The Molecular Basis of Inheritance Chapter 16 The Molecular Basis of Inheritance

Overview: Life’s Operating Instructions In 1953, James Watson and Francis Crick introduced an elegant double-helical model for the structure of deoxyribonucleic acid, or DNA

The Search for the Genetic Material: Scientific Inquiry When Morgan’s group showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material

Evidence That DNA Can Transform Bacteria The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928 Living S cells (control) Living R cells Heat-killed S cells (control) Mixture of heat-killed S cells and living R cells Mouse dies are found in blood sample Mouse healthy RESULTS

Evidence That Viral DNA Can Program Cells Viruses, called bacteriophages (or phages), are widely used in molecular genetics research Animation: Phage T2 Reproductive Cycle

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

Animation: Hershey-Chase Experiment The 1952, Alfred Hershey & Martha Chase Experiment Bacterial cell Phage DNA Radioactive protein Empty protein shell Radioactivity (phage protein) in liquid Batch 1: Sulfur (35S) Centrifuge Pellet (bacterial cells and contents) Pellet (phage DNA) in pellet Batch 2: Phosphorus (32P) Animation: Hershey-Chase Experiment

Additional Evidence That DNA Is the Genetic Material Sugar–phosphate backbone 5 end Nitrogenous bases Thymine (T) Adenine (A) Cytosine (C) DNA nucleotide Phosphate 3 end Guanine (G) Sugar (deoxyribose) In 1947, Erwin Chargaff reported that DNA composition varies from one species to the next. Animation: DNA and RNA Structure

Building a Structural Model of DNA: Scientific Inquiry Rosalind Franklin was using a technique called X-ray crystallography and produced a picture of DNA Franklin’s X-ray diffraction photograph of DNA Rosalind Franklin

Animation: DNA Double Helix Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical 5 end 3 end Space-filling model Partial chemical structure Hydrogen bond Key features of DNA structure 0.34 nm 3.4 nm 1 nm

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

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

Animation: DNA Replication Overview Concept 16.2: Many proteins work together in DNA replication and repair Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material 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. Animation: DNA Replication Overview

Watson and Crick’s semiconservative model 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. Parent cell First replication Second Watson and Crick’s semiconservative model Competing models: conservative model dispersive model

LE 16-11 Bacteria cultured in medium containing 15N Bacteria transferred to medium containing 14N Experiments by Meselson and Stahl supported the semiconservative model. 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

DNA Replication: A Closer Look More than a dozen enzymes and other proteins participate in DNA replication

Getting Started: Origins of Replication Replication begins at special sites called origins of replication At the end of each replication bubble is a replication fork

Animation: Origins of Replication 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). Animation: Origins of Replication

Elongating a New DNA Strand Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork New strand 5¢ end Phosphate Base Sugar Template strand 3¢ end Nucleoside triphosphate DNA polymerase Pyrophosphate

Antiparallel Elongation Parental DNA 5¢ 3¢ Leading strand Okazaki fragments Lagging strand DNA pol III Template strand DNA ligase Overall direction of replication Terms: Leading Strand Lagging Strand Okazaki Fragments DNA Polymerase DNA Ligase Animation: Leading Strand

Priming DNA Synthesis DNA polymerases cannot initiate synthesis of a polynucleotide The initial nucleotide strand is a short one called an RNA or DNA primer An enzyme called primase produces the primer.

LE 16-15_1 Primase joins RNA nucleotides into a primer. 3¢ 5¢ 5¢ 3¢ Template strand Overall direction of replication

LE 16-15_2 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 5¢ 3¢ 5¢ Overall direction of replication

LE 16-15_3 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¢ Overall direction of replication

LE 16-15_4 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¢ Overall direction of replication

LE 16-15_5 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¢ Overall direction of replication

Animation: Lagging Strand 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¢ Animation: Lagging Strand Overall direction of replication

Other Proteins That Assist DNA Replication Helicase Primase DNA pol III DNA pol I DNA ligase

Animation: DNA Replication 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¢ Animation: DNA Replication Review

Proofreading and Repairing DNA DNA polymerases proofread newly made DNA, correcting most mistakes. Mismatch repair of DNA - repair enzymes correct errors in base pairing Nucleotide excision repair - enzymes cut out and replace damaged stretches of DNA DNA ligase polymerase DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete. Repair synthesis by a DNA polymerase fills in the missing nucleotides. A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease A thymine dimer distorts the DNA molecule.

Replicating the Ends of DNA Molecules End of parental DNA strands 5¢ 3¢ Lagging strand Last fragment RNA primer Leading strand Previous fragment 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 Second round of replication Further rounds New leading strand Shorter and shorter daughter molecules Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes

Telomeres Do not prevent the shortening of DNA molecules, but postpone the erosion of genes near the ends of DNA molecules

LE 16-19 1 µm

An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells