1. DNA Structure. Genetic Material: Protein or DNA? 10/28/2015. Chapter 16: DNA Structure & Replication. 1. DNA Structure. 2.

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1 hapter 6: DN Structure & Replication. DN Structure 2. DN Replication. DN Structure hapter Reading pp enetic Material: Protein or DN? Until the early 950 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 DN 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

2 00 nm 0/28/205 ransformation of Bacteria EXPERIMEN Living S cells (control) RESULS 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. he nature of that genetic material was still unknown. Mouse dies Mouse healthy Mouse healthy Mouse dies (Frederick riffith, 928) Living S cells What is the enetic Material of Bacteriophages? Bacteriophages are viruses that infect bacteria. consist of a protein capsid which contains DN What enters the bacterial host cell, viral protein, DN, or both? whatever enters the host cell should be the genetic material Bacterial cell Phage head ail sheath ail fiber DN Bacteriophage enetic Material is DN EXPERIMEN Phage Bacterial cell Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Batch : Radioactive sulfur ( 35 S) DN Phage DN entrifuge Radioactive DN Pellet (bacterial cells and contents) Batch 2: Radioactive phosphorus ( 32 P) entrifuge (lfred Hershey & Martha hase, 952) Pellet Radioactivity (phage DN) in pellet 2

3 he Discovery of DN Structure Using the technique of x-ray crystallography, Rosalind Franklin, James Watson & Francis rick figured out the structure of DN Watson & rick used the X-ray diffraction data of Rosalind Franklin to deduce the structure of DN (a) Rosalind Franklin (b) Franklin s X-ray diffraction photograph of DN DN: a Polymer of 4 Nucleotides pyrimidines purines phosphate backbone end Nitrogenous bases hymine () denine () ytosine () Structure of a DN Strand single DN polymer or consists of a sugar-phosphate backbone with the bases project out. Phosphate (deoxyribose) DN Nitrogenous base nucleotide end uanine () he ends of a DN are different, with one end having a free 5 phosphate, and the other having a free 3 hydroxyl group 3

4 Structure of Double-ed DN the 2 s are anti-parallel and interact via base pairs end Hydrogen bond end 3.4 nm nm end 0.34 nm end (a) Key features of DN structure (b) Partial chemical structure (c) Space-filling DN Base-Pairing Base pairs are held together by hydrogen bonds. Why only : and :? denine () hymine () the position of chemical groups involved in H-Bonds the size of the bases (purine & pyrimidine) Purine purine: too wide uanine () ytosine () Pyrimidine pyrimidine: too narrow Purine pyrimidine: width consistent with X-ray data he DN Sequence he DN sequence is the linear order of nucleotides in a DN : each DN in the double helix has its own sequence the sequences in each are considered as complementary to each other e.g. they differ, but fit just right with each other ea will fit with only complementary

5 2. DN Replication hapter Reading pp How is DN Replicated? Every time a cell reproduces (i.e., divides) it must replicate its chromosomes (DN) during S phase. he process of DN was originally proposed to depend on the rules of base pairing: : & :, : & : the sequence of one dictates the sequence of the other each of the double helix could serve as a template to make a complementary Model for DN Replication (a) Parent molecule (b) Separation of s (c) Daughter DN molecules, each consisting of one parental and one new he semiconservative of DN proposed that each original serves as a template to produce a new complementary. note that ea original ends up in a different molecule 5

6 (a) onservative Parent cell First Second Other Models ONSERVIVE he original DN s stay together (b) Semiconservative SEMIONSERVIVE he original DN s remain intact in separate molecules (c) Dispersive DISPERSIVE he original DN s are dispersed among the all daughter s esting the Models In this experiment, bacteria with DN containing the heavy isotope 5 N were allowed to reproduce in medium containing lighter 4 N. Density-gradient centrifugation revealed that DN is semiconservative. Matthew Meselson & Franklin Stahl, 958 EXPERIMEN Bacteria cultured in medium with 5 N (heavy isotope) RESULS 3 DN sample 4 DN sample centrifuged centrifuged after first after second Bacteria transferred to medium with 4 N (lighter isotope) Less dense More dense ONLUSION Predictions: First Second onservative Semiconservative Dispersive 2 (a) Origin of in an E. coli cell Origin of DN Replication in Bacteria Parental (template) Daughter (new) Initiation of DN requires an origin of Doubleed DN molecule Replication bubble Replication fork wo daughter DN molecules 0.5 m 6

7 DN Replication in Eukaryotes (b) Origins of in a eukaryotic cell Origin of Parental (template) Double-ed DN molecule Daughter (new) Eukaryotic DN requires multiple origins of Bubble Replication fork wo daughter DN molecules 0.25 m Overview of DN Replication Leading Overview Origin of Lagging Primer Lagging Overall directions of Leading DN Replication proceeds 5 to 3 New emplate DN polymerase Phosphate Base can add only to the 3 end OH DN polymerase P P i Pyrophosphate OH Nucleoside triphosphate 2 P i 7

8 Enzymes involved in DN Replication DN Polymerase synthesizes new DN Helicase unwinds DN double helix opoisomerase relieves tension due to DN unwinding opoisomerase Helicase Primase Single- binding proteins RN primer Primase makes short RN primers DN Ligase connects DN fragments Leading Strand DN Synthesis Proceeds toward unwinding fork: Parental DN Origin of RN primer Sliding clamp DN pol III DN synthesis is continuous on the leading only DN polymerase can only synthesize DN in a 5 to 3 direction. DN polymerase requires an RN primer which it can extend in a continuous manner toward the unwinding fork emplate Lagging Strand DN Synthesis RN primer for fragment 2 Okazaki fragment 2 DN synthesis is discontinuous on the lagging Okazaki fragment 2 RN primer for fragment 2 Proceeds away from the unwinding fork: 2 Overall direction of DN polymerase synthesizes DN in Okazaki fragments each fragment requires an RN primer another DN polymerase will replace the RN with DN DN ligase will link the fragments together 8

9 Summary of DN Replication Leading Overview Origin of Lagging Leading Leading Lagging Overall directions of DN pol III Parental DN Primer Primase DN pol III 4 Lagging DN pol I DN ligase 3 2 DN proceeds in this manner in LL living organisms. urrent Model of DN Replication DN pol III Parental DN Leading onnecting protein DN pol III Helicase Lagging Lagging template Nuclease DN polymerase DN Repair When DN is damaged it is essential that the DN is repaired so it can be replicated and expressed properly. special enzymes recognize and remove the damaged portion of DN DN ligase a DN polymerase will fill in the gap DN ligase will then connect the newly made DN to the adjacent 9

10 he Problem with elomeres he ends of linear chromosomes, the telomeres, cannot be completely copied on the lagging. his results in progressive shortening of the chromosome every time it is replicated. Ends of parental DN s Lagging Parental Last fragment RN primer New leading Leading Lagging Next-to-last fragment Removal of primers and replacement with DN where a end is available Second round of elomerase will solve this problem in certain cell types New lagging Further rounds of Shorter and shorter daughter molecules more on hromatin hromatin refers to the complex of DN and histone proteins in eukaryotic nuclei: chromosomal DN 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 EUHROMIN loosely packed DN HEEROHROMIN tightly packed DN 0

11 Key erms for hapter 6 bacterial transformation, bacteriophage x-ray crystallography pyrimidine, purine, base-pair, complementary double helix, anti-parallel, sugar-phosphate backbone DN, template conservative, semiconservative, dispersive DN polymerase, helicase, topoisomerase, primase, DN ligase leading, lagging ; continuous, discontinuous Relevant hapter Questions -7, 9 Okazaki fragment, telomere, telomerase chromatin, nucleosome, euchromatin, heterochromatin