Chapter 16: The Molecular Basis of Inheritance (DNA) Zooming in on DNA

Mixture of heat-killed S cells and living R cells Living S cells (control) Living R cells (control) Heat-killed S cells (control) RESULTS Mouse dies Mouse healthy Mouse healthy Mouse dies Living S cells are found in blood sample

LE 16-3 Phage head Tail Tail fiber DNA 100 nm Bacterial cell

Activity: Hersey Chase Experiment LE 16-4 Empty protein shell Radioactive protein Radioactivity (phage protein) in liquid Phage Bacterial cell Batch 1: Sulfur (35S) DNA Phage DNA Centrifuge Radioactive DNA Pellet (bacterial cells and contents) Batch 2: Phosphorus (32P) Centrifuge Radioactivity (phage DNA) in pellet Pellet Activity: Hersey Chase Experiment

LE 16-5 Sugar–phosphate backbone Nitrogenous bases 5 end Thymine (T) Adenine (A) Cytosine (C) Phosphate DNA nucleotide Sugar (deoxyribose) 3 end Guanine (G)

Chargaff DNAi.org

Franklin’s X-ray diffraction photograph of DNA LE 16-6 Rosalind Franklin Franklin’s X-ray diffraction photograph of DNA DNAi.org

Figure 16-01 DNAi.org

Purine + purine: too wide LE 16-UN298 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data DNAi.org

Sugar Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G) LE 16-8 Sugar Sugar Adenine (A) Thymine (T) Sugar Sugar Guanine (G) Cytosine (C)

Activity: DNA & RNA Structure Activity: DNA Double Helix 5 end Hydrogen bond 3 end 1 nm 3.4 nm 3 end 0.34 nm 5 end Key features of DNA structure Partial chemical structure Space-filling model Activity: DNA & RNA Structure DNAi.org Activity: DNA Double Helix

LE 16-9_4 The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C. The first step in replication is separation of the two DNA strands. Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand. The nucleotides are connected to form the sugar-phosphate back- bones of the new strands. Each “daughter” DNA molecule consists of one parental strand and one new strand.

LE 16-10 First Second replication replication Parent cell Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, comple-mentary strand. Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA.

Meselson & Stahl LE 16-11 Bacteria cultured in medium containing 15N transferred to medium containing 14N Meselson & Stahl DNA sample centrifuged after 20 min (after first replication) DNA sample centrifuged after 40 min (after second replication) Less dense More dense First replication Second replication Conservative model Semiconservative model Dispersive model

Activity: DNA Replication: An Overview LE 16-12 Parental (template) strand 0.25 µm Origin of replication Daughter (new) strand Bubble Replication fork Two daughter DNA molecules In eukaryotes, DNA replication begins at may sites along the giant DNA molecule of each chromosome. In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM). Activity: DNA Replication: An Overview

Activity: DNA Replication: A Closer Look LE 16-13 New strand Template strand 5¢ end 3¢ end 5¢ end 3¢ end Sugar Base Phosphate DNA polymerase 3¢ end 3¢ end Pyrophosphate Nucleoside triphosphate 5¢ end 5¢ end Activity: DNA Replication: A Closer Look

Overall direction of replication LE 16-14 3¢ Parental DNA 5¢ Leading strand 5¢ 3¢ Okazaki fragments Lagging strand 3¢ 5¢ DNA pol III Template strand Leading strand Lagging strand Template strand DNA ligase Overall direction of replication

LE 16-15_6 Primase joins RNA nucleotides into a primer. 3¢ 5¢ 5¢ 3¢ Template strand DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 3¢ RNA primer 3¢ 5¢ 5¢ After reaching the next RNA primer (not shown), DNA pol III falls off. Okazaki fragment 3¢ 3¢ 5¢ 5¢ After the second fragment is primed, DNA pol III adds DNA nucleotides until it reaches the first primer and falls off. 5¢ 3¢ 3¢ 5¢ DNA pol I replaces the RNA with DNA, adding to the 3¢ end of fragment 2. 5¢ 3¢ 3¢ 5¢ DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. The lagging strand in the region is now complete. 5¢ 3¢ 3¢ 5¢ Overall direction of replication

Activity: DNA Replication: A Review LE 16-16 Overall direction of replication Leading strand Lagging strand Origin of replication Lagging strand Leading strand OVERVIEW DNA pol III Leading strand DNA ligase Replication fork 5¢ DNA pol I 3¢ Primase Parental DNA DNA pol III Lagging strand Primer 3¢ 5¢ DNAi.org Activity: DNA Replication: A Review

Proofreading and Repair LE 16-17 A thymine dimer distorts the DNA molecule. Proofreading and Repair A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease Repair synthesis by a DNA polymerase fills in the missing nucleotides. DNA polymerase DNA ligase DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete.

Telomeres LE 16-18 5¢ End of parental DNA strands Leading strand Lagging strand 3¢ Last fragment Previous fragment RNA primer Telomeres Lagging strand 5¢ 3¢ Primer removed but cannot be replaced with DNA because no 3¢ end available for DNA polymerase 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 leading strand 5¢ 3¢ Further rounds of replication Shorter and shorter daughter molecules