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The Varied Effects of Tetraalkylammonium Cations on the Properties of DNA
Monday, February 13, 2017
213a
Though the mRNAs in the cytoplasm are well imaged in fixed or living cells, the detection of the export event has been difficult due to the lack of adequate technique to distinguish nuclear and cytoplasmic mRNAs, while it is crucial in elucidating the mechanism of controlling their life cycle. We developed a novel fluorescent labeling tool that will mark the nuclear and cytoplasmic mRNAs differently. Using the tool, we can potentially trace the full life cycle of mRNAs from transcription, splicing, to translation. We present our preliminary results on developing the labeling tool and tracking the live mRNA export dynamics.
lytes except the synthetic copolymers exhibit a common dependence on the logarithm of fractional charge. The different results observed for the synthetic copolymers can probably be attributed to the flexibility and hydrophobicity of the copolymer backbone. By contrast, the other polyelectrolytes have hydrophilic and relatively rigid backbones. The mobilities observed for the charge variants of the DNA oligomers are well predicted by the Manning electrophoresis equation, while the mobilities predicted by zeta potential theories are too high. However, the mobility ratios observed for the protein charge variants are better described by zeta potential theories.
1051-Pos Board B119 Structural Visualization of the p53/RNA Polymerase II Assembly Sameer Singh, Zhen Qiao, Robert Coleman, Wei-Li Liu. Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA. The master tumor suppressor p53 activates transcription in response to various cellular stresses, in part by facilitating recruitment of the transcription machinery to DNA. Recent studies have documented a direct, yet poorly characterized interaction between p53 and RNA Polymerase II (Pol II). Therefore, we have dissected the human p53/Pol II interaction via single particle cryo-electron microscopy, structural docking and biochemical analyses. This study reveals that p53 binds Pol II via the Rpb1 and Rpb2 subunits, bridging the DNA binding cleft of Pol II proximal to the upstream DNA entry site. In addition, the key DNA binding surface of p53, frequently disrupted in various cancers, remains exposed within the assembly. This suggests that p53 may retain its ability to bind DNA when associated with Pol II. Furthermore, the p53/Pol II cocomplex displays a closed conformation as defined by position of the Pol II clamp domain. Notably, the interaction of p53 and Pol II leads to increased Pol II elongation activity. These findings indicate that p53 may structurally regulate DNA binding functions of Pol II via the clamp domain, thereby providing insights on p53-regulated Pol II transcription.
1054-Pos Board B122 Observation of Flexibility Reversal in DNA Bending Jiyoun Jeong, Harold D. Kim. School of Physics, Georgia Tech, Atlanta, GA, USA. Several experiments on DNA looping show that DNA looping probability at short length scales is higher than the prediction of the wormlike chain model. This observation suggests that DNA becomes more flexible at large bending angles and hints at the possibility that sequence dependence of flexibility may not be universal across different bending regimes. Using a FRET-based DNA looping assay, we measured flexibilities of various DNA sequences (some of which include base pair mismatches) from the looping and unlooping rates. Surprisingly, we find a strong correlation between the measured looping and unlooping rates, which points to an apparent flexibility reversal: more flexible sequences in the unlooped state are more rigid in the looped state. To explain this counterintuitive finding, we present a few hypotheses that challenge our coarsegrained level understanding of DNA.
Nucleic Acid Structure and Dynamics II 1052-Pos Board B120 MD Simulations and CD Spectroscopies of (Benz)Acridine: rDNA G-Quadruplex Complexes Manal Ahmidouch. Wake Forest University, Greensboro, NC, USA. In both prokaryotic and eukaryotic cells, guanine-rich stretches of DNA sequences are found in gene regulatory regions and telomeric DNAs to form G-quadruplexes, and they are involved in many fundamental biological processes and have been implicated in a number of diseases including cancer. While most previous G-quadruplex studies have focused on telomeric G-quadruplexes for validating targets for anticancer therapy, the nucleolar G-quadruplexes may be an even more promising target for chemotherapeutic intervention. The nucleolus plays a central role in tumor pathology because it controls the high level of ribosomal RNA (rRNA) synthesis necessary to sustain ribosome biogenesis during rapid cancer cell growth. Ribosome biogenesis occurs in the nucleolus and is tightly regulated by many cell signaling pathways that converge on the RNA polymerase I complex (Pol I). Using small molecules (drugs) to disrupt ribosome biogenesis represents an attractive strategy for selectively killing cancer cells. Aromatic acridines that sit on top of a G-quadruplex tetrad via p-p stacking interactions have been extensively explored as scaffolds for developing G-quadruplex-specific binders. In this present study, we performed MD simulations of 13 modeled putative rDNA G-quadruplexes that have been characterized via CD spectra to form G-quadruplexes. In addition, we simulated their interactions with designed acridines and benzacridines and analyzed them using a set of novel order parameters to quantify their binding selectivities. We are currently validating our computational predictions via CD spectra. In one G-quadruplex we studied, NUC 23, CD spectra shows that it adopts a parallel topology structure that is further stabilized upon interactions with benzacridine. In excellent agreement, we also observe the same phenomenon in our MD simulations. 1053-Pos Board B121 The Charge Variants of DNA and other Polyelectrolytes: Comparison of Experimental Mobilities with Electrophoresis Theories Nancy C. Stellwagen. Biochemistry, University of Iowa, Iowa City, IA, USA. The free solution mobilities of the charge variants of single- and doublestranded DNA oligomers, small aromatic molecules, peptides, proteins and synthetic copolymers depend on the logarithm of the number of charged residues, not the first power of the charge as predicted by classic electrophoresis theories. The mobility ratios observed for the charge variants of all polyelectro-
1055-Pos Board B123 The Varied Effects of Tetraalkylammonium Cations on the Properties of DNA Earle Stellwagen, Nancy Stellwagen. Department of Biochemistry, University of Iowa, Iowa City, IA, USA. We have been examining the effect of monovalent cations on the melting transition of short model DNA hairpins using capillary electrophoresis. Here we focus on the effects contributed by two disparate monovalent cations, Na and tetrabutylammonium (TBA). Each melting transition yields two parameters, the melting temperature and the amplitude. The dependence of the melting temperature on cation concentration indicates the number of cations released upon melting. Our measurements indicate that more Na is released than TBA upon melting. By contrast, the amplitude of the transition is greater in TBA than in Na. We interpret these observations to indicate that the condensed counter ion cloud about DNA contains fewer TBA than Na, owing to the larger size and apolar surface of TBA. Consequently, the net charge of DNA is greater in TBA than in Na, increasing mobility but lowering the Tm by about 20o due to charge repulsion. This accounts for the ability of TBA to function as a DNA denaturant. A second feature of TBA is that it appears to associate preferentially with AT base pairs and that such association is diminished in sequences containing A-tracts, four or more contiguous AT base pairs 1056-Pos Board B124 Binding of the Carcinogen 4-Nitroquinoline-1-Oxide to phiX174 DNA Selectivity and Distortion Stephen A. Winkle, Jessie Limonta, Juan Medina, Elizabeth Tinoco. Chemistry and Biochemistry, Florida International University, Miami, FL, USA. Optical titration studies, NMR studies and studies with DNA oligonucleotides have suggested that the carcinogen 4-nitroquinoline-1-oxide [NQO] binds to DNA with marked sequence selectivity and cooperativity and that the binding affects the reactivity of enzymes with DNA. To further examine the sequence selectivity of NQO binding to DNA, we have examined the binding of NQO [at NQO/DNA bp ratios 0.1-3] to various locations on phiX174RF DNA using restriction enzyme activity assays employing seven restriction enzymes chosen to have different reaction sequences and flanking sequences. Alterations in restriction enzyme activity were observed with the restriction enzymes Alw44, BssH II, Dra I, Stu I and Xho I while no effect was observed for cleavage by Mlu I and Pst I. While the cleavage sites for the affected enzymes differ, the flanking sequences contain similar motifs [TCTT, TGATTG, TGTTG, TCCTTG, TGTTG]. This motif type is not found in the vicinity of the unaffected enzymes. This motif type was previous shown, with oligomers, to be a hot spot for NQO binding and to be a hot spot for the binding of the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. The alterations in DNA cleavage [enhanced cleavage by Alw44 and inhibition by the other enzymes] provide further indication that NQO alters DNA structure upon binding. The effects of NQO binding on DNA structure were probed using E.coli
213a
Though the mRNAs in the cytoplasm are well imaged in fixed or living cells, the detection of the export event has been difficult due to the lack of adequate technique to distinguish nuclear and cytoplasmic mRNAs, while it is crucial in elucidating the mechanism of controlling their life cycle. We developed a novel fluorescent labeling tool that will mark the nuclear and cytoplasmic mRNAs differently. Using the tool, we can potentially trace the full life cycle of mRNAs from transcription, splicing, to translation. We present our preliminary results on developing the labeling tool and tracking the live mRNA export dynamics.
lytes except the synthetic copolymers exhibit a common dependence on the logarithm of fractional charge. The different results observed for the synthetic copolymers can probably be attributed to the flexibility and hydrophobicity of the copolymer backbone. By contrast, the other polyelectrolytes have hydrophilic and relatively rigid backbones. The mobilities observed for the charge variants of the DNA oligomers are well predicted by the Manning electrophoresis equation, while the mobilities predicted by zeta potential theories are too high. However, the mobility ratios observed for the protein charge variants are better described by zeta potential theories.
1051-Pos Board B119 Structural Visualization of the p53/RNA Polymerase II Assembly Sameer Singh, Zhen Qiao, Robert Coleman, Wei-Li Liu. Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY, USA. The master tumor suppressor p53 activates transcription in response to various cellular stresses, in part by facilitating recruitment of the transcription machinery to DNA. Recent studies have documented a direct, yet poorly characterized interaction between p53 and RNA Polymerase II (Pol II). Therefore, we have dissected the human p53/Pol II interaction via single particle cryo-electron microscopy, structural docking and biochemical analyses. This study reveals that p53 binds Pol II via the Rpb1 and Rpb2 subunits, bridging the DNA binding cleft of Pol II proximal to the upstream DNA entry site. In addition, the key DNA binding surface of p53, frequently disrupted in various cancers, remains exposed within the assembly. This suggests that p53 may retain its ability to bind DNA when associated with Pol II. Furthermore, the p53/Pol II cocomplex displays a closed conformation as defined by position of the Pol II clamp domain. Notably, the interaction of p53 and Pol II leads to increased Pol II elongation activity. These findings indicate that p53 may structurally regulate DNA binding functions of Pol II via the clamp domain, thereby providing insights on p53-regulated Pol II transcription.
1054-Pos Board B122 Observation of Flexibility Reversal in DNA Bending Jiyoun Jeong, Harold D. Kim. School of Physics, Georgia Tech, Atlanta, GA, USA. Several experiments on DNA looping show that DNA looping probability at short length scales is higher than the prediction of the wormlike chain model. This observation suggests that DNA becomes more flexible at large bending angles and hints at the possibility that sequence dependence of flexibility may not be universal across different bending regimes. Using a FRET-based DNA looping assay, we measured flexibilities of various DNA sequences (some of which include base pair mismatches) from the looping and unlooping rates. Surprisingly, we find a strong correlation between the measured looping and unlooping rates, which points to an apparent flexibility reversal: more flexible sequences in the unlooped state are more rigid in the looped state. To explain this counterintuitive finding, we present a few hypotheses that challenge our coarsegrained level understanding of DNA.
Nucleic Acid Structure and Dynamics II 1052-Pos Board B120 MD Simulations and CD Spectroscopies of (Benz)Acridine: rDNA G-Quadruplex Complexes Manal Ahmidouch. Wake Forest University, Greensboro, NC, USA. In both prokaryotic and eukaryotic cells, guanine-rich stretches of DNA sequences are found in gene regulatory regions and telomeric DNAs to form G-quadruplexes, and they are involved in many fundamental biological processes and have been implicated in a number of diseases including cancer. While most previous G-quadruplex studies have focused on telomeric G-quadruplexes for validating targets for anticancer therapy, the nucleolar G-quadruplexes may be an even more promising target for chemotherapeutic intervention. The nucleolus plays a central role in tumor pathology because it controls the high level of ribosomal RNA (rRNA) synthesis necessary to sustain ribosome biogenesis during rapid cancer cell growth. Ribosome biogenesis occurs in the nucleolus and is tightly regulated by many cell signaling pathways that converge on the RNA polymerase I complex (Pol I). Using small molecules (drugs) to disrupt ribosome biogenesis represents an attractive strategy for selectively killing cancer cells. Aromatic acridines that sit on top of a G-quadruplex tetrad via p-p stacking interactions have been extensively explored as scaffolds for developing G-quadruplex-specific binders. In this present study, we performed MD simulations of 13 modeled putative rDNA G-quadruplexes that have been characterized via CD spectra to form G-quadruplexes. In addition, we simulated their interactions with designed acridines and benzacridines and analyzed them using a set of novel order parameters to quantify their binding selectivities. We are currently validating our computational predictions via CD spectra. In one G-quadruplex we studied, NUC 23, CD spectra shows that it adopts a parallel topology structure that is further stabilized upon interactions with benzacridine. In excellent agreement, we also observe the same phenomenon in our MD simulations. 1053-Pos Board B121 The Charge Variants of DNA and other Polyelectrolytes: Comparison of Experimental Mobilities with Electrophoresis Theories Nancy C. Stellwagen. Biochemistry, University of Iowa, Iowa City, IA, USA. The free solution mobilities of the charge variants of single- and doublestranded DNA oligomers, small aromatic molecules, peptides, proteins and synthetic copolymers depend on the logarithm of the number of charged residues, not the first power of the charge as predicted by classic electrophoresis theories. The mobility ratios observed for the charge variants of all polyelectro-
1055-Pos Board B123 The Varied Effects of Tetraalkylammonium Cations on the Properties of DNA Earle Stellwagen, Nancy Stellwagen. Department of Biochemistry, University of Iowa, Iowa City, IA, USA. We have been examining the effect of monovalent cations on the melting transition of short model DNA hairpins using capillary electrophoresis. Here we focus on the effects contributed by two disparate monovalent cations, Na and tetrabutylammonium (TBA). Each melting transition yields two parameters, the melting temperature and the amplitude. The dependence of the melting temperature on cation concentration indicates the number of cations released upon melting. Our measurements indicate that more Na is released than TBA upon melting. By contrast, the amplitude of the transition is greater in TBA than in Na. We interpret these observations to indicate that the condensed counter ion cloud about DNA contains fewer TBA than Na, owing to the larger size and apolar surface of TBA. Consequently, the net charge of DNA is greater in TBA than in Na, increasing mobility but lowering the Tm by about 20o due to charge repulsion. This accounts for the ability of TBA to function as a DNA denaturant. A second feature of TBA is that it appears to associate preferentially with AT base pairs and that such association is diminished in sequences containing A-tracts, four or more contiguous AT base pairs 1056-Pos Board B124 Binding of the Carcinogen 4-Nitroquinoline-1-Oxide to phiX174 DNA Selectivity and Distortion Stephen A. Winkle, Jessie Limonta, Juan Medina, Elizabeth Tinoco. Chemistry and Biochemistry, Florida International University, Miami, FL, USA. Optical titration studies, NMR studies and studies with DNA oligonucleotides have suggested that the carcinogen 4-nitroquinoline-1-oxide [NQO] binds to DNA with marked sequence selectivity and cooperativity and that the binding affects the reactivity of enzymes with DNA. To further examine the sequence selectivity of NQO binding to DNA, we have examined the binding of NQO [at NQO/DNA bp ratios 0.1-3] to various locations on phiX174RF DNA using restriction enzyme activity assays employing seven restriction enzymes chosen to have different reaction sequences and flanking sequences. Alterations in restriction enzyme activity were observed with the restriction enzymes Alw44, BssH II, Dra I, Stu I and Xho I while no effect was observed for cleavage by Mlu I and Pst I. While the cleavage sites for the affected enzymes differ, the flanking sequences contain similar motifs [TCTT, TGATTG, TGTTG, TCCTTG, TGTTG]. This motif type is not found in the vicinity of the unaffected enzymes. This motif type was previous shown, with oligomers, to be a hot spot for NQO binding and to be a hot spot for the binding of the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. The alterations in DNA cleavage [enhanced cleavage by Alw44 and inhibition by the other enzymes] provide further indication that NQO alters DNA structure upon binding. The effects of NQO binding on DNA structure were probed using E.coli