Chapter 16: DNA Structure & Replication
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1 hapter 16: DN Structure & Replication 1. DN Structure 2. DN Replication
2 1. DN Structure hapter Reading pp
3 enetic Material: Protein or DN? 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 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
4 ransformation of Bacteria EXPERIMEN Living S cells (control) Living R cells (control) Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells RESULS 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, 1928) Living S cells
5 100 nm 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? Phage head ail sheath whatever enters the host cell should be the genetic material Bacterial cell ail fiber DN
6 Bacteriophage enetic Material is DN EXPERIMEN Phage Bacterial cell Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Batch 1: 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, 1952) Pellet Radioactivity (phage DN) in pellet
7 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
8 DN: a Polymer of 4 Nucleotides pyrimidines purines
9 Sugar phosphate backbone end Nitrogenous bases hymine () denine () ytosine () Structure of a DN Strand single DN polymer or strand consists of a sugar-phosphate backbone with the bases project out. Phosphate Sugar (deoxyribose) DN nucleotide end Nitrogenous base uanine () he ends of a DN strand are different, with one end having a free 5 phosphate, and the other having a free 3 hydroxyl group
10 Structure of Double-stranded DN the 2 strands are anti-parallel and interact via base pairs end Hydrogen bond end 3.4 nm 1 nm end 0.34 nm end (a) Key features of DN structure (b) Partial chemical structure (c) Space-filling model
11 DN Base-Pairing Base pairs are held together by hydrogen bonds. Why only : and :? Sugar denine () Sugar hymine () the position of chemical groups involved in H-Bonds the size of the bases (purine & pyrimidine) Purine purine: too wide Sugar uanine () Sugar ytosine () Pyrimidine pyrimidine: too narrow Purine pyrimidine: width consistent with X-ray data
12 he DN Sequence he DN sequence is the linear order of nucleotides in a DN strand: each DN strand in the double helix has its own sequence the sequences in each strand are considered as complementary to each other e.g. they differ, but fit just right with each other ea strand will fit with only 1 complementary strand
13 2. DN Replication hapter Reading pp
14 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 replication was originally proposed to depend on the rules of base pairing: : & :, : & : 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
15 Model for DN Replication (a) Parent molecule (b) Separation of strands (c) Daughter DN molecules, each consisting of one parental strand and one new strand he semiconservative model of DN 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
16 Parent cell First replication Second replication Other (a) onservative model Models ONSERVIVE he original DN strands stay together (b) Semiconservative model SEMIONSERVIVE he original DN strands remain intact in separate molecules (c) Dispersive model DISPERSIVE he original DN strands are dispersed among the all daughter strands
17 esting the Models In this experiment, bacteria with DN containing the heavy isotope 15 N were allowed to reproduce in medium containing lighter 14 N. Density-gradient centrifugation revealed that DN replication is semiconservative. Matthew Meselson & Franklin Stahl, 1958 EXPERIMEN 1 Bacteria cultured in medium with 15 N (heavy isotope) RESULS 3 DN sample centrifuged after first replication 4 DN sample centrifuged after second replication Bacteria transferred to medium with 14 N (lighter isotope) Less dense More dense ONLUSION Predictions: First replication Second replication onservative model Semiconservative model Dispersive model 2
18 DN Replication in Bacteria (a) Origin of replication in an E. coli cell Origin of replication Parental (template) strand Daughter (new) strand Initiation of DN replication requires an origin of replication Doublestranded DN molecule Replication bubble Replication fork wo daughter DN molecules 0.5 m
19 DN Replication in Eukaryotes (b) Origins of replication in a eukaryotic cell Origin of replication Parental (template) strand Double-stranded DN molecule Daughter (new) strand Eukaryotic DN replication requires multiple origins of replication Bubble Replication fork wo daughter DN molecules 0.25 m
20 Overview of DN Replication Leading strand Overview Origin of replication Lagging strand Primer Lagging strand Overall directions of replication Leading strand
21 DN Replication proceeds 5 to 3 New strand Sugar Phosphate Base emplate strand DN polymerase can add only to the 3 end OH DN polymerase P P i Pyrophosphate OH Nucleoside triphosphate 2 P i
22 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-strand binding proteins RN primer Primase makes short RN primers DN Ligase connects DN fragments
23 Leading Strand DN Synthesis Proceeds toward unwinding replication fork: Origin of replication RN primer Sliding clamp DN polymerase can only synthesize DN in a 5 to 3 direction. Parental DN DN pol III DN synthesis is continuous on the leading strand only DN polymerase requires an RN primer which it can extend in a continuous manner toward the unwinding replication fork
24 Lagging Strand DN Synthesis emplate strand RN primer for fragment 2 Okazaki fragment 2 DN synthesis is discontinuous on the lagging strand 1 Okazaki fragment 1 1 RN primer for fragment Proceeds away from the unwinding replication fork: Overall direction of replication 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
25 Summary of DN Replication Overview Leading strand Origin of replication Lagging strand Leading strand Lagging strand Overall directions of replication Leading strand DN pol III Parental DN Primer Primase DN pol III 4 Lagging strand DN pol I DN ligase DN replication proceeds in this manner in LL living organisms.
26 urrent Model of DN Replication DN pol III Parental DN Leading strand onnecting protein Helicase DN pol III Lagging strand Lagging strand template
27 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 strand
28 he Problem with elomeres he ends of linear chromosomes, the telomeres, cannot be completely copied on the lagging strand. his results in progressive shortening of the chromosome every time it is replicated. elomerase will solve this problem in certain cell types Ends of parental DN strands Lagging strand Parental strand Last fragment RN primer New leading strand New lagging strand Leading strand Lagging strand Next-to-last fragment Removal of primers and replacement with DN where a end is available Second round of replication Further rounds of replication Shorter and shorter daughter molecules
29
30 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
31 Key erms for hapter 16 bacterial transformation, bacteriophage x-ray crystallography pyrimidine, purine, base-pair, complementary double helix, anti-parallel, sugar-phosphate backbone DN replication, template conservative, semiconservative, dispersive DN polymerase, helicase, topoisomerase, primase, DN ligase Relevant hapter Questions 1-7, 9 leading, lagging strand; continuous, discontinuous Okazaki fragment, telomere, telomerase chromatin, nucleosome, euchromatin, heterochromatin
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