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Byong-Seok Choi

Publications and source records attributed to Byong-Seok Choi.

At least 19 recordsLinked to original sources
PubMed2006

Intrinsically unstructured N-terminal domain of bZIP transcription factor HY5.

The Arabidopsis HY5 protein is a basic leucine zipper (bZIP) transcription factor that promotes photomorphogenesis. HY5 binds directly to the promoters of light responsible element containing the G-box and thus regulates their transcriptional activity. The level and activity of HY5 are negatively regulated, in a light-dependent manner, by interaction with the COP1 protein, which targets HY5 for proteasome-mediated degradation in the nucleus. Despite its essential roles in plant development, no structural information exists for HY5. In this article, we report the first structural and biophysical characterization of HY5. Using limited proteolysis in combination with mass spectrometry, circular dichroism, and nuclear magnetic resonance spectroscopy, we have deduced that the N-terminal 77 amino acids of HY5 form a premolten globular structure, while amino acids 78-110, which constitute the basic region (BR) of the protein, exist in a molten globule state. Our studies also revealed that the overall structural features of full-length HY5 are dominated largely by the disordered N-terminal domain, despite the existence of a bZIP domain at its C-terminus. We propose that HY5 is a member of the intrinsically unstructured protein (IUP) family, and that HY5 functions as an unstructured protein and benefits from being the same, in vivo.

Amino Acid Sequence↗
PubMed2006

Structural and dynamic basis of a supercoiling-responsive DNA element.

In both eukaryotes and prokaryotes, negative supercoiling of chromosomal DNA acts locally to regulate a variety of cellular processes, such as transcription, replication, recombination and response to environmental stresses. While studying the interaction between the Hin recombinase and mutated versions of its cognate DNA-binding site, we identified a mutated DNA site that binds Hin only when the DNA is supercoiled. To understand the mechanism of this supercoiling-responsive DNA site, we used NMR spectroscopy and fluorescence resonance energy transfer to determine the solution structures and dynamics of three related DNA oligonucleotides. The supercoiling-responsive DNA site formed a partially unwound and stretched helix and showed significant flexibility and base pair opening kinetics. The single CAG/CTG triplet contained in this DNA sequence displayed the same characteristics as do multiple CAG/CTG repeats, which are associated with several hereditary neuromuscular diseases. It is known that short DNA sequence motifs that have either very high or low bending flexibility occur preferentially at supercoiling-sensitive bacterial and eukaryotic promoters. From our results and these previous data, we propose a model in which supercoiling utilizes the intrinsic flexibility of a short DNA site to switch the local DNA structure from an inefficient conformation for protein binding to an efficient one, or vice versa.

Base Pairing↗
PubMed2006

The protein shuffle. Sequential interactions among components of the human nucleotide excision repair pathway.

Xeroderma pigmentosum (XP) is an inherited disease in which cells from patients exhibit defects in nucleotide excision repair (NER). XP proteins A-G are crucial in the processes of DNA damage recognition and incision, and patients with XP can carry mutations in any of the genes that specify these proteins. In mammalian cells, NER is a dynamic process in which a variety of proteins interact with one another, via modular domains, to carry out their functions. XP proteins are key players in several steps of the NER process, including DNA strand discrimination (XPA, in complex with replication protein A), repair complex formation (XPC, in complex with hHR23B; XPF, in complex with ERCC1) and repair factor recruitment (transcription factor IIH, in complex with XPG). Through these protein-protein interactions, various types of bulky DNA adducts can be recognized and repaired. Communication between the NER system and other cellular pathways is also achieved by selected binding of the various structural domains. Here, we summarize recent studies on the domain structures of human NER components and the regulatory networks that utilize these proteins. Data provided by these studies have helped to illuminate the complex molecular interactions among NER factors in the context of DNA repair.

DNA Damage↗
PubMed2005

Subcellular in vivo 1H MR spectroscopy of Xenopus laevis oocytes.

In vivo magnetic resonance (MR) spectra are typically obtained from voxels whose spatial dimensions far exceed those of the cells they contain. This study was designed to evaluate the potential of localized MR spectroscopy to investigate subcellular phenomena. Using a high magnetic field and a home-built microscopy probe with large gradient field strengths, we achieved voxel sizes of (180 microm)3. In the large oocytes of the frog Xenopus laevis, this was small enough to allow the recording of the first compartment-selective in vivo MR spectra from the animal and vegetal cytoplasm as well as the nucleus. The two cytoplasmic regions differed in their lipid contents and NMR lineshape characteristics-differences that are not detectable with whole-cell NMR techniques. In the nucleus, the signal appeared to be dominated by water, whereas other contributions were negligible. We also used localized spectroscopy to monitor the uptake of diminazene acturate, an antitrypanosomal agent, into compartments of a single living oocyte. The resulting spectra from the nucleus and cytoplasm revealed different uptake kinetics for the two components of the drug and demonstrate that MR technology is on the verge of becoming a tool for cell biology.

Animals↗
PubMed2005

Solution structure of the DNA-binding domain of RPA from Saccharomyces cerevisiae and its interaction with single-stranded DNA and SV40 T antigen.

Replication protein A (RPA) is a three-subunit complex with multiple roles in DNA metabolism. DNA-binding domain A in the large subunit of human RPA (hRPA70A) binds to single-stranded DNA (ssDNA) and is responsible for the species-specific RPA-T antigen (T-ag) interaction required for Simian virus 40 replication. Although Saccharomyces cerevisiae RPA70A (scRPA70A) shares high sequence homology with hRPA70A, the two are not functionally equivalent. To elucidate the similarities and differences between these two homologous proteins, we determined the solution structure of scRPA70A, which closely resembled the structure of hRPA70A. The structure of ssDNA-bound scRPA70A, as simulated by residual dipolar coupling-based homology modeling, suggested that the positioning of the ssDNA is the same for scRPA70A and hRPA70A, although the conformational changes that occur in the two proteins upon ssDNA binding are not identical. NMR titrations of hRPA70A with T-ag showed that the T-ag binding surface is separate from the ssDNA-binding region and is more neutral than the corresponding part of scRPA70A. These differences might account for the species-specific nature of the hRPA70A-T-ag interaction. Our results provide insight into how these two homologous RPA proteins can exhibit functional differences, but still both retain their ability to bind ssDNA.

Amino Acid Sequence↗
PubMed2005

Biophysical characterization of the interaction domains and mapping of the contact residues in the XPF-ERCC1 complex.

XPF and ERCC1 exist as a heterodimer to be stable and active in cells and catalyze DNA cleavage on the 5'-side of a lesion during nucleotide excision repair. To characterize the specific interaction between XPF and ERCC1, we expressed the human ERCC1 binding domain of XPF (XPF-EB) and the XPF binding domain of ERCC1 (ERCC1-FB) in Escherichia coli. Milligram quantities of a heterodimer were characterized with gel filtration chromatography, an Ni(2+)-NTA binding assay, and analytical ultracentrifugation. Cross-linking experiments at high salt concentrations revealed that XPF interacts with ERCC1 mainly through hydrophobic interactions. XPF-EB was also shown to homodimerize in the absence of ERCC1. NMR cross-saturation methods were applied to map the residues involved in formation of the XPF-EB.XPF-EB homodimer and the XPF-EB.ERCC1-FB heterodimer. Helix H3 and the C-terminal region of XPF-EB were either within or in close proximity to the homodimer interface, whereas the ERCC1-FB binding site of XPF-EB was distributed across helix H1, a small part of H2, H3, and the C-terminal region, most of which exhibited large changes in chemical shift upon ERCC1 binding. The XPF-EB heterodimeric interface is larger than the XPF-EB homodimeric one, which could explain why XPF has a stronger affinity for ERCC1 than for a second molecule of XPF. The XPF binding sites of ERCC1 were located in helices H1 and H3 and in the C-terminal region, similar to the involved surface of XPF. We used cross-saturation data and the crystal structure of related proteins to model the two complexes.

Amino Acid Sequence↗
PubMed2005

Structural insights into the monosaccharide specificity of Escherichia coli rhamnose mutarotase.

The crystal structure of Escherichia coli rhamnose mutarotase (YiiL) is completely different from the previously reported structures of the Lactococcus lactis galactose mutarotase and the Bacillus subtilis RbsD (pyranase). YiiL exists as a locally asymmetric dimer, which is stabilized by an intermolecular beta-sheet, various hydrophobic interactions, and a cation-pi interaction with a salt-bridge. The protein folds of YiiL are similar to those of a Streptomyces coelicolor mono-oxygenase and a hypothetical Arabidopsis thaliana protein At3g17210. By assaying the enzymatic activity of six active-site mutants and by comparing the crystal structure-derived active site conformations of YiiL, RbsD, and a galactose mutarotase, we were able to define the amino acid residues required for catalysis and suggest a possible catalytic mechanism for YiiL. Although the active-site amino acid residues of YiiL (His, Tyr, and Trp) differ greatly from those of galactose mutarotase (His, Glu, and Asp), their geometries, which determine the structures of the preferred monosaccharide substrates, are conserved. In addition, the in vivo function of YiiL was assessed by constructing a mutant E.coli strain that carries a yiiL deletion. The presence of the yiiL gene is critical for efficient cell growth only when concentrations of l-rhamnose are limited.

Amino Acid Sequence↗
PubMed2005

Solution structure and backbone dynamics of the XPC-binding domain of the human DNA repair protein hHR23B.

Human cells contain two homologs of the yeast RAD23 protein, hHR23A and hHR23B, which participate in the DNA repair process. hHR23B houses a domain (residues 277-332, called XPCB) that binds specifically and directly to the xeroderma pigmentosum group C protein (XPC) to initiate nucleotide excision repair (NER). This domain shares sequence homology with a heat shock chaperonin-binding motif that is also found in the stress-inducible yeast phosphoprotein STI1. We determined the solution structure of a protein fragment containing amino acids 275-342 of hHR23B (termed XPCB-hHR23B) and compared it with the previously reported solution structures of the corresponding domain of hHR23A. The periodic positioning of proline residues in XPCB-hHR23B produced kinked alpha helices and assisted in the formation of a compact domain. Although the overall structure of the XPCB domain was similar in both XPCB-hHR23B and XPCB-hHR23A, the N-terminal part (residues 275-283) of XPCB-hHR23B was more flexible than the corresponding part of hHR23A. We tried to infer the characteristics of this flexibility through (15)N-relaxation studies. The hydrophobic surface of XPCB-hHR23B, which results from the diverse distribution of N-terminal region, might give rise to the functional pleiotropy observed in vivo for hHR23B, but not for hHR23A.

Amino Acid Sequence↗
PubMed2005

Cnu, a novel oriC-binding protein of Escherichia coli.

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

Amino Acid Sequence↗
PubMed2004

NMR analysis of enzyme-catalyzed and free-equilibrium mutarotation kinetics of monosaccharides.

We have exploited the saturation difference (SD) NMR technique to investigate the mutarotations of two sugars, l-fucose and d-ribose, at equilibrium. The KM and kcat values of Escherichia coli FucU-catalyzed mutarotation of l-fucose have been determined by this technique, and the values of KM for alpha- and beta-forms are shown to be the same. Similarly, the spontaneous mutarotation of d-ribose was investigated by using the same method, and it was found that d-ribose has an exceptionally high spontaneous alpha-to-beta conversion rate only between the furan forms.

Carbohydrate Conformation↗
PubMed2004

Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography.

Knowledge of the three-dimensional structures of RNA and its complexes is important for understanding the molecular mechanism of RNA recognition by proteins or ligands. Enzymatic synthesis using T7 bacteriophage RNA polymerase is used to prepare samples for NMR spectroscopy and X-ray crystallography. However, this run-off transcription method results in heterogeneity at the RNA 3-terminus. For structural studies, RNA purification requires a single nucleotide resolution. Usually PAGE purification is used, but it is tedious, time-consuming and cost ineffective. To overcome these problems in high-throughput RNA synthesis, we devised a method of RNA preparation that uses trans-acting DNAzyme and sequence-specific affinity column chromatography. A tag sequence is added at the 3' end of RNA, and the tagged RNA is picked out using an affinity column that contains the complementary DNA sequence. The 3' end tag is then removed by sequence-specific cleavage using trans-acting DNAzyme, the arm lengths of which are optimized for turnover number. This purification method is simpler and faster than the conventional method.

Base Sequence↗
PubMed2004

Synthesis and characterization of tri(ethylene oxide)-attached poly(amidoamine) dendrimer layers on gold.

This paper describes the synthesis of a tri(ethylene oxide)-attached fourth-generation poly(amidoamine) dendrimer (EO3-dendrimer) and the characterization of its layers on gold. NMR analysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry revealed that about 61 amine groups of a G4 PAMAM dendrimer were covalently conjugated with tri(ethylene oxide) units, accounting for a 95% modification level. Layers of the EO3-dendrimer were formed on gold, and the resulting surface was characterized by infrared reflection absorption spectroscopy, ellipsometry, and contact angle goniometry. The EO3-dendrimer resulted in more hydrophilic and less compact layers with no substantial deformation of the molecule during layer formation by virtue of the EO3 units, compared to a PAMAM dendrimer. Interestingly, the specific binding of avidin to the biotinylated layers of the EO3-dendrimer approached a surface density of 5.2 +/- 0.2 ngmm-2, showing about 92% of full surface coverage. The layers of the EO3-dendrimer were found to be more resistant to nonspecific adsorption of proteins than PAMAM dendrimer layers when bovine serum albumin and serum proteins were tested.

Adsorption↗
PubMed2004

NMR structure of the DNA decamer duplex containing double T*G mismatches of cis-syn cyclobutane pyrimidine dimer: implications for DNA damage recognition by the XPC-hHR23B complex.

The cis-syn cyclobutane pyrimidine dimer (CPD) is a cytotoxic, mutagenic and carcinogenic DNA photoproduct and is repaired by the nucleotide excision repair (NER) pathway in mammalian cells. The XPC-hHR23B complex as the initiator of global genomic NER binds to sites of certain kinds of DNA damage. Although CPDs are rarely recognized by the XPC-hHR23B complex, the presence of mismatched bases opposite a CPD significantly increased the binding affinity of the XPC-hHR23B complex to the CPD. In order to decipher the properties of the DNA structures that determine the binding affinity for XPC-hHR23B to DNA, we carried out structural analyses of the various types of CPDs by NMR spectroscopy. The DNA duplex which contains a single 3' T*G wobble pair in a CPD (CPD/GA duplex) induces little conformational distortion. However, severe distortion of the helical conformation occurs when a CPD contains double T*G wobble pairs (CPD/GG duplex) even though the T residues of the CPD form stable hydrogen bonds with the opposite G residues. The helical bending angle of the CPD/GG duplex was larger than those of the CPD/GA duplex and properly matched CPD/AA duplex. The fluctuation of the backbone conformation and significant changes in the widths of the major and minor grooves at the double T*G wobble paired site were also observed in the CPD/GG duplex. These structural features were also found in a duplex that contains the (6-4) adduct, which is efficiently recognized by the XPC-hHR23B complex. Thus, we suggest that the unique structural features of the DNA double helix (that is, helical bending, flexible backbone conformation, and significant changes of the major and/or minor grooves) might be important factors in determining the binding affinity of the XPC-hHR23B complex to DNA.

Adenine↗
PubMed2004

NMR application probes a novel and ubiquitous family of enzymes that alter monosaccharide configuration.

By exploiting nuclear magnetic resonance (NMR) techniques along with novel applications of saturation difference analysis, we deciphered the functions of the previously uncharacterized products of three bacterial genes, rbsD, fucU, and yiiL, which are part of the ribose, fucose, and rhamnose operons of Escherichia coli, respectively. We show that RbsD catalyzes the pyran to furan conversion of ribose, whereas FucU and YiiL are involved in the catalysis of the anomeric conversion of their respective sugars. It was observed that the anomeric exchange of only ribofuranose, not ribopyranose, occurs spontaneously in solution rationalizing its evolutionary incorporation into the nucleic acid. The RbsD and FucU proteins share sequence homology and belong to the same protein family that is found from eubacteria to human, whereas the YiiL homologues exist in archaebacteria and lower eukaryotes. These enzymes, including the galactose mutarotase, exhibit a certain degree of cross-specificity to structurally analogous sugars thereby encompassing all existing monosaccharides in terms of their reactivities. The ubiquitous presence of enzymes involved in the anomeric changes of monosaccharides highlights an importance of these activities in various cellular processes requiring efficient monosaccharide utilization.

Carbohydrate Epimerases↗
PubMed2003

Structure and dynamics of hemimethylated GATC sites: implications for DNA-SeqA recognition.

DNA methylation occurs in most organisms from bacteria to mammals and provides a mechanism for epigenetic control of a variety of cellular processes. In Escherichia coli, most of the N6 positions in adenines found in the sequence GATC are methylated by DNA adenine methyltransferase. After DNA replication, the GATC sites exist transiently in the hemimethylated state, and the specific recognition of these hemimethylated GATC sites is essential for several processes, including sequestration of the site of replication initiation by the SeqA protein, strand discrimination in DNA mismatch repair by the MutH protein, and transcription of several genes. Here, we characterize the solution structure and dynamics of two dodecamer DNA duplexes that each contains a single GATC site in either unmethylated or hemimethylated state. We found that the N6-methylated adenine of a hemimethylated GATC site undergoes a slow trans-cis interconversion. The release of a tightly bound cation from hemimethylated DNA explains the instability of this structure. In addition, quantitative structural analysis revealed that hemimethylated DNA has unusual backbone structures and a remarkably narrow major groove. These dynamic and structural features provide insights into the specific recognition of hemimethylated GATC sites by the SeqA protein.

Bacterial Outer Membrane Proteins↗
PubMed2003

NMR study on the interaction between RPA and DNA decamer containing cis-syn cyclobutane pyrimidine dimer in the presence of XPA: implication for damage verification and strand-specific dual incision in nucleotide excision repair.

In mammalian cells, nucleotide excision repair (NER) is the major pathway for the removal of bulky DNA adducts. Many of the key NER proteins are members of the XP family (XPA, XPB, etc.), which was named on the basis of its association with the disorder xerodoma pigmentosum. Human replication protein A (RPA), the ubiquitous single-stranded DNA-binding protein, is another of the essential proteins for NER. RPA stimulates the interaction of XPA with damaged DNA by forming an RPA-XPA complex on damaged DNA sites. Binding of RPA to the undamaged DNA strand is most important during NER, because XPA, which directs the excision nucleases XPG and XPF, must bind to the damaged strand. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to assess the binding of the tandem high affinity DNA-binding domains, RPA-AB, and of the isolated domain RPA-A, to normal DNA and damaged DNA containing the cyclobutane pyrimidine dimer (CPD) lesion. Both RPA-A and RPA-AB were found to bind non- specifically to both strands of normal and CPD- containing DNA duplexes. There were no differences observed when binding to normal DNA duplex was examined in the presence of the minimal DNA-binding domain of XPA (XPA-MBD). However, there is a drastic difference for CPD-damaged DNA duplex as both RPA-A and RPA-AB bind specifically to the undamaged strand. The strand-specific binding of RPA and XPA to the damaged duplex DNA shows that RPA and XPA play crucial roles in damage verification and guiding cleavage of damaged DNA during NER.

DNA↗