DNA Structure and Sequencing

Solution structure and backbone dynamics of the XPC-binding domain of the human DNA repair protein hHR23B Byoungkook Kim 1, *, Kyoung-Seok Ryu 2, *, Hyun-Jin Kim 1 , Sung-Jae Cho 1 and Byong-Seok Choi 1 1 Department of Chemistry, and National Creative Research Initiative Center for the Repair System of Damaged DNA, Korea Advanced Institute of Science and Technology, South Korea 2 Korea Basic Science Institute, Daejon, South Korea Nucleotide excision repair (NER) is an important pathway for the removal of DNA lesions caused by diverse environmental factors, such as UV irradiation and chemical modifications [1,2]. There are two human homologs (A and B) of the yeast Rad23 protein (hHR23A and hHR23B), both of which can form a complex with the xeroderma pigmentosum group C protein (XPC) [1,3]. Recent in vitro and in vivo studies point to a role for the XPC–hHR23B complex as the initiator of global genomic NER [1,4]. Although the precise functions performed by hHR23A and hHR23B alone in human NER have not yet been determined, Keywords hHR23B; nucleotide excision repair; stress- inducible; structure; xeroderma pigmentosum group C protein Correspondence B S. Choi, Department of Chemistry and National Creative Research Initiative Center for Repair System of Damaged DNA, Korea Advanced Institute of Science and Technology, Yusong-Gu, Gusong-Dong 373-1, Daejon 305-701, South Korea Fax: +82 42 869 2810 Tel: +82 42 869 2868 E-mail: byongseok.choi@kaist.ac.kr *Byoungkook Kim and Kyoung-Seok Ryu contributed equally to this work Note The atomic coordinates of the bundle of 20 conformers have been deposited in the RCSB Protein Data Bank with entry code 1PVE (Received 9 November 2004, revised 4 March 2005, accepted 17 March 2005) doi:10.1111/j.1742-4658.2005.04667.x Human cells contain two homologs of the yeast RAD23 protein, hHR23A and hHR23B, which participate in the DNA repair process. hHR23B hou- ses 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 position- ing of proline residues in XPCB–hHR23B produced kinked a helices and assisted in the formation of a compact domain. Although the overall struc- ture 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 func- tional pleiotropy observed in vivo for hHR23B, but not for hHR23A. Abbreviations hHR23B, human homolog B of yeast Rad23; NER, nucleotide excision repair; RMSD, root mean square deviation; STI1, stress-inducible, heat shock chaperonin-binding motif; UBA, ubiquitin-associated domains; UbL, ubiquitin-like domain; XPC, xeroderma pigmentosum group DNA Structure and Sequencing DNA Structure and Sequencing Bởi: OpenStaxCollege The building blocks of DNA are nucleotides The important components of the nucleotide are a nitrogenous base, deoxyribose (5-carbon sugar), and a phosphate group ([link]) The nucleotide is named depending on the nitrogenous base The nitrogenous base can be a purine such as adenine (A) and guanine (G), or a pyrimidine such as cytosine (C) and thymine (T) Each nucleotide is made up of a sugar, a phosphate group, and a nitrogenous base The sugar is deoxyribose in DNA and ribose in RNA The nucleotides combine with each other by covalent bonds known as phosphodiester bonds or linkages The purines have a double ring structure with a six-membered ring fused to a five-membered ring Pyrimidines are smaller in size; they have a single sixmembered ring structure The carbon atoms of the five-carbon sugar are numbered 1', 2', 3', 4', and 5' (1' is read as “one prime”) The phosphate residue is attached to the hydroxyl group of the 5' carbon of one sugar of one nucleotide and the hydroxyl group of the 3' carbon of the sugar of the next nucleotide, thereby forming a 5'-3' phosphodiester bond In the 1950s, Francis Crick and James Watson worked together to determine the structure of DNA at the University of Cambridge, England Other scientists like Linus 1/10 DNA Structure and Sequencing Pauling and Maurice Wilkins were also actively exploring this field Pauling had discovered the secondary structure of proteins using X-ray crystallography In Wilkins’ lab, researcher Rosalind Franklin was using X-ray diffraction methods to understand the structure of DNA Watson and Crick were able to piece together the puzzle of the DNA molecule on the basis of Franklin's data because Crick had also studied X-ray diffraction ([link]) In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Medicine Unfortunately, by then Franklin had died, and Nobel prizes are not awarded posthumously The work of pioneering scientists (a) James Watson, Francis Crick, and Maclyn McCarty led to our present day understanding of DNA Scientist Rosalind Franklin discovered (b) the X-ray diffraction pattern of DNA, which helped to elucidate its double helix structure (credit a: modification of work by Marjorie McCarty, Public Library of Science) Watson and Crick proposed that DNA is made up of two strands that are twisted around each other to form a right-handed helix Base pairing takes place between a purine and pyrimidine; namely, A pairs with T and G pairs with C Adenine and thymine are complementary base pairs, and cytosine and guanine are also complementary base pairs The base pairs are stabilized by hydrogen bonds; adenine and thymine form two hydrogen bonds and cytosine and guanine form three hydrogen bonds The two strands are anti-parallel in nature; that is, the 3' end of one strand faces the 5' end of the other strand The sugar and phosphate of the nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside Each base pair is separated from the other base pair by a distance of 0.34 nm, and each turn of the helix measures 3.4 nm Therefore, ten base pairs are present per turn of the helix The diameter of the DNA double helix is nm, and it is uniform throughout Only the pairing between a purine and pyrimidine can explain the uniform diameter The twisting of the two strands around each other results in the formation of uniformly spaced major and minor grooves ([link]) 2/10 DNA Structure and Sequencing DNA has (a) a double helix structure and (b) phosphodiester bonds The (c) major and minor grooves are binding sites for DNA binding proteins during processes such as transcription (the copying of RNA from DNA) and replication DNA Sequencing Techniques Until the 1990s, the sequencing of DNA (reading the sequence of DNA) was a relatively expensive and long process Using radiolabeled nucleotides also compounded the problem through safety concerns With currently available technology and automated machines, the process is cheap, safer, and can be completed in a matter of hours Fred Sanger developed the sequencing method used for the human genome sequencing project, which is widely used today ([link]) Link to Learning Visit this site to watch a video explaining the DNA sequence reading technique that resulted from Sanger’s work The method is known as the dideoxy chain termination method The sequencing method is based on the use of chain terminators, the dideoxynucleotides (ddNTPs) The dideoxynucleotides, or ddNTPSs, differ from the deoxynucleotides by the lack of a free 3' OH group on the five-carbon sugar If a ddNTP is added to a growing a DNA strand, the chain is not extended any further because the free 3' OH group needed to add another nucleotide is not available By using a predetermined ratio of deoxyribonucleotides to dideoxynucleotides, it is possible to generate DNA fragments of different sizes 3/10 DNA Structure ...Structure–activity relation for synthetic phenoxazone drugs Evidence for a direct correlation between DNA binding and pro-apoptotic activity Alexei N. Veselkov 1 , Vladimir Ya. Maleev 2 , Evgenie N. Glibin 3 , Leonid Karawajew 4 and David B. Davies 5 1 Department of Physics and Chemistry, Sevastopol National Technical University, Crimea, Ukraine; 2 Department of Biophysical and Medical Physics, Kharkov National University, Ukraine; 3 Department of Chemistry, St Petersburg State Technological University, Russia; 4 Department of Haematology, Oncology, and Tumour Immunology, Robert-Ro ¨ ssle Clinic, Charite ´ , Humboldt-University of Berlin, Germany; 5 School of Biological and Chemical Sciences, Birkbeck College, University of London, UK The structure–activity relations of a series of synthetic phenoxazone drugs with aminoalkyl side chains of variable length and different terminal groups were investigated by examining their biological activity and DNA complexation affinity. Biological activity was determined from their ability to induce apoptosis and cell cycle perturbations (activation of cell cycle checkpoints) using the human malignant MOLT-3 cell line. The thermodynamic parameters of drug– DNA complexation were determined by differential scan- ning calorimetry. By comparing the activities of compounds with different terminal groups (amino, dimethylamino and diethylamino), we found that the existence of a terminal dimethylamino group in the alkylamino side chain is an important factor for anti-tumour activity. Minor modifica- tions in the dimethylaminoalkyl side chain (e.g. elongation by one methylene group) led to notable changes in both the anti-tumour activity and DNA-binding properties of the drug, providing unambiguous evidence of a marked struc- ture–activity relation. Keywords: apoptotic activity; differential scanning calori- metry (DSC); drug–DNA binding; phenoxazone drugs; structure–activity relationship. Many anti-tumour drugs are thought to exert their cytotoxic effect through DNA-specific interactions, resulting in geno- toxic stress and consequent induction of programmed cell death (apoptosis) [1–3]. Clinically important drugs belong to structurally different families, reflecting the range of possible anchoring mechanisms and their different activities with nucleic acids [4]. These drugs include intercalators, groove binders, and those binding with a combination of the two mechanisms. The antibiotic actinomycin D consists of a planar phenoxazone chromophore with two identical side chains consisting of pentapeptide lactone rings. It is an example of an aromatic drug with both intercalative and groove-binding mechanisms of complexation with DNA. Although the structural significance of the phenoxazone chromophore is well established, the role of the side chains is still under discussion. One hypothesis suggested [5] that actinomycin D may be characterized as an ionophore- antibiotic, because it shows significant complexation of the side chains with sodium ions but not with potassium ions; this, in turn, suggested that the activity of actinomycin D may only be manifested when the pentapeptide rings form complexes with sodium ions. As crown ethers are well known to exhibit selective binding with metal cations [6], this hypothesis was tested on actinomycin D derivatives with crown-like structures in the side chains [7]. None of the derivatives showed significant activity with human leukemia MOLT-3 cell lines, even though the crown side groups had different specificities for metal cation binding, different lengths of spacers in the side chains, etc. [7]. On the other hand, it was found that the rather simple Open Access Available online http://arthritis-research.com/content/10/2/R40 Page 1 of 14 (page number not for citation purposes) Vol 10 No 2 Research article Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing Mariam Siala 1 , Benoit Jaulhac 2 , Radhouane Gdoura 1 , Jean Sibilia 2 , Hela Fourati 3 , Mohamed Younes 4 , Sofien Baklouti 3 , Naceur Bargaoui 4 , Slaheddine Sellami 5 , Abir Znazen 1 , Cathy Barthel 2 , Elody Collin 2 , Adnane Hammami 1 and Abdelghani Sghir 6,7 1 Laboratoire de Recherche 'Micro-organismes et Pathologie Humaine', EPS Habib Bourguiba, Rue El Ferdaous, 3029 Sfax, Tunisie 2 Laboratoire de Physiopathologie des Interactions Hôte-bactérie, UPRES-EA 3432, Faculté de Médecine, Université Louis-Pasteur, rue Koeberlé, 67000 Strasbourg, France 3 Service de Rhumatologie Hôpital Hedi Chaker, Avenue Majida Boulila, 3029 Sfax, Tunisie 4 Service de Rhumatologie, EPS Fattouma Bourguiba, Rue 1er Juin, 5019 Monastir, Tunisie 5 Service de Rhumatologie, EPS La Rabta, rue 7051 Centre Urbain Nord, 1082 Tunis, Tunisie 6 CNRS-UMR 8030, CEA-Genoscope, rue Gaston Crémieux, 91000 Évry, France 7 University of Evry Val d'Essonne, Boulevard François Mitterrand, 91025 Évry Cedex, 91000 Évry, France Corresponding author: Adnane Hammami, adnene.hammami@rns.tn Received: 27 Dec 2007 Revisions requested: 6 Feb 2008 Revisions received: 18 Mar 2008 Accepted: 14 Apr 2008 Published: 14 Apr 2008 Arthritis Research & Therapy 2008, 10:R40 (doi:10.1186/ar2398) This article is online at: http://arthritis-research.com/content/10/2/R40 © 2008 Siala et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Introduction Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing. Methods We examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database. Results Bacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei, were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples. Conclusion This study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The Open Access Available online http://arthritis-research.com/content/11/4/R102 Page 1 of 11 (page number not for citation purposes) Vol 11 No 4 Research article Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis Mariam Siala 1 , Radhouane Gdoura 1 , Hela Fourati 2 , Markus Rihl 3 , Benoit Jaulhac 4 , Mohamed Younes 5 , Jean Sibilia 4 , Sofien Baklouti 2 , Naceur Bargaoui 5 , Slaheddine Sellami 6 , Abdelghani Sghir 7,8 and Adnane Hammami 1 1 Laboratoire de Recherche 'Micro-organismes et Pathologie Humaine', EPS Habib Bourguiba, Rue El Ferdaous, 3029 Sfax, Tunisie 2 Service de Rhumatologie Hôpital Hedi Chaker, Avenue Majida Boulila, 3029 Sfax, Tunisie 3 Hannover Medical School (MHH), Clinic for Immunology and Rheumatology, 30625 Hannover; Germany 4 Laboratoire de Physiopathologie des Interactions Hôte-bactérie, UPRES-EA 3432, Faculté de Médecine, Université Louis-Pasteur, rue Koeberlé, 67000 Strasbourg, France 5 Service de Rhumatologie, EPS Fattouma Bourguiba, Rue 1er Juin, 5019 Monastir, Tunisie 6 Service de Rhumatologie, EPS La Rabta, rue 7051 Centre Urbain Nord, 1082 Tunis, Tunisie 7 CNRS-UMR 8030, CEA-Genoscope, rue Gaston Crémieux, 91000 Évry, France 8 University of Evry Val d'Essonne, Boulevard François Mitterrand, 91025 Évry Cedex, 91000 Évry, France Corresponding author: Adnane Hammami, adnene.hammami@rns.tn Received: 8 Apr 2009 Revisions requested: 21 May 2009 Revisions received: 29 May 2009 Accepted: 1 Jul 2009 Published: 1 Jul 2009 Arthritis Research & Therapy 2009, 11:R102 (doi:10.1186/ar2748) This article is online at: http://arthritis-research.com/content/11/4/R102 © 2009 Siala et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Introduction Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples. Methods We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database. Results Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium REVIE W Open Access A comparative approach for the investigation of biological information processing: An examination of the structure and function of computer hard drives and DNA David J D’Onofrio 1,2* , Gary An 3 * Correspondence: davidj@email. phoenix.edu 1 College of Arts and Science, Math Department, University of Phoenix, 5480 Corporate Drive, Suite 240, Troy, Michigan, 48098, USA Abstract Background: The robust storage, updating and utilization of information are necessary for the maintenance and perpetuation of dynamic systems. These systems can exist as constructs of metal-oxide semiconductors and silicon, as in a digital computer, or in the “wetware” of organic compounds, proteins and nucleic acids that make up biological organisms. We propose that there are essential functional properties of centralized information-processing systems; for digital computers these properties reside in the computer’s hard drive, and for eukaryotic cells they are manifest in the DNA and associated structures. Methods: Presented herein is a descriptive framework that compares DNA and its associated proteins and sub-nuclear structure with the structure and function of the computer hard drive. We identify four essential properties of information for a centralized storage and processing system: (1) orthogonal uniqueness, (2) low level formatting, (3) high level formatting and (4) translation of stored to usable form. The corresponding aspects of the DNA compl ex and a computer hard drive are categorized using this classification. This is intended to demonstrate a functional equivalence between the components of the two systems, and thus the systems themselves. Results: Both the DNA complex and the computer hard drive contain components that fulfill the essential properties of a centralized information storage and processing system. The functional equivalence of these components provides insight into both the design process of engineered systems and the evolved solutions addressing similar system requirements. However, there are points where the comparison breaks down, particularly when there are externally imposed information-organizing structures on the computer hard drive. A specific example of this is the imposition of the File Allocation Table (FAT) during high level formatting of the computer hard drive and the subsequent loading of an operating system (OS). Biological systems do not have an external source for a map of their stored information or for an operational instruction set; rather, they must contain an organizational template conserved within their intra-nuclear architecture that “manipulates” the laws of chemistry and physics into a highly robust instruction set. We propose that the epigenetic structure of the intra-nuclear environment and the non-coding RNA may play the roles of a Biological File Allocation Table (BFAT) and biological operating system (Bio-OS) in eukaryotic cells. D’Onofrio and An Theoretical Biology and Medical Modelling 2010, 7:3 http://www.tbiomed.com/content/7/1/3 © 2010 D’Onofrio and An; licensee BioMed Central Ltd. This is an Open Access arti cle distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any me dium, pr ovided the original work is properly cited. Conclusions: The comparison of functional and structural characteristics of the DNA complex and the computer hard drive leads to a new descriptive paradigm that identifies the DNA as a dynamic storage system of biological information. This system is embodied in an autonomous operating system that inductively follows organizational structures, data hierarchy and executable operations that are well understood in the computer science industry. Characterizing the “DNA hard drive” in this fashion can lead to insights arising from discrepancies in the descriptive framework, particularly with respect to positing the role of ... major and minor grooves ([link]) 2/10 DNA Structure and Sequencing DNA has (a) a double helix structure and (b) phosphodiester bonds The (c) major and minor grooves are binding sites for DNA binding... detector to determine the sequence of bases 9/10 DNA Structure and Sequencing Describe the structure and complementary base pairing of DNA DNA has two strands in anti-parallel orientation The sugar-phosphate... (the copying of RNA from DNA) and replication DNA Sequencing Techniques Until the 1990s, the sequencing of DNA (reading the sequence of DNA) was a relatively expensive and long process Using radiolabeled