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Chapter 16: DNA Structure & Replication 1. DNA Structure 2. DNA Replication 1. DNA Structure Chapter Reading – pp. 313-318 Genetic Material: Protein or DNA? Until the early 1950’s no one knew for sure, but it was generally thought that protein was the genetic material. Why? • protein is made of 20 different amino acids • DNA is made of only 4 different nucleotides • protein could theoretically store more info • a “20 letter alphabet” vs a “4 letter alphabet” • it was assumed that life was so complex, therefore a “bigger alphabet” was necessary to somehow encode it! Some classic experiments would prove otherwise… Transformation of Bacteria EXPERIMENT Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Demonstrated the transfer of a genetic trait between different bacteria. The nature of that genetic material was still unknown. RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies (Frederick Griffith, 1928) Living S cells What is the Genetic Material of Bacteriophages? Bacteriophages are viruses that infect bacteria. • consist of a protein capsid which contains DNA What enters the bacterial host cell, viral protein, DNA, or both? Tail sheath Tail fiber DNA Bacterial cell 100 nm • whatever enters the host cell should be the genetic material Phage head Bacteriophage Genetic Material is DNA EXPERIMENT Phage Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Bacterial cell Batch 1: Radioactive sulfur (35S) DNA Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: Radioactive phosphorus (32P) Centrifuge (Alfred Hershey & Martha Chase, Radioactivity Pellet (phage DNA) in pellet 1952) The Discovery of DNA Structure Using the technique of x-ray crystallography, Rosalind Franklin, James Watson & Francis Crick figured out the structure of DNA • Watson & Crick used the X-ray diffraction data of Rosalind Franklin to deduce the structure of DNA (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA DNA: a Polymer of 4 Nucleotides pyrimidines purines A T C G Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T) Adenine (A) Cytosine (C) Phosphate Guanine (G) Sugar (deoxyribose) DNA nucleotide 3 end Nitrogenous base Structure of a DNA Strand A single DNA polymer or “strand” consists of a sugar-phosphate backbone with the bases project out. The ends of a DNA strand are different, with one end having a free 5’ phosphate, and the other having a free 3’ hydroxyl group Structure of Double-stranded DNA • the 2 strands are anti-parallel and interact via base pairs C 5 end G C Hydrogen bond G C G 3 end C G A T 3.4 nm A T C G C G A T 1 nm C A G C G A G A T 3 end T A T G C T C C G T 0.34 nm 5 end A (a) Key features of DNA structure (b) Partial chemical structure (c) Space-filling model DNA “Base-Pairing” Base pairs are held together by hydrogen bonds. Why only A:T and C:G? • the position of chemical groups involved in H-Bonds Sugar Sugar Adenine (A) Thymine (T) • the size of the bases (purine & pyrimidine) Purine purine: too wide Sugar Pyrimidine pyrimidine: too narrow Sugar Guanine (G) Cytosine (C) Purine pyrimidine: width consistent with X-ray data The DNA “Sequence” The DNA sequence is the linear order of nucleotides in a DNA strand: • each DNA strand in the double helix has its own sequence • the sequences in each strand are considered as complementary to each other • they differ, but “fit just right” with each other • ea strand will “fit” with only 1 complementary strand e.g. 5’ 3’ A–C–A–C–A–C–A–C–A–C 3’ T–G–T–G–T–G–T–G–T–G 5’ 2. DNA Replication Chapter Reading – pp. 318-330 How is DNA Replicated? Every time a cell reproduces (i.e., divides) it must replicate its chromosomes (DNA) during S phase. The process of DNA replication was originally proposed to depend on the rules of base pairing: • A:T & T:A , C:G & G:C • the sequence of one strand dictates the sequence of the other • each strand of the double helix could serve as a template to make a complementary strand Model for DNA Replication A T A T A T A T C G C G C G C G T A T A T A T A A T A T A T A T G C G C G C G C (a) Parent molecule (b) Separation of strands (c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand The semiconservative model of DNA replication proposed that each original strand serves as a template to produce a new complementary strand. • note that ea original strand ends up in a different molecule Parent cell (a) Conservative model First Second replication replication Other Models CONSERVATIVE The original DNA strands stay together (b) Semiconservative model SEMICONSERVATIVE The original DNA strands remain intact in separate molecules (c) Dispersive model DISPERSIVE The original DNA strands are dispersed among the all daughter strands Testing the Models In this experiment, bacteria with DNA containing the “heavy” isotope 15N were allowed to reproduce in medium containing lighter 14N. Density-gradient centrifugation revealed that DNA replication is semiconservative. Matthew Meselson & Franklin Stahl, 1958 EXPERIMENT 1 Bacteria cultured in medium with 15N (heavy isotope) 2 Bacteria transferred to medium with 14N (lighter isotope) RESULTS 3 DNA sample centrifuged after first replication CONCLUSION Predictions: First replication Conservative model Semiconservative model Dispersive model 4 DNA sample centrifuged after second replication Less dense More dense Second replication DNA Replication in Bacteria (a) Origin of replication in an E. coli cell Origin of replication Parental (template) strand Daughter (new) strand Doublestranded DNA molecule Replication bubble Initiation of DNA replication requires an origin of replication Replication fork Two daughter DNA molecules 0.5 m DNA Replication in Eukaryotes (b) Origins of replication in a eukaryotic cell Origin of replication Parental (template) strand Bubble Double-stranded DNA molecule Daughter (new) strand Eukaryotic DNA replication requires multiple origins of replication Replication fork Two daughter DNA molecules 0.25 m Overview of DNA Replication Leading strand Overview Origin of replication Lagging strand Primer Leading strand Lagging strand Overall directions of replication DNA Replication proceeds 5’ to 3’ Template strand 3 New strand 5 Sugar Phosphate A Base T C G G C DNA polymerase can add only to the 3’ end A P C Nucleoside triphosphate 3 A T C G G C T A DNA polymerase OH 3 5 OH Pi Pyrophosphate 3 C 2Pi 5 5 Enzymes involved in DNA Replication DNA Polymerase – synthesizes new DNA Helicase – unwinds DNA double helix Topoisomerase – relieves tension due to DNA unwinding Primase Topoisomerase 3 5 5 3 Primase – 3 makes short RNA RNA primers primer Helicase 5 Single-strand binding proteins DNA Ligase – connects DNA fragments Leading Strand DNA Synthesis Proceeds toward unwinding replication fork: Origin of replication 3 5 RNA primer 5 3 3 Sliding clamp DNA pol III Parental DNA 5 3 5 5 3 3 5 DNA synthesis is continuous on the leading strand only DNA polymerase can only synthesize DNA in a 5’ to 3’ direction. DNA polymerase requires an RNA primer which it can extend in a continuous manner toward the unwinding replication fork Lagging Strand DNA Synthesis 3 5 Template strand Proceeds away from the unwinding replication fork: 3 5 3 RNA primer for fragment 1 5 1 • DNA polymerase synthesizes DNA in Okazaki fragments 3 5 Okazaki fragment 1 3 5 RNA primer for fragment 2 1 • each fragment requires an RNA primer 3 5 5 3 2 Okazaki fragment 2 1 5 3 DNA synthesis is discontinuous on the lagging strand • another DNA polymerase will replace the RNA with DNA 3 5 2 1 5 3 3 5 • DNA ligase will link the fragments together 2 1 3 5 Overall direction of replication Summary of DNA Replication Overview Origin of replication Leading strand Lagging strand Leading strand Lagging strand Overall directions of replication Leading strand DNA pol III 5 3 3 Parental DNA Primer 5 3 Primase 5 DNA pol III 4 Lagging strand DNA pol I 35 3 2 DNA ligase 1 3 5 DNA replication proceeds in this manner in ALL living organisms. Current Model of DNA Replication DNA pol III Parental DNA 5 3 5 3 5 5 Connecting protein 3 Helicase 3 DNA pol III Leading strand 3 5 3 5 Lagging strand Lagging strand template 5 3 3 5 Nuclease 5 3 3 5 DNA polymerase DNA Repair When DNA is damaged it is essential that the DNA is repaired so it can be replicated and expressed properly. • special enzymes recognize and remove the damaged portion of DNA 5 3 3 5 • a DNA polymerase will fill in the gap 5 3 3 5 • DNA ligase will then connect the newly made DNA to the adjacent strand DNA ligase The Problem with Telomeres The ends of linear chromosomes, the telomeres, cannot be completely copied on the lagging strand. 5 Leading strand Lagging strand Ends of parental DNA strands 3 Last fragment RNA primer Lagging strand 5 3 Parental strand This results in progressive shortening of the chromosome every time it is replicated. Next-to-last fragment Removal of primers and replacement with DNA where a 3 end is available 5 3 Second round of replication 5 New leading strand 3 New lagging strand 5 Telomerase will solve this problem in certain cell types 3 Further rounds of replication Shorter and shorter daughter molecules …more on Chromatin Chromatin refers to the complex of DNA and histone proteins in eukaryotic nuclei: • chromosomal DNA wraps around histone proteins to form structures called nucleosomes that look like “beads on a string” • different parts of a chromosome can be in various states of “packing” EUCHROMATIN – loosely packed DNA HETEROCHROMATIN – tightly packed DNA Key Terms for Chapter 16 • bacterial transformation, bacteriophage • x-ray crystallography • pyrimidine, purine, base-pair, complementary • double helix, anti-parallel, sugar-phosphate backbone • DNA replication, template • conservative, semiconservative, dispersive • DNA polymerase, helicase, topoisomerase, primase, DNA ligase Relevant Chapter Questions 1-7, 9 • leading, lagging strand; continuous, discontinuous • Okazaki fragment, telomere, telomerase • chromatin, nucleosome, euchromatin, heterochromatin