other amino acid in the cytoplasmic 22-mer (Phe802) was important for internalization. A review of published data revealed NPXY sequences in cytoplasmic domains of at least 10 other cell surface proteins, including tyrosine kinase-linked receptors of the epidermal growth factor and insulin receptor family, the beta-subunits of three integrin receptors, and the amyloid A4 precursor protein. This occurrence is much more frequent than would be expected by chance alone. The possibility of a conditional role for the NPXY sequence in ligand-independent internalization of these proteins is discussed. 相似文献
5.
The carboxyl-terminal sequence of the lac repressor protein contains heptad repeats of leucines at positions 342, 349, and 356 that are required for tetramer assembly, as substitution of these leucine residues yields solely dimeric species (Chakerian, A. E., Tesmer, V. M., Manly, S. P., Brackett, J. K., Lynch, M. J., Hoh, J. T., and Matthews, K. S. (1991) J. Biol. Chem. 266, 1371-1374; Alberti, S., Oehler, S., von Wilcken-Bergmann, B., Kr?mer, H., and Müller-Hill, B. (1991) New Biol. 3, 57-62). To further investigate this region, which may form a leucine zipper motif, a family of lac repressor carboxyl-terminal deletion mutants eliminating the last 4, 5, 11, 18, and 32 amino acids (aa) has been constructed. The -4 aa mutant, in which all of the leucines in the presumed leucine zipper are intact, is tetrameric and displays operator and inducer binding properties similar to wild-type repressor. The -5 aa, -11 aa, -18 aa, and -32 aa deletion mutants, depleted of 1, 2, or all 3 of the leucines in the heptad repeats, are all dimeric, as demonstrated by gel filtration chromatography. Circular dichroism spectra and protease digestion studies indicate similar secondary/tertiary structures for the mutant and wild-type proteins. Differences in reaction with a monoclonal antibody specific for a subunit interface are observed for the dimeric versus tetrameric proteins, indicative of exposure of the target epitope as a consequence of deletion. Inducer binding properties of the deletion mutants are similar to wild-type tetrameric repressor at neutral pH. Only small differences in affinity and cooperativity from wild-type are evident at elevated pH; thus, the cooperative unit within the tetramer appears to be the dimer. "Apparent" operator binding affinity for the dimeric proteins is diminished, although minimal change in operator dissociation rate constants was observed. The diminution in apparent operator affinity may therefore derive from either 1) dissociation of the dimeric mutants to monomer generating a linked equilibrium or 2) alterations in intrinsic operator affinity of the dimers; the former explanation is favored. This detailed characterization of the purified mutant proteins confirms that the carboxyl-terminal region is involved in the dimer-dimer interface and demonstrates that cooperativity for inducer binding is contained within the dimer unit of the tetramer structure. 相似文献
6.
Aichi virus 2A protein is involved in viral RNA replication 总被引:1,自引:0,他引:1
The Aichi virus 2A protein is not a protease, unlike many other picornavirus 2A proteins, and it is related to a cellular protein, H-rev107. Here, we examined the replication properties of two 2A mutants in Vero cells and a cell-free translation/replication system. In one mutant, amino acids 36 to 126 were replaced with an unrelated amino acid sequence. In the other mutant, the NC motif conserved in the H-rev107 family of proteins was changed to alanine residues. The two mutations abolished virus replication in cells. The mutations affected both negative- and positive-strand synthesis, the defect in positive-strand synthesis being more severe than that in negative-strand synthesis. 相似文献
7.
Specific binding of the replication protein of plasmid pPS10 to direct and inverted repeats is mediated by an HTH motif. 总被引:3,自引:0,他引:3
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The initiator protein of the plasmid pPS10, RepA, has a putative helix-turn-helix (HTH) motif at its C-terminal end. RepA dimers bind to an inverted repeat at the repA promoter (repAP) to autoregulate RepA synthesis. [D. García de Viedma, et al. (1996) EMBO J. in press]. RepA monomers bind to four direct repeats at the origin of replication (oriV) to initiate pPS10 replication This report shows that randomly generated mutations in RepA, associated with defficiencies in autoregulation, map either at the putative HTH motif or in its vicinity. These mutant proteins do not promote pPS10 replication and are severely affected in binding to both the repAP and oriV regions in vitro. Revertants of a mutant that map in the vicinity of the HTH motif have been obtained and correspond to a second amino acid substitution far upstream of the motif. However, reversion of mutants that map in the helices of the motif occurs less frequently, at least by an order of magnitude. All these data indicate that the helices of the HTH motif play an essential role in specific RepA-DNA interactions, although additional regions also seem to be involved in DNA binding activity. Some mutations have slightly different effects in replication and autoregulation, suggesting that the role of the HTH motif in the interaction of RepA dimers or monomers with their respective DNA targets (IR or DR) is not the same. 相似文献
8.
Characterization of the antigenic structure of herpes simplex virus type 1 glycoprotein C through DNA sequence analysis of monoclonal antibody-resistant mutants.
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Earlier studies of a group of monoclonal antibody-resistant (mar) mutants of herpes simplex virus type 1 glycoprotein C (gC) operationally defined two distinct antigenic sites on this molecule, each consisting of numerous overlapping epitopes. In this report, we further define epitopes of gC by sequence analysis of the mar mutant gC genes. In 18 mar mutants studied, the mar phenotype was associated with a single nucleotide substitution and a single predicted amino acid change. The mutations were localized to two regions within the coding sequence of the external domain of gC and correlated with the two previously defined antigenic sites. The predicted amino acid substitutions of site I mutants resided between residues Gln-307 and Pro-373, whereas those of site II mutants occurred between amino acids Arg-129 and Glu-247. Of the 12 site II mutations, 9 induced amino acid substitutions within an arginine-rich segment of 8 amino acids extending from residues 143 to 151. The clustering of the majority of substituted residues suggests that they contribute to the structure of the affected sites. Moreover, the patterns of substitutions which affected recognition by antibodies with similar epitope specificities provided evidence that epitope structures are physically linked and overlap within antigenic sites. Of the nine epitopes defined on the basis of mutations, three were located within site I and six were located within site II. Substituted residues affecting the site I epitopes did not overlap substituted residues of site II, supporting our earlier conclusion that sites I and II reside in spatially distinct antigenic domains. A computer analysis of the distribution of charged residues and the predicted secondary structural features of wild-type gC revealed that the two antigenic sites reside within the most hydrophilic regions of the molecule and that the antigenic residues are likely to be organized as beta sheets which loop out from the surface of the molecule. Together, these data and our previous studies support the conclusion that the mar mutations identified by sequence analysis very likely occur within or near the epitope structures themselves. Thus, two highly antigenic regions of gC have now been physically and genetically mapped to well-defined domains of the protein molecule. 相似文献
9.
The YXXL motif, but not the two NPXY motifs, serves as the dominant endocytosis signal for low density lipoprotein receptor-related protein 总被引:1,自引:0,他引:1
Li Y Marzolo MP van Kerkhof P Strous GJ Bu G 《The Journal of biological chemistry》2000,275(22):17187-17194
All members of the low density lipoprotein (LDL) receptor family contain at least one copy of the NPXY sequence within their cytoplasmic tails. For the LDL receptor, it has been demonstrated that the NPXY motif serves as a signal for rapid endocytosis through coated pits. Thus, it is generally believed that the NPXY sequences function as endocytosis signals for all the LDL receptor family members. The primary aim of this study is to define the endocytosis signal(s) within the cytoplasmic tail of LDL receptor-related protein (LRP). By using LRP minireceptors, which mimic the function and trafficking of full-length endogenous LRP, we demonstrate that the YXXL motif, but not the two NPXY motifs, serves as the dominant signal for LRP endocytosis. We also found that the distal di-leucine motif within the LRP tail contributes to its endocytosis, and its function is independent of the YXXL motif. Although the proximal NPXY motif and the proximal di-leucine motif each play a limited role in LRP endocytosis in the context of the full-length tail, these motifs were functional within the truncated receptor tail. In addition, we show that LRP minireceptor mutants defective in endocytosis signal(s) accumulate at the cell surface and are less efficient in delivery of ligand for degradation. 相似文献
10.
The YIPP (tyrosine-isoleucine-proline-proline, amino acids 319-322) motif within the C-terminal part of the human AT(1) receptor is associated with angiotensin II (AII)-induced activation of the Jak-STAT pathway and phospholipase Cgamma1 phosphorylation. We report here that mutations of the YIPP motif strongly affect ligand-binding to the receptor. We analysed AT(1) receptors of the wild type (WT) and 11 mutants with a FLAG-epitope-tag within their C-terminal portion. Mutations of the "P-P" amino acid sequence of this motif decreased both AII binding and the AII-induced intracellular Ca(2+) transients. Mutant and WT receptors were expressed equally in the cell membrane and were localized within the plasma membrane. These results suggest that the "P-P" amino acid sequence within the YIPP motif is important for AII binding to the AT(1) receptor. 相似文献
11.
Matthew Rienzo Angela R. Rocchi Stephanie D. Threatt Dennis A. Dougherty Sarah C. R. Lummis 《The Journal of biological chemistry》2016,291(12):6272-6280
Gloeobacter violaceus ligand-gated ion channel (GLIC) has served as a valuable structural and functional model for the eukaryotic Cys-loop receptor superfamily. In Cys-loop and other receptors, we have previously demonstrated the crucial roles played by several conserved prolines. Here we explore the role of prolines in the gating transitions of GLIC. As conventional substitutions at some positions resulted in nonfunctional proteins, we used in vivo non-canonical amino acid mutagenesis to determine the specific structural requirements at these sites. Receptors were expressed heterologously in Xenopus laevis oocytes, and whole-cell electrophysiology was used to monitor channel activity. Pro-119 in the Cys-loop, Pro-198 and Pro-203 in the M1 helix, and Pro-299 in the M4 helix were sensitive to substitution, and distinct roles in receptor activity were revealed for each. In the context of the available structural data for GLIC, the behaviors of Pro-119, Pro-203, and Pro-299 mutants are consistent with earlier proline mutagenesis work. However, the Pro-198 site displays a unique phenotype that gives evidence of the importance of the region surrounding this residue for the correct functioning of GLIC. 相似文献
12.
Sueo Urushizaki Yasuo Ota Tetsuya Haruyama Hiromasa Kano Kuniomi Matsumoto Minoru Haga 《Bioscience, biotechnology, and biochemistry》2013,77(12):3159-3161
To find amino acid residues which are required for glucoamylase activity, mutant glucoamylase genes were constructed by in vitro mutations of GLU1 DNA encoding Saccharomycopsis fibuligera glucoamylase and introduced into Saccharomyces cerevisiae, and the resulting mutant proteins were assayed for enzymatic activities. Eighteen mutant proteins were obtained by random insertions of a BamHX linker DNA. Six out of 7 proteins with mutations in conserved regions among divergent glucoamylases showed no activities, while 8 out of 11 proteins with mutations in unconserved regions had similar specific activities to a wild-type value, suggesting that the conserved regions are important to the activity. A series of amino-terminal deletion mutants were also constructed. A mutant protein with a deletion of only two amino acid residues from the amino terminus had a significant reduction in the activity, suggesting an essential role for the amino-terminal peptide. Ten mutant proteins with single amino acid replacements were produced by site-directed mutagenesis. Analyses for thermal stability and temperature dependency of these mutant proteins revealed that Ala81, Asp89, Trp94, Arg96, Asp97, and Trp166 are required for wild-type levels of activities, and that at least Ala81 and Asp89 are not essential to catalytic activities, but act in thermal stability. 相似文献
13.
The Bacillus subtilis spo0J gene: evidence for involvement in catabolite repression of sporulation. 总被引:5,自引:2,他引:3
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Previous observations concerning the ability of the Bacillus subtilis bacteriophages SP10 and PMB12 to suppress mutations in spo0J and to make wild-type sporulation catabolite resistant suggested that spo0J had a role in catabolite repression of sporulation. This suggestion was supported in the present report by the ability of the catabolite-resistant sporulation mutation crsF4 to suppress a Tn917 insertion mutation of the B. subtilis spo0J locus (spo0J::Tn917 omega HU261) in medium without glucose. Although crsF4 and SP10 made wild-type B. subtilis sporulation catabolite resistant, neither crsF4 nor SP10 caused a mutant with spo0J::Tn917 omega HU261 to sporulate in medium with glucose. Sequencing the spo0J locus revealed an open reading frame that was 179 codons in length. Disruption of the open reading frame resulted in a sporulation-negative (Spo-) phenotype that was similar to those of other spo0J mutations. Analysis of the deduced amino acid sequence of the spo0J locus indicated that the spo0J gene product contains an alpha-helix-turn-alpha-helix unit similar to the motif found in lambda Cro-like DNA-binding proteins. 相似文献
14.
Structural significance of the GTP-binding domain of ras p21 studied by site-directed mutagenesis. 总被引:7,自引:3,他引:4
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Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, or 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding. 相似文献
15.
PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intra- peroxisomal protein that is a member of the WD repeat family and is essential for the import of thiolase into peroxisomes 总被引:9,自引:6,他引:3
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《The Journal of cell biology》1995,129(1):65-80
We have previously described mutant S. cerevisiae that are defective in peroxisome biogenesis (peb mutants) (Zhang, J. W., Y. Han, and P. B. Lazarow. 1993. J. Cell Biol. 123:1133-1147.). In some mutants, peroxisomes are undetectable. Other mutants contain normal-looking peroxisomes but fail to package subsets of peroxisomal proteins into the organelle (Zhang, J. W., C. Luckey, and P. B. Lazarow. 1993. Mol. Biol. Cell. 4:1351-1359.). In peb1 (pas7) cells, for example, the peroxisomes contain proteins that are targeted by COOH-terminal tripeptides and contain acyl-CoA oxidase (which is probably targeted by internal oligopeptides), but fail to import thiolase (which is targeted by an NH(2)-terminal 16-amino acid sequence). These and other data suggest that there are three branches in the pathway for the import of proteins into peroxisomes, each of which contains a receptor for one type of peroxisomal topogenic information. Here, we report the cloning and characterization of the PEB1 gene, that encodes a 42,320-Da hydrophilic protein with no predicted transmembrane segment. The protein contains six WD repeats, a motif which has been found in 27 proteins involved in diverse cellular functions. The PEB1 gene product was tagged with the hemagglutinin epitope and found to rescue thiolase import in the peb1 null mutant. The epitope-tagged protein was shown to be inside of peroxisomes by immunofluorescence, digitonin permeabilization, equilibrium density centrifugation, immunoelectron microscopy, and proteinase K protection studies. The PEB1 gene product does not cleave the thiolase-targeting sequence. It may function to draw thiolase into peroxisomes. 相似文献
16.
A series of mutant lac repressor proteins at positions 281 or 282 was isolated for detailed characterization. Although Cys281 modification by sulfhydryl reagents abrogates pH effects on inducer binding and diminishes operator binding (Daly, T. J., Olson, J. S., and Matthews, K. S. (1986) Biochemistry 25, 5468-5474), substitution at this site by alanine, serine, phenylalanine, isoleucine, or methionine did not abolish completely the pH shift nor affect operator affinity. Thus, ionization of the sulfhydryl residue does not account fully for the alterations in inducer affinity and cooperativity of binding observed with elevated pH. Substitution for Cys281 did, however, alter the kinetic parameters for inducer association with the protein. The polarity of the side chain at 281 influenced the rates of sugar binding, presumably by altering the rate of opening/closing of the binding site. Furthermore, the presence of the branched side chain of isoleucine at position 281 disrupted oligomerization of the repressor. In contrast to the tolerance for substitution at 281, the only amino acid side chain exchanges for Tyr282 which yielded tetrameric protein with near normal operator binding characteristics were phenylalanine and leucine; this result is consistent with studies of suppressed nonsense mutations at position 282 which indicated repression occurred only for the corresponding substitutions (Kleina, L. G., and Miller, J. H. (1990) J. Mol. Biol. 221, 295-318). Despite the tetrameric character of the Y282F mutant protein, the pH dependence and cooperativity of inducer binding for this mutant protein were altered. All amino acid substitutions other than phenylalanine and leucine at this position resulted in either monomeric protein or no detectable repressor in the cell. Thus, the hydrophobic character of the side chain at position 282 is essential for tetramer formation, and the phenyl ring alone alters inducer binding parameters. The monomeric mutant proteins with substitutions for Tyr282 exhibited lower stability than their tetrameric counterparts, and the absence of dimer formation suggests alterations at this site affect both dimer and tetramer interfaces. Based on previous genetic studies and our detailed mutant characterization, the region encompassing 281 and 282, indicated by secondary structure prediction to be a turn or coil, is essential for oligomer formation and additionally exerts a strong influence on the dynamic properties of the protein, presumably mediated by interactions at the subunit interface which regulate the rate of opening and closing of the inducer binding cleft. 相似文献
17.
Mutant Lrp1 knock-in mice generated by recombinase-mediated cassette exchange reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development
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Roebroek AJ Reekmans S Lauwers A Feyaerts N Smeijers L Hartmann D 《Molecular and cellular biology》2006,26(2):605-616
Lrp1 knock-in mice carrying either a wild-type allele or three different mutated alleles encoding the multifunctional endocytic receptor LRP1 were generated by recombinase-mediated cassette exchange (RMCE). Reinsertion by RMCE of a wild-type allele led to a normal pattern and level of gene expression and a completely normal phenotype, indicating that the RMCE procedure itself is neutral with respect to the function of the gene locus. In contrast, reinsertion of mutated LRP1 alleles carrying either inactivating mutations in the proximal NPXY motif (NPTY-->AATA) of the cytoplasmic domain or in the furin cleavage site (RHRR-->AHAA) caused distinctive liver phenotypes: respectively, either a late fetal destruction of the organ causing perinatal death or a selective enlargement of von-Kupffer cell lysosomes reminiscent of a mild lysosomal storage without an apparent negative effect on animal survival. Notably, mutation of the distal NPXY motif overlapping with an YXXL motif (NPVYATL-->AAVAATL) did not cause any obvious pathological effect. The mutations showed no effect on the LRP1 expression level; however, as expected, the proteolytic maturation of LRP1 into its two subunits was significantly impaired, although not completely abolished, in the furin cleavage mutant. These data demonstrate that RMCE is a reliable and efficient approach to generate multiple mutant knock-in alleles for in vivo functional analysis of individual domains or motifs of large multidomain proteins. Its application in Lrp1 reveals dramatically variant phenotypes, of which further characterization will definitively contribute to our understanding of the biology of this multifunctional receptor. 相似文献
18.
Mutational analysis of polyomavirus small-T-antigen functions in productive infection and in transformation. 总被引:10,自引:8,他引:2
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The function of polyomavirus small T antigen in productive infection and in transformation was studied. Transfection of permissive mouse cells with mixtures of mutants that express only one type of T antigen showed that small T antigen increased large-T-antigen-dependent viral DNA synthesis approximately 10-fold. Under the same conditions, small T antigen was also essential for the formation of infectious virus particles. To analyze these activities of small T antigen, mutants producing protein with single amino acid replacements were constructed. Two mutants, bc1073 and bc1075, were characterized. Although both mutations led to the substitution of amino acid residues of more than one T antigen, the phenotype of both mutants was associated with alterations of the small T antigen. Both mutant proteins had lost their activity in the maturation of infectious virus particles. The bc1075 but not the bc1073 small T antigen had also lost its ability to stimulate viral DNA synthesis in mouse 3T6 cells. Finally, both mutants retained a third activity of small T antigen: to confer on rat cells also expressing middle T antigen the ability to grow efficiently in semisolid medium. The phenotypes of the mutants in these three assays suggest that small T antigen has at least three separate functions. 相似文献
19.
To localize the DNA binding domain of the Saccharomyces cerevisiae Ars binding factor 1 (ABF1), a multifunctional DNA binding protein, plasmid constructs carrying point mutations and internal deletions in the ABF1 gene were generated and expressed in Escherichia coli. Normal and mutant ABF1 proteins were purified by affinity chromatography and their DNA binding activities were analyzed. The substitution of His61, Cys66 and His67 respectively, located in the zinc finger motif in the N-terminal region (amino acids 40-91), eliminated the DNA binding activity of ABF1 protein. Point mutations in the middle region of ABF1, specifically at Leu353, Leu399, Tyr403, Gly404, Phe410 and Lys434, also eliminated or reduced DNA binding activity. However, the DNA binding activity of point mutants of Ser307, Ser496 and Glu649 was the same as that of wild-type ABF1 protein and deletion mutants of amino acids 200-265, between the zinc finger region and the middle region (residues 323-496) retained DNA binding activity. As a result, we confirmed that the DNA binding domain of ABF1 appears to be bipartite and another DNA binding motif, other than the zinc finger motif, is situated between amino acid residues 323 and 496. 相似文献
20.
María A. Blasco José A. Esteban Juan Méndez Luis Blanco Margarita Salas 《Chromosoma》1992,102(1):S32-S38
TheBacillus subtilis phage ø29 DNA polymerase, involved in protein-primed viral DNA replication, contains several amino acid consensus sequences common to other eukaryotic-type DNA polymerases. Using site-directed mutagenesis, we have studied the functional significance of a C-terminal conserved region, represented by the Lys-X-Tyr (“K-Y”) motif. Single point mutants have been constructed and the corresponding proteins have been overproduced and characterized. Measurements of the activity of the mutant proteins indicated that the invariant Lys and Tyr residues play a critical role in DNA polymerization. Interestingly, substitution of the invariant Lys either by Arg or Thr, produced enzymes with an increased or a largely reduced, respectively, capability to use a protein as primer, an intrinsic property of TP-priming DNA polymerases. On the other hand, the viral protein p6, which stimulates initiation of ø29 DNA replication by formation of a nucleoprotein complex at both DNA replication origins, increased (about 5-fold) the insertion fidelity of ø29 DNA polymerase during the formation of the TP-dAMP initiation complex. We propose a model in which the special strategy to maintain the integrity of the ø29 DNA ends, by means of a “sliding-back” mechanism, could also contribute to increase the fidelity of ø29 DNA replication. 相似文献