* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download DNA is Composed of Complementary Strands
Survey
Document related concepts
Eukaryotic DNA replication wikipedia , lookup
DNA sequencing wikipedia , lookup
DNA repair protein XRCC4 wikipedia , lookup
Zinc finger nuclease wikipedia , lookup
Homologous recombination wikipedia , lookup
DNA replication wikipedia , lookup
DNA profiling wikipedia , lookup
DNA polymerase wikipedia , lookup
DNA nanotechnology wikipedia , lookup
Microsatellite wikipedia , lookup
Transcript
Hydrogen bond donors and acceptors in DNA grooves facilitate its recognition by proteins The edges of base pairs displayed to DNA major and minor groove contain potential H-bond donors and acceptors: Major groove N n O H 2N h o h To n= Nitrogen hydrogen bond acceptor o= Oxygen hydrogen bond acceptor h= Amino hydrogen bond donor O N H 2N o de xy N se o rib H2N NH N N N NH2 O Minor groove To d eox yri bo se Hydrogen bond donors and acceptors on each edge of a base pair Major groove To o de xy os b i r e To d Minor groove eox yri bo se Structural characteristics of DNA facilitating DNA-Protein Recogtnition 1. Major and major groove of DNA contain sequencedependent patterns of H-bond donors and acceptors. 2. Sequence-dependent duplex structure (A, B, Z, bent DNA). 3. Hydrophobic interactions via intercalation. 4. Ionic interactions with phosphates. Groove binding proteins and drugs NH3 Leucine zipper proteins bind DNA major groove H2N H N NH3 NH2 4,6-diamidino-2-phenylindole (DAPI) 5’-ATT-3’ Others: netropsin, distamycin, Hoechst 33258 Triple helix and Antigene approach N N G O N N H NH2 O N N G H2N NH N C N N NH2 O G:GC Hoogsteen base pairing = parallel Reversed Hoogsteen = antiparallel Biophysical properties of DNA • Facile denaturation (melting) and re-association of the duplex are important for DNA’s biological functions. In the laboratory, melting can be induced by heating. • A260 Single strands T° TM duplex 70 • 80 90 100 T, C Hybridization techniques are based on the affinity of complementary DNA strands for each other. • Duplex stability is affected by DNA length, % GC base pairs, ionic strength, the presence of organic solvents, pH • Negative charge – can be separated by gel electrophoresis Separation of DNA fragments by gel electrophoresis Polyacrylamide gel: O H2C CH-C-NH2 O O H2C CH-C-NH-CH2 HN-C-C CH2 SO4- H2N O C H2C CH- • DNA strands are negatively charged – migrate towards the (+) electrode (anode) • Migration time ~ ln (number of base pairs) DNA Topology DNA has to be coiled to fit inside the cell Organism Number of base pairs Contour length, m E. Coli bacteria 4,600,000 1,360 SV40 virus 5,100 1.7 Human chromosomes 48,000,000240,000,000 1.6 – 8.2 cm DNA polymers must be folded to fit into the cell or nucleus (tertiary structure). DNA Topology: • Negative supercoiling: DNA is twisted in the direction opposite to the direction of the double helix (underwound) • Positive supercoiling: DNA is twisted in the same direction as the direction of the double helix (overwound) DNA Topology: linking number • Topoisomers can be quantitatively defined by the linking number (Lk). • Lk is the number of times a strand of DNA winds in the right handed direction around the helix axis when the axis is constrained. • Tw (twist) is the helical winding of the strands around each other (# b.p./10.4 for B form DNA). • Wr (writh) is the number of superhelical turns Lk = Tw + Wr, if Lk = const., Tw = - Wr Consider a 260 bp B-duplex: Connect the ends to make a circular DNA: Tw = 260/10.4 = 25 An electron micrograph of negatively supercoiled and relaxed DNA Stryer Fig. 27.20 Organization of chromosomal DNA • Chromosomal DNA is organized in loops (no free ends) • It is negatively supercoiled: 1 (-) supercoil per 200 nucleotides 145 bp duplex Histone octamer (H2A, H2B, H3, H4)2 H1 is bound to the linker region Enzymes that control DNA supercoiling: DNA Topoisomerases Change the linking number (Lk) of DNA duplex by concerted breakage and re-joining DNA strands Topoisomerase enzymes Topoisomerases I Topoisomerases II Relax DNA supercoiling by increments of 1 (cleave one strand) Change DNA supercoiling by the increments of 2 (cleave both strands) Usually introduce negative supercoiling Human DNA Topoisomerase I: DNA: side view 20Å Stryer Fig. 27.21 Mechanism of DNA Topoisomerases I -O O Base H H H H OH H 723 OH P-Topo Wr = 1 Drugs that inhibit DNA Topoisomerase I 9 O 10 C-10 C-9 N Camptothecin H Topotecan OH N H (CH3)2NHCH2 O CH3CH2 OH O • Camptothecin, topotecan and analogs • Antitumor activity correlates with interference with topoisomerase activity • Stabilizes topoisomerase I-DNA intermediate, preventing DNA strand re-ligation • Used in treatment of colorectal, ovarian, and small cell lung tumors Enzymes that control DNA supercoiling: DNA Topoisomerases Change the linking number (Lk) of DNA duplex by concerted breakage and re-joining DNA strands Topoisomerase enzymes Topoisomerases I Topoisomerases II Relax DNA supercoiling by increments of 1 (cleave one strand) Change DNA supercoiling by the increments of 2 (cleave both strands) Usually introduce negative supercoiling Topoisomerases II • • • • • Most of Topoisomerases II introduce negative supercoils (e.g. E. coli DNA Gyrase) Require energy (ATP) Each round introduces two supercoils ( Wr = - 2) Necessary for DNA synthesis Form a covalent DNA-protein complex similar to Topoisomerases I Yeast DNA Topoisomerase II Stryer Fig. 27.23 Topoisomerase II - mechanism Stryer Fig. 27.24 Drugs that inhibit bacterial Topoisomerase II (DNA gyrase) Interfere with breakage and rejoining DNA ends: H3C N Et N NH N N COOH F O Nalidixic acid COOH O Ciprofloxacin Inhibit ATP binding: CH3 O CH3 H3CO O CH3 O O OH CH3 CH3 O O OH N OH H NH2 Novobiocin O Enzymes that cut DNA: exonucleases 5’ HO A 5’ 3’ 3’ 5’ OH + dNMPs • Degrade DNA in a stepwise manner by removing deoxynucleotides in 5’ 3’ (A) or 3’ 5’ direction (B) • Require a free OH • Most exonucleases are active on both single- and double-stranded DNA • Used for degrading foreign DNA and in proofreading during DNA synthesis B HO H 3’ Phosphate group Nucleobase 2’-deoxyribose DNA Endonucleases • Cleave internal phosphodiester bonds resulting in 3’-OH and 5’-phosphate ends 5’ 3’-OH 5’-P 5’-P 3’-OH • some endonucleases cleave randomly (DNase I, II) • Type II Restriction endonucleases are highly sequence specific EcoRI recognition site: Cleavage Site G A A T T C C T T A A G Palindromic site (inverted repeat) Cleavage Site • RE are found in bacteria where they are used for protection against foreign DNA Recognition sequences of some common restriction endonucleases DNA Restriction Enzyme EcoR V Applications of Restriction Endonucleases in Molecular Biology 1. DNA fingerprinting (restriction fragment length polymorphism). 2. Molecular cloning (isolation and amplification of genes). Southern blotting Restriction fragment length polymorphisms are used to compare DNA from different sources DNA Ligase AMP + PPi O O OH -O P O- O O DNA Ligase + (ATP or NAD+) P O O- • Forms phosphodiester bonds between 3’ OH and 5’ phosphate • Requires double-stranded DNA • Activates 5’phosphate to nucleophilic attack by transesterification with activated AMP DNA Cloning: recombinant DNA technology Human Genetic Polymorphisms • Human genome size: 3.2 x 109 base pairs • 30,000 genes • 2-4 % of total sequence codes for proteins • Human genetic variation: 1 sigle nucleotide polymorphism (SNP) per 1,300 bp Examples of genetic polymorphisms of drug metabolizing enzymes Enzyme cytochrome 2B6 substrate examples cyclophosphamide tamoxifen benzodiazepines DNA regions involved exons 1,4,5, and 9 cytochrome 2D6 cytochrome 1A2 debrisoquine caffein phenacetin internal base changes 5' flanking region N-acetyltransferase aromatic amines DNA Structure: Take Home Message 1. Genetic information is stored in DNA. 2. DNA is a double stranded biopolymer containing repeating units of nitrogen base, deoxyribose sugar, and phosphate. 3. DNA can be arranged in 3 types of duplexes which contain major and minor grooves. 4. DNA can adopt several topological forms. 5. There are enzymes that will cut DNA, ligate DNA, and change the topology of DNA. 6. Human genome contains about 3.2 billion base pairs. Interindividual differences are observed at about 1 per 1,000 nucleotides.