DNA Topology DNA has to be coiled to fit inside the cell OrganismNumber of base pairs Contour length,  m E. Coli bacteria 4,600,0001,360 SV40 virus5, Human chromosomes 48,000, ,000, – 8.2 cm DNA polymers must be folded to fit into the cell or nucleus (tertiary structure).

DNA Topology :

DNA Topology: linking number

Consider a 260 bp B-duplex:

Connect the ends to make a circular DNA: Tw = 260/10.4 = 25

Stryer Fig An electron micrograph of negatively supercoiled and relaxed DNA

Organization of chromosomal DNA Chromosomal DNA is organized in loops (no free ends) It is negatively supercoiled: 1 (-) supercoil per 200 nucleotides Histone octamer (H2A, H2B, H3, H4) bp duplex 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 Relax DNA supercoiling by increments of 1 (cleave one strand) Topoisomerases II Change DNA supercoiling by the increments of 2 (break both strands) Usually introduce negative supercoiling

Human DNA Topoisomerase I: DNA: side view 20Å Stryer Fig

Mechanism of DNA Topoisomerases I OH P-Topo  Wr = 1 723

Drugs that inhibit DNA Topoisomerase I 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 Relax DNA supercoiling by increments of 1 (cleave one strand) Topoisomerases II Change DNA supercoiling by the increments of 2 (break 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

Topoisomerase II - mechanism Stryer Fig

Drugs that inhibit bacterial Topoisomerase II (DNA gyrase) Interfere with breakage and rejoining DNA ends: Inhibit ATP binding:

Enzymes that cut DNA and RNA : nucleases (Dnases and Rnase) HO Degrade DNA in a stepwise manner by removing deoxynucleotides in 5’  3’ (A) or 3’  5’ direction (B) Require a free OH Exonucleases can be active on both single- and double-stranded DNA Used for degrading foreign DNA and in proofreading during DNA synthesis 5’ 3’ + dNMPs 3’ 5’ A Nucleobase Phosphate group 2’-deoxyribose Exonucleases B Examples: B: snake venom phosphodiesterase A : calf intestinal phosphodiesterase

DNA Endonucleases EcoRI recognition site : Cleave internal phosphodiester bonds resulting in 3’-OH and 5’-phosphate ends 3’-OH 5’ 5’-P Type II Restriction endonucleases are highly sequence specific RE are found in bacteria where they are used for protection against foreign DNA some endonucleases cleave randomly (DNase I, II) Palindromic site (inverted repeat)

Recognition sequences of some common restriction endonucleases

DNA Restriction Enzyme EcoR V 5’-GAT ATC-3’ 3’-CTA TAG-5’ Asn185 Thr186 HN N O O N N N N NH 2 AT N H O N O

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 OH P O -OO O- OP O O DNA Ligase + (ATP or NAD+) AMP + PPi 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 10 9 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

Enzymesubstrate examples DNA regions involved cytochrome 2B6cyclophosphamideexons 1,4,5, and 9 tamoxifen benzodiazepines cytochrome 2D6 debrisoquineinternal base changes cytochrome 1A2caffein5' flanking region phenacetin N-acetyltransferasearomatic amines Examples of genetic polymorphisms of drug metabolizing enzymes

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. Inter- individual differences are observed at about 1 per 1,000 nucleotides.