Watson-Crick Model of B-DNA

Transcription

1 Reading: Ch8; Ch24; Problems: Ch8 (text); 9 Ch8 (study-guide: facts); 3 Ch24 (text); 5,7,9,10,14,16 Ch24 (study-guide: applying); 1 Ch24 (study-guide: facts); 1,2,4 NEXT Reading: Ch1; Ch8; Ch9; , 346 Problems: Ch8 (text); 6,7,8,10 Ch8 (study-guide: applying); 1,3 Ch8 (study-guide: facts); 10,11 Ch9 (text); 1,2,3,4 Ch9 (study-guide: facts); 1,2,3,4,5 Ch24 (study-guide: facts); 3,5,6 Ch26 (text); 3 Ch26 (study-guide: applying); 2,3 Ch26 (study-guide: facts); 7 Ch27 (text); 1,2,3,4 Lecture 23 (11/10/17) Nucleic Acids A. The 4 S s 1. Size 2. Solubility 3. Shape a. A-DNA b. Z-DNA c. Topology i. Packaging ii. Supercoiling iii.topoisomerases 4. Stability a. Nucleotides i. Tautomers ii. Acid/base b. Nucleic Acids i. Chemistry ii. Denaturation iii.stability iv.nucleases Watson-Crick Model of B-DNA 1

2 Nucleic Acids: Shape Sterically Allowed Base Orientations B-DNA B-DNA Notice the bp interface Nucleic Acids: Shape Nucleotide Sugar Conformations RNA RNA-DNA hybrid A-DNA B-DNA What is this A-DNA? 2

3 Nucleic Acids: Shape Nucleic Acids: Shape 29 Å 6 Are there other shapes of DNA? 3

4 Nucleic Acids: Shape Nucleic Acids: Shape Structural Features of A-, B-, & Z-DNA 29 Å 6 4

5 Other Forms of DNA 9 5

6 Nucleic Acids: Shape Nucleotide Sugar Conformations Metaphase Chromosome How do we get something that is 2-10 cm long into one of these, which is only 10 µm (10,000x)? 6

7 Metaphase Chromatin Organization What is this nucleosome? (2 nm) Histone H1 Bound to Nucleosome 8 Histones make up the core 7

8 Histones Are Highly Conserved Nucleosome Core Particle Chicken nucleosome core particle PDBid 1EQZ 8

9 Interactions between Nucleosomes Front and Side Views of Histone Amino-Terminal Tails Amino-Terminal Tails 30-nm Chromatin Fiber 300 Å 30-nm chromatin fiber PDBid 1ZBB 9

10 Histone-Depleted Chromosome DNA Loops Attached to Scaffold All this looping leads to a supertwist of the DNA Nucleosomal DNA Is Underwound Wrapping DNA around the histone core requires removal of one helical turn. The underwinding occurs without a strand break, so a compensatory (+) supercoil forms. This (+) supercoil is relaxed by a topoisomerase, leaving DNA with net negative supercoil. 10

11 What is Supercoiling: the Coiling of a Coil All this looping leads to a supertwist of the DNA Topology L = T + W (for B-DNA: T=#bp/10) Does this occur in biology? 11

12 Topology Is supercoiling only possible with circular DNA? Eukaryotic chromosomes are linear.. Supercoiling of Circular Duplex DNA from the adenovirus, SV40 Topology Nonsupercoiled DNA is called relaxed. Many circular DNAs are supercoiled. Supercoiling has great influence on transcription and replication of DNA. Supercoiling can be highly regulated. 12

13 Consequences of supercoiling: 1) Required for information retrieval; must be negative 2) All circular extra-chromosomal DNAs are negatively supercoiled 3) Can be used for isolation of these DNAs in the laboratory Techniques to Detect Topology Separate DNAs with different topology based on density Supercoiled (more dense) Linear/relaxed (less dense) 13

14 Techniques to Detect Topology Modify topology with topoisomerase I Gel Electrophoresis/Ethidium-Bromide Staining What is Topo I? Type IA Topoisomerase Activity Can generate catemers of circular ss-dnas Type I Topoisomerase also called nickingclosing enzyme Can take supercoiled DNA and make it less supercoiled; usually less negative (L = +1) 14

15 Topoisomerase Y723F Topoisomerase I Mutant Y723F mutant of human topoisomerase I PDBid 1A36 Monomeric Creates single-stranded breaks Use of covalent Tyr intermediate Changes L by +1 (if W 0) Uses energy in supercoils (W# 9 kcal/mole) IA & IB types differ in how they relax the cleaved strand Topoisomerase Covalent Topoisomerase-DNA Adduct

16 Topoisomerase Type IA Topoisomerase Mechanism Topoisomerase Type II Topoisomerase Mechanism Type II Topoisomerase also called gyrase Tetrameric (a 2 b 2 ; a=105 kda, b=95 kda (ATPase)) Creates double-stranded breaks Use of covalent Tyr intermediate Changes L by 2 Uses ATP for energy to put in supercoils (DL = 2) 16

17 Topoisomerase Topoisomerase II Related to Daunorubicin, which nhibits re-joining in (step #5 on previous slide) of Topo II (DNA gyrase) Yeast topoisomerase II PDBids 1PVG and 2RGR X-Ray structures of yeast topoisomerase II. Topoisomerase Inhibitors as Antibiotics/Anticancer Chemotherapeutics 17