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Aneel K. Aggarwal - One of the best experts on this subject based on the ideXlab platform.
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Asymmetric DNA recognition by the OkrAI endonuclease, an isoschizomer of BamHI
Nucleic Acids Research, 2010Co-Authors: Eva Vanamee, Hector Viadiu, Siu-hong Chan, Ajay Ummat, Adrian M. Hartline, Aneel K. AggarwalAbstract:Restriction enzymes share little or no sequence homology with the exception of isoschizomers, or enzymes that recognize and cleave the same DNA sequence. We present here the structure of a BamHI isoschizomer, OkrAI, bound to the same DNA sequence (TATGGATCCATA) as that cocrystallized with BamHI. We show that OkrAI is a more minimal version of BamHI, lacking not only the N- and C-terminal helices but also an internal 3(10) helix and containing β-strands that are shorter than those in BamHI. Despite these structural differences, OkrAI recognizes the DNA in a remarkably similar manner to BamHI, including asymmetric contacts via C-terminal 'arms' that appear to 'compete' for the minor groove. However, the arms are shorter than in BamHI. We observe similar DNA-binding affinities between OkrAI and BamHI but OkrAI has higher star activity (at 37°C) compared to BamHI. Together, the OkrAI and BamHI structures offer a rare opportunity to compare two restriction enzymes that work on exactly the same DNA substrate.
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Crystal Structure of BstYI at 1.85 Å Resolution: A Thermophilic Restriction Endonuclease with Overlapping Specificities to BamHI and BglII
Journal of Molecular Biology, 2004Co-Authors: Sharon A. Townson, Eva Vanamee, James C. Samuelson, Thomas A. Edwards, Carlos R. Escalante, Aneel K. AggarwalAbstract:Abstract We report here the structure of BstYI, an “intermediate” type II restriction endonuclease with overlapping sequence specificities to BamHI and BglII. BstYI, a thermophilic endonuclease, recognizes and cleaves the degenerate hexanucleotide sequence 5′-RGATCY-3′ (where R=A or G and Y=C or T), cleaving DNA after the 5′-R on each strand to produce four-base (5′) staggered ends. The crystal structure of free BstYI was solved at 1.85 A resolution by multiwavelength anomalous dispersion (MAD) phasing. Comparison with BamHI and BglII reveals a strong structural consensus between all three enzymes mapping to the α/β core domain and residues involved in catalysis. Unexpectedly, BstYI also contains an additional “arm” substructure outside of the core protein, which enables the enzyme to adopt a more compact, intertwined dimer structure compared with BamHI and BglII. This arm substructure may underlie the thermostability of BstYI. We identify putative DNA recognition residues and speculate as to how this enzyme achieves a “relaxed” DNA specificity.
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Energetic and Structural Considerations for the Mechanism of Protein Sliding along DNA in the Nonspecific BamHI-DNA Complex
Biophysical Journal, 2003Co-Authors: Jian Sun, Hector Viadiu, Aneel K. Aggarwal, Harel WeinsteinAbstract:The molecular mechanism by which DNA-binding proteins find their specific binding sites is still unclear. To gain insights into structural and energetic elements of this mechanism, we used the crystal structure of the nonspecific BamHI-DNA complex as a template to study the dominant electrostatic interaction in the nonspecific association of protein with DNA, and the possible sliding pathways that could be sustained by such an interaction. Based on calculations using the nonlinear Poisson-Boltzmann method and Brownian dynamics, a model is proposed for the initial nonspecific binding of BamHI to B-form DNA that differs from that seen in the crystal structure of the nonspecific complex. The model is electrostatically favorable, and the salt dependence as well as other thermodynamic parameters calculated for this model are in good agreement with experimental results. Several residues in BamHI are identified for their important contribution to the energy in the nonspecific binding model, and specific mutagenesis experiments are proposed to test the model on this basis. We show that a favorable sliding pathway of the protein along DNA is helical.
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Crystallization of restriction endonuclease BamHI with nonspecific DNA.
Journal of Structural Biology, 2000Co-Authors: Hector Viadiu, Ira Schildkraut, Rebecca Kucera, Aneel K. AggarwalAbstract:Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a million-fold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 5′-ATGAATCCATA-3′, yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 A with synchrotron radiation. The cocrystals belong to the space group P212121 with unit cell dimensions of a = 114.8 A, b = 91.1 A, c = 66.4 A, α = 90°, β = 90°, γ = 90°. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA–protein complexes.
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CRYSTALLIZATION NOTE Crystallization of Restriction Endonuclease BamHI with Nonspecific DNA
2000Co-Authors: Hector Viadiu, Ira Schildkraut, Rebecca Kucera, Aneel K. AggarwalAbstract:Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a millionfold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 58-ATGAATCCATA-38, yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 A with synchrotron radiation. The cocrystals belong to the space group P212121 with unit cell dimensions of a 5 114.8 A, b 5 91.1 A, c 5 66.4 A, a 5 90°, b5 90°, g5 90°. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA‐ protein complexes. r 2000 Academic Press
Ira Schildkraut - One of the best experts on this subject based on the ideXlab platform.
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Crystallization of restriction endonuclease BamHI with nonspecific DNA.
Journal of Structural Biology, 2000Co-Authors: Hector Viadiu, Ira Schildkraut, Rebecca Kucera, Aneel K. AggarwalAbstract:Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a million-fold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 5′-ATGAATCCATA-3′, yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 A with synchrotron radiation. The cocrystals belong to the space group P212121 with unit cell dimensions of a = 114.8 A, b = 91.1 A, c = 66.4 A, α = 90°, β = 90°, γ = 90°. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA–protein complexes.
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CRYSTALLIZATION NOTE Crystallization of Restriction Endonuclease BamHI with Nonspecific DNA
2000Co-Authors: Hector Viadiu, Ira Schildkraut, Rebecca Kucera, Aneel K. AggarwalAbstract:Restriction endonucleases show extraordinary specificity in distinguishing specific from nonspecific DNA sequences. A single basepair change within the recognition sequence results in over a millionfold loss in activity. To understand the basis of this sequence discrimination, it is just as important to study the nonspecific complex as the specific complex. We describe here the crystallization of restriction endonuclease BamHI with several nonspecific oligonucleotides. The 11-mer, 58-ATGAATCCATA-38, yielded cocrystals with BamHI, in the presence of low salt, that diffracted to 1.9 A with synchrotron radiation. The cocrystals belong to the space group P212121 with unit cell dimensions of a 5 114.8 A, b 5 91.1 A, c 5 66.4 A, a 5 90°, b5 90°, g5 90°. This success in the cocrystallization of BamHI with a nonspecific DNA provides insights for future attempts at crystallization of other nonspecific DNA‐ protein complexes. r 2000 Academic Press
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A mutant of BamHI restriction endonuclease which requires N6-methyladenine for cleavage.
Journal of Molecular Biology, 1999Co-Authors: Richard D. Whitaker, Lydia F Dorner, Ira SchildkrautAbstract:Amino acid residues Asn116 and Ser118 of the restriction endonuclease BamHI make several sequence-specific and water-bridged contacts to the DNA bases. An in vivo selection was used to isolate BamHI variants at position 116, 118 and 122 which maintained sequence specificity to GGATCC sites. Here, the variants N116H, N116H/S118G and S118G were purified and characterized. The variants N116H and N116H/S118G were found to have lost their ability to cleave unmethylated GGATCC sequences by more than two orders of magnitude, while maintaining nearly wild-type levels of activity on the N6-methyladenine-containing sequence, GGmATCC. In contrast, wild-type BamHI and variant S118G have only a three- to fourfold lower activity on unmethylated GGATCC sequences compared with GGmATCC sequences. The N116 to H116 mutation has effectively altered the specificity of BamHI from an endonuclease which recognizes and cleaves GGATCC and GGmATC, to an endonuclease which only cleaves GGmATCC. The N116H change of specificity is due to the lowered binding affinity for the unmethylated sequence because of the loss of two asparagine-DNA hydrogen bonds and the introduction of a favorable van der Waals contact between the imidazole group of histidine and the N6-methyl group of adenine.
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structure of restriction endonuclease BamHI and its relationship to ecori
Nature, 1994Co-Authors: M Newman, Ira Schildkraut, T Strzelecka, Lydia F Dorner, Aneel K. AggarwalAbstract:Type II restriction endonucleases are characterized by the remarkable specificity with which they cleave specific DNA sequences. Surprisingly, their protein sequences are in most cases unrelated, and no recurring structural motif has yet been identified. We have determined the structure of restriction endonuclease BamHI at 1.95 A resolution. BamHI shows striking resemblance to the structure of endonuclease EcoRI (refs 3, 4), despite the lack of sequence similarity between them. We also observe some curious differences between the two structures, and propose an evolutionary scheme that may explain them. The active site of BamHI is structurally similar to the active sites of EcoRI and EcoRV (ref. 5), but the mechanism by which BamHI activates a water molecule for nucleophilic attack may be different.
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direct selection of binding proficient catalytic deficient variants of BamHI endonuclease
Nucleic Acids Research, 1994Co-Authors: Lydia F Dorner, Ira SchildkrautAbstract:Variants of BamHI endonuclease in which the glutamate 113 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was demonstrated by placing a BamHI recognition sequence, GGATCC, positioned as an operator sequence in an antisense promoter for the aadA gene (spectinomycin resistance). Repression of this promoter relieved the inhibition of expression of spectinomycin resistance. This system was then used to select new binding proficient/cleavage deficient BamHI variants. The BamHI endonuclease gene was mutagenized either by exposure to hydroxylamine or by PCR. The mutagenized DNA was reintroduced into E. coli carrying the aadA gene construct, and transformants that conferred spectinomycin resistance were selected. Twenty Spr transformants were sequenced. Thirteen of these were newly isolated variants of the previously identified D94 and E113 residues which are known to be involved in catalysis. The remaining seven variants were all located at residue 111 and the glutamate 111 residue was shown to be involved with catalysis.
Yasuaki Harabuchi - One of the best experts on this subject based on the ideXlab platform.
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clinical usefulness of serum ebv dna levels of BamHI w and lmp1 for nasal nk t cell lymphoma
Journal of Medical Virology, 2007Co-Authors: Hideyuki Ishii, Takeshi Ogino, Christoph Berger, Toshihiro Nagato, Miki Takahara, David Nadal, Nicole Kochlischmitz, Yasuaki HarabuchiAbstract:Quantitative real-time polymerase chain reaction (PCR) was utilized to measure serum EBV DNA levels of BamHI W fragment and latent membrane protein 1 (LMP1) in 20 nasal natural killer (NK)/T-cell lymphoma patients. Both serum EBV DNAs were detected at high levels in all patients, but the levels were below the limit of detection in all healthy controls. The BamHI Z fragment, Epstein-Barr-replication activator (ZEBRA) expression was detected in a small proportion (0.1-3%) of lymphoma cells from 10 (50%) of the patients. Patients with ZEBRA expression showed significantly higher DNA levels of BamHI W and LMP1 (P = 0.0081, P = 0.004), suggesting that EBV DNA may be caused by EBV replication from lymphoma cells. Kaplan-Meier and univariate analyses revealed that high DNA levels of BamHI W and LMP1 at pre-treatment and high BamHI W DNA level at post-treatment were associated with short disease-free survival and overall survival (P < 0.05, each). Although the DNA levels of BamHI W and LMP1 correlated significantly, their dynamics were not always parallel. Patients with low pre-treatment level of both EBV DNAs showed a favorable course, in contrast to patients with high pre-treatment level of both EBV DNAs who showed an aggressive course (P = 0.0085). More importantly, the high pre-treatment level of both EBV DNAs was determined as the only independent prognostic factor among various prognostic factors. These data suggest that simultaneous measurement of serum levels of both BamHI W and LMP1 DNAs may be useful for diagnosis, disease monitoring, and prediction of prognosis for nasal NK/T-cell lymphoma patients.
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Clinical usefulness of serum EBV DNA levels of BamHI W and LMP1 for Nasal NK/T‐cell lymphoma
Journal of Medical Virology, 2007Co-Authors: Hideyuki Ishii, Takeshi Ogino, Christoph Berger, Nicole Köchli-schmitz, Toshihiro Nagato, Miki Takahara, David Nadal, Yasuaki HarabuchiAbstract:Quantitative real-time polymerase chain reaction (PCR) was utilized to measure serum EBV DNA levels of BamHI W fragment and latent membrane protein 1 (LMP1) in 20 nasal natural killer (NK)/T-cell lymphoma patients. Both serum EBV DNAs were detected at high levels in all patients, but the levels were below the limit of detection in all healthy controls. The BamHI Z fragment, Epstein–Barr-replication activator (ZEBRA) expression was detected in a small proportion (0.1–3%) of lymphoma cells from 10 (50%) of the patients. Patients with ZEBRA expression showed significantly higher DNA levels of BamHI W and LMP1 (P = 0.0081, P = 0.004), suggesting that EBV DNA may be caused by EBV replication from lymphoma cells. Kaplan–Meier and univariate analyses revealed that high DNA levels of BamHI W and LMP1 at pre-treatment and high BamHI W DNA level at post-treatment were associated with short disease-free survival and overall survival (P
Hideyuki Ishii - One of the best experts on this subject based on the ideXlab platform.
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clinical usefulness of serum ebv dna levels of BamHI w and lmp1 for nasal nk t cell lymphoma
Journal of Medical Virology, 2007Co-Authors: Hideyuki Ishii, Takeshi Ogino, Christoph Berger, Toshihiro Nagato, Miki Takahara, David Nadal, Nicole Kochlischmitz, Yasuaki HarabuchiAbstract:Quantitative real-time polymerase chain reaction (PCR) was utilized to measure serum EBV DNA levels of BamHI W fragment and latent membrane protein 1 (LMP1) in 20 nasal natural killer (NK)/T-cell lymphoma patients. Both serum EBV DNAs were detected at high levels in all patients, but the levels were below the limit of detection in all healthy controls. The BamHI Z fragment, Epstein-Barr-replication activator (ZEBRA) expression was detected in a small proportion (0.1-3%) of lymphoma cells from 10 (50%) of the patients. Patients with ZEBRA expression showed significantly higher DNA levels of BamHI W and LMP1 (P = 0.0081, P = 0.004), suggesting that EBV DNA may be caused by EBV replication from lymphoma cells. Kaplan-Meier and univariate analyses revealed that high DNA levels of BamHI W and LMP1 at pre-treatment and high BamHI W DNA level at post-treatment were associated with short disease-free survival and overall survival (P < 0.05, each). Although the DNA levels of BamHI W and LMP1 correlated significantly, their dynamics were not always parallel. Patients with low pre-treatment level of both EBV DNAs showed a favorable course, in contrast to patients with high pre-treatment level of both EBV DNAs who showed an aggressive course (P = 0.0085). More importantly, the high pre-treatment level of both EBV DNAs was determined as the only independent prognostic factor among various prognostic factors. These data suggest that simultaneous measurement of serum levels of both BamHI W and LMP1 DNAs may be useful for diagnosis, disease monitoring, and prediction of prognosis for nasal NK/T-cell lymphoma patients.
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Clinical usefulness of serum EBV DNA levels of BamHI W and LMP1 for Nasal NK/T‐cell lymphoma
Journal of Medical Virology, 2007Co-Authors: Hideyuki Ishii, Takeshi Ogino, Christoph Berger, Nicole Köchli-schmitz, Toshihiro Nagato, Miki Takahara, David Nadal, Yasuaki HarabuchiAbstract:Quantitative real-time polymerase chain reaction (PCR) was utilized to measure serum EBV DNA levels of BamHI W fragment and latent membrane protein 1 (LMP1) in 20 nasal natural killer (NK)/T-cell lymphoma patients. Both serum EBV DNAs were detected at high levels in all patients, but the levels were below the limit of detection in all healthy controls. The BamHI Z fragment, Epstein–Barr-replication activator (ZEBRA) expression was detected in a small proportion (0.1–3%) of lymphoma cells from 10 (50%) of the patients. Patients with ZEBRA expression showed significantly higher DNA levels of BamHI W and LMP1 (P = 0.0081, P = 0.004), suggesting that EBV DNA may be caused by EBV replication from lymphoma cells. Kaplan–Meier and univariate analyses revealed that high DNA levels of BamHI W and LMP1 at pre-treatment and high BamHI W DNA level at post-treatment were associated with short disease-free survival and overall survival (P
Lydia F Dorner - One of the best experts on this subject based on the ideXlab platform.
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A mutant of BamHI restriction endonuclease which requires N6-methyladenine for cleavage.
Journal of Molecular Biology, 1999Co-Authors: Richard D. Whitaker, Lydia F Dorner, Ira SchildkrautAbstract:Amino acid residues Asn116 and Ser118 of the restriction endonuclease BamHI make several sequence-specific and water-bridged contacts to the DNA bases. An in vivo selection was used to isolate BamHI variants at position 116, 118 and 122 which maintained sequence specificity to GGATCC sites. Here, the variants N116H, N116H/S118G and S118G were purified and characterized. The variants N116H and N116H/S118G were found to have lost their ability to cleave unmethylated GGATCC sequences by more than two orders of magnitude, while maintaining nearly wild-type levels of activity on the N6-methyladenine-containing sequence, GGmATCC. In contrast, wild-type BamHI and variant S118G have only a three- to fourfold lower activity on unmethylated GGATCC sequences compared with GGmATCC sequences. The N116 to H116 mutation has effectively altered the specificity of BamHI from an endonuclease which recognizes and cleaves GGATCC and GGmATC, to an endonuclease which only cleaves GGmATCC. The N116H change of specificity is due to the lowered binding affinity for the unmethylated sequence because of the loss of two asparagine-DNA hydrogen bonds and the introduction of a favorable van der Waals contact between the imidazole group of histidine and the N6-methyl group of adenine.
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structure of restriction endonuclease BamHI and its relationship to ecori
Nature, 1994Co-Authors: M Newman, Ira Schildkraut, T Strzelecka, Lydia F Dorner, Aneel K. AggarwalAbstract:Type II restriction endonucleases are characterized by the remarkable specificity with which they cleave specific DNA sequences. Surprisingly, their protein sequences are in most cases unrelated, and no recurring structural motif has yet been identified. We have determined the structure of restriction endonuclease BamHI at 1.95 A resolution. BamHI shows striking resemblance to the structure of endonuclease EcoRI (refs 3, 4), despite the lack of sequence similarity between them. We also observe some curious differences between the two structures, and propose an evolutionary scheme that may explain them. The active site of BamHI is structurally similar to the active sites of EcoRI and EcoRV (ref. 5), but the mechanism by which BamHI activates a water molecule for nucleophilic attack may be different.
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direct selection of binding proficient catalytic deficient variants of BamHI endonuclease
Nucleic Acids Research, 1994Co-Authors: Lydia F Dorner, Ira SchildkrautAbstract:Variants of BamHI endonuclease in which the glutamate 113 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was demonstrated by placing a BamHI recognition sequence, GGATCC, positioned as an operator sequence in an antisense promoter for the aadA gene (spectinomycin resistance). Repression of this promoter relieved the inhibition of expression of spectinomycin resistance. This system was then used to select new binding proficient/cleavage deficient BamHI variants. The BamHI endonuclease gene was mutagenized either by exposure to hydroxylamine or by PCR. The mutagenized DNA was reintroduced into E. coli carrying the aadA gene construct, and transformants that conferred spectinomycin resistance were selected. Twenty Spr transformants were sequenced. Thirteen of these were newly isolated variants of the previously identified D94 and E113 residues which are known to be involved in catalysis. The remaining seven variants were all located at residue 111 and the glutamate 111 residue was shown to be involved with catalysis.
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Direct selection of binding proficient/catalytic deficient variants of BamHI endonuclease
Nucleic Acids Research, 1994Co-Authors: Lydia F Dorner, Ira SchildkrautAbstract:Variants of BamHI endonuclease in which the glutamate 113 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was demonstrated by placing a BamHI recognition sequence, GGATCC, positioned as an operator sequence in an antisense promoter for the aadA gene (spectinomycin resistance). Repression of this promoter relieved the inhibition of expression of spectinomycin resistance. This system was then used to select new binding proficient/cleavage deficient BamHI variants. The BamHI endonuclease gene was mutagenized either by exposure to hydroxylamine or by PCR. The mutagenized DNA was reintroduced into E. coli carrying the aadA gene construct, and transformants that conferred spectinomycin resistance were selected. Twenty Spr transformants were sequenced. Thirteen of these were newly isolated variants of the previously identified D94 and E113 residues which are known to be involved in catalysis. The remaining seven variants were all located at residue 111 and the glutamate 111 residue was shown to be involved with catalysis.
