The Molecular Basis of Inheritance Chapter 16 Overview

The Molecular Basis of Inheritance Chapter # 16

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 • DNA, the substance of inheritance, is the most celebrated molecule of our time • Hereditary information is encoded in DNA and reproduced in all cells of the body • This DNA program directs the development of biochemical, anatomical, physiological, and (to some extent) behavioral traits

Fig. 16 -1 Francis Crick James Watson

Concept 16. 1: DNA is the genetic material • Early in the 20 th century, the identification of the molecules of inheritance loomed as a major challenge to biologists Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

The Search for the Genetic Material: Scientific Inquiry • When T. H. Morgan’s group (started in 1907 with drosophila- fruit flies) showed that genes are located on chromosomes, the two components of chromosomes—DNA and protein—became candidates for the genetic material • The key factor in determining the genetic material was choosing appropriate experimental organisms • The role of DNA in heredity was first discovered by studying bacteria and the viruses that infect them Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Evidence That DNA Can Transform Bacteria • The discovery of the genetic role of DNA began with research by Frederick Griffith in 1928 • Griffith worked with two strains of a bacterium, one pathogenic and one harmless Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• When he mixed heat-killed remains of the pathogenic strain with living cells of the harmless strain, some living cells became pathogenic • He called this phenomenon transformation, now defined as a change in genotype and phenotype due to assimilation of foreign DNA Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -2 Mixture of heat-killed Living S cells Living R cells Heat-killed S cells and (control) S cells (control) living R cells EXPERIMENT capsule Griffith’s experiment in 1928 Living bacteria had been transformed into pathogenic bacteria by an unknown heritable substance from the dead S cells. RESULTS Mouse dies Mouse healthy Mouse dies The “S” strain of bacteria causes pneumonia in mice. Living S cells

• In 1944, Oswald Avery, Maclyn Mc. Carty, and Colin Mac. Leod announced that the transforming substance was DNA • Their conclusion was based on experimental evidence that only DNA worked in transforming harmless bacteria into pathogenic bacteria • Many biologists remained skeptical, mainly because little was known about DNA Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Evidence That Viral DNA Can Program Cells • More evidence for DNA as the genetic material came from studies of viruses that infect bacteria • Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research Animation: Phage T 2 Reproductive Cycle Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -3 Phage head Tail sheath Tail fiber Bacterial cell 100 nm DNA

• In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T 2 • To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T 2 (DNA or protein) enters an E. coli cell during infection • They concluded that the injected DNA of the phage provides the genetic information Animation: Hershery-Chase Experiment Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Alfred Hershey and Martha Chase experiment to see if protein or DNA carried genetic information Phage Radioactive protein Bacterial cell Batch 1: radioactive sulfur (35 S) DNA Radioactive DNA Batch 2: radioactive phosphorus ( 32 P)

Fig. 16 -4 -2 EXPERIMENT Phage Empty protein Radioactive shell protein Bacterial cell Batch 1: radioactive sulfur (35 S) DNA Phage DNA Radioactive DNA Batch 2: radioactive phosphorus ( 32 P) A blender was used to separate the bacteria from the empty protein shells (that are attached to the outside of the bacteria).

The bacteria are denser than the protein coats of the bacteriophages. As a result the bacteriophage coats remain suspended in the liquid while the bacteria form a pellet at the bottom. Phage Empty protein Radioactive shell protein Radioactivity (phage protein) in liquid Bacterial cell Batch 1: radioactive sulfur (35 S) DNA Phage DNA Centrifuge Pellet (bacterial cells and contents) Radioactive DNA Batch 2: radioactive phosphorus ( 32 P) Centrifuge Pellet Conclusion: Phage DNA enters bacterial cells, phage protein does not. DNA functions as genetic material. Radioactivity (phage DNA) in pellet

Additional Evidence That DNA Is the Genetic Material • It was known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group • In 1950, Erwin Chargaff reported that DNA composition varies from one species to the next. 30. 3% of human DNA have the base Adenine (A) while E. coli have 26. 0% (A). • This evidence of diversity made DNA a more credible candidate for the genetic material Animation: DNA and RNA Structure Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• Chargaff found that in every species he studied, the number of Adenine (A) and Thymine (T) bases were equal. Also the number of Guanine (G) and Cytosine (C) base pairs was equal. In humans (A) = 30. 3% (T) = 30. 3 % (G) = 19. 5% and (C) = 19. 9% • Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases. The reason for this rule was unknown until the double helix was discovered

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

Building a Structural Model of DNA: Scientific Inquiry • After most biologists became convinced that DNA was the genetic material, the challenge was to determine how its structure accounts for its role • Maurice Wilkins and Rosalind Franklin were using a technique called X-ray crystallography to study molecular structure • Franklin produced a picture of the DNA molecule using this technique Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -6 (a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

Fig. 16 -6 a (a) Rosalind Franklin

Fig. 16 -6 b (b) Franklin’s X-ray diffraction photograph of DNA

• Franklin’s X-ray crystallographic images of DNA enabled Watson to deduce that DNA was helical • The X-ray images also enabled Watson to deduce the width of the helix and the spacing of the nitrogenous bases • The width suggested that the DNA molecule was made up of two strands, forming a double helix Animation: DNA Double Helix Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -7 5 end Hydrogen bond 3 end 1 nm 3. 4 nm 3 end 0. 34 nm (a) Key features of DNA structure (b) Partial chemical structure 5 end (c) Space-filling model

Fig. 16 -7 a 5 end Hydrogen bond 3 end 1 nm 3. 4 nm 3 end 0. 34 nm (a) Key features of DNA structure (b) Partial chemical structure 5 end

Fig. 16 -7 b (c) Space-filling model

• Watson and Crick built models of a double helix to conform to the X-rays and chemistry of DNA • Franklin had concluded that there were two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• At first, Watson and Crick thought the bases paired like with like (A with A, and so on), but such pairings did not result in a uniform width • Instead, pairing a purine with a pyrimidine resulted in a uniform width consistent with the X -ray Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -UN 1 Purine + purine: too wide Pyrimidine + pyrimidine: too narrow Purine + pyrimidine: width consistent with X-ray data

• Watson and Crick reasoned that the pairing was more specific, dictated by the base structures • They determined that adenine (A) paired only with thymine (T), and guanine (G) paired only with cytosine (C) • The Watson-Crick model explains Chargaff’s rules: in any organism the amount of A = T, and the amount of G = C Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -8 Adenine (A) Thymine (T) Guanine (G) Cytosine (C)

Concept 16. 2: Many proteins work together in DNA replication and repair • The relationship between structure and function is manifest in the double helix • Watson and Crick noted that the specific base pairing suggested a possible copying mechanism for genetic material Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

The Basic Principle: Base Pairing to a Template Strand • Since the two strands of DNA are complementary, each strand acts as a template for building a new strand in replication • In DNA replication, the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules Animation: DNA Replication Overview Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -9 -1 A T C G T A A T G C (a) Parent molecule

Fig. 16 -9 -2 A T C G T A A T G C (a) Parent molecule (b) Separation of strands

Fig. 16 -9 -3 A T A T C G C G T A T A T G C G C (a) Parent molecule (b) Separation of strands (c) “Daughter” DNA molecules, each consisting of one parental strand one new strand

• Watson and Crick’s semiconservative model of replication predicts that when a double helix replicates, each daughter molecule will have one old strand (derived or “conserved” from the parent molecule) and one newly made strand • Competing models were the conservative model (the two parent strands rejoin) and the dispersive model (each strand is a mix of old and new) Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -10 Parent cell (a) Conservative model (b) Semiconservative model (c) Dispersive model First replication Second replication

• Experiments by Matthew Meselson and Franklin Stahl supported the semiconservative model • They labeled the nucleotides of the old strands with a heavy isotope of nitrogen, while any new nucleotides were labeled with a lighter isotope Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• The first replication produced a band of hybrid DNA, eliminating the conservative model • A second replication produced both light and hybrid DNA, eliminating the dispersive model and supporting the semiconservative model Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -11 EXPERIMENT 1 Bacteria cultured in medium containing 15 N 2 Bacteria transferred to medium containing 14 N RESULTS 3 DNA sample centrifuged after 20 min (after first application) 4 DNA sample centrifuged after 40 min (after second replication) CONCLUSION First replication Conservative model Semiconservative model Dispersive model Second replication Less dense More dense

Fig. 16 -11 a EXPERIMENT 1 Bacteria cultured in medium containing 15 N 2 Bacteria transferred to medium containing 14 N RESULTS 3 DNA sample centrifuged after 20 min (after first application) 4 DNA sample centrifuged after 20 min (after second replication) Less dense More dense

Fig. 16 -11 b CONCLUSION First replication Conservative model Semiconservative model Dispersive model Second replication

DNA Replication: A Closer Look • The copying of DNA is remarkable in its speed and accuracy • More than a dozen enzymes and other proteins participate in DNA replication Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Getting Started • Replication begins at special sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble” • A eukaryotic chromosome may have hundreds or even thousands of origins of replication • Replication proceeds in both directions from each origin, until the entire molecule is copied Animation: Origins of Replication Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -12 Origin of replication Parental (template) strand Daughter (new) strand Doublestranded DNA molecule Replication fork Replication bubble 0. 5 µm Two daughter DNA molecules (a) Origins of replication in E. coli Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand 0. 25 µm Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

Fig. 16 -12 a Origin of replication Parental (template) strand Daughter (new) strand Doublestranded DNA molecule Replication fork Replication bubble 0. 5 µm Two daughter DNA molecules (a) Origins of replication in E. coli

Fig. 16 -12 b Origin of replication Double-stranded DNA molecule Parental (template) strand Daughter (new) strand 0. 25 µm Bubble Replication fork Two daughter DNA molecules (b) Origins of replication in eukaryotes

• At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating • Helicases are enzymes that untwist the double helix at the replication forks • Single-strand binding protein binds to and stabilizes single-stranded DNA until it can be used as a template • Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -13 Primase Single-strand binding proteins 3 Topoisomerase 5 3 5 Helicase 5 RNA primer 3

Fig. 16 -7 a 5 end Hydrogen bond 3 end 1 nm 3. 4 nm 3 end 0. 34 nm (a) Key features of DNA structure (b) Partial chemical structure 5 end

• DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end • The initial nucleotide strand is a short RNA primer Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• An enzyme called primase can start an RNA chain from scratch and adds RNA nucleotides one at a time using the parental DNA as a template • The primer is short (5– 10 nucleotides long), and the 3 end serves as the starting point for the new DNA strand Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Synthesizing a New DNA Strand • Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork • Most DNA polymerases require a primer and a DNA template strand • The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate • d. ATP supplies adenine to DNA and is similar to the ATP of energy metabolism • The difference is in their sugars: d. ATP has deoxyribose while ATP has ribose • As each monomer of d. ATP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -14 New strand 5 end Sugar 5 end 3 end T A T C G G C T A A Base Phosphate Template strand 3 end DNA polymerase 3 end A T Pyrophosphate 3 end C Nucleoside triphosphate 5 end C 5 end

Antiparallel Elongation • The antiparallel structure of the double helix (two strands oriented in opposite directions) affects replication • DNA polymerases add nucleotides only to the free 3 end of a growing strand; therefore, a new DNA strand can elongate only in the 5 to 3 direction Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -7 a 5 end Hydrogen bond 3 end 1 nm 3. 4 nm 3 end 0. 34 nm (a) Key features of DNA structure (b) Partial chemical structure 5 end

• Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork Animation: Leading Strand Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -15 Overview Origin of replication Leading strand Lagging strand Primer Lagging strand Leading strand Overall directions of replication Origin of replication 3 5 RNA primer 5 “Sliding clamp” 3 5 Parental DNA poll III 3 5 5 3 5

Fig. 16 -15 a Overview Origin of replication Leading strand Lagging strand Primer Leading strand Lagging strand Overall directions of replication

Fig. 16 -15 b Origin of replication 3 5 RNA primer 5 “Sliding clamp” 3 5 Parental DNA pol III 3 5 5 3 5

• To elongate the other new strand, called the lagging strand, DNA polymerase must work in the direction away from the replication fork • The lagging strand is synthesized as a series of segments called Okazaki fragments, which are joined together by DNA ligase Animation: Lagging Strand Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -16 Overview Origin of replication Lagging strand Leading strand Lagging strand 2 1 Leading strand Overall directions of replication 3 5 5 Template strand 3 RNA primer 3 5 3 1 5 3 5 Okazaki fragment 3 1 5 3 5 2 3 3 5 1 5 2 1 3 5 Overall direction of replication

Fig. 16 -16 a Overview Origin of replication Leading strand Lagging strand 2 1 Leading strand Overall directions of replication

Fig. 16 -16 b 1 3 Template strand 5 5 3

Fig. 16 -16 b 2 3 Template strand 3 5 5 RNA primer 5 3 1 3 5

Fig. 16 -16 b 3 3 Template strand 3 5 5 RNA primer 5 3 3 1 Okazaki fragment 3 1 5 5 3 5

Fig. 16 -16 b 4 3 5 5 Template strand 3 RNA primer 5 3 1 5 3 5 Okazaki fragment 3 3 3 1 5 5 2 1 3 5

Fig. 16 -16 b 5 3 5 5 Template strand 3 RNA primer 5 3 1 3 5 1 5 5 2 3 5 Okazaki fragment 3 3 3 1 3 5 5 2 1 3 5

Fig. 16 -16 b 6 3 5 5 Template strand 3 RNA primer 5 3 1 5 2 3 5 1 5 2 3 3 5 1 5 3 5 Okazaki fragment 3 3 3 3 5 1 5 2 1 Overall direction of replication 3 5

Table 16 -1

Fig. 16 -17 Overview Origin of replication Lagging strand Leading strand Lagging strand Overall directions of replication Single-strand binding protein Helicase 5 3 Parental DNA Leading strand 3 DNA pol III Primer 5 Primase 3 5 DNA pol III 4 3 5 Lagging strand DNA pol I 3 2 DNA ligase 1 3 5

The DNA Replication Complex • The proteins that participate in DNA replication form a large complex, a “DNA replication machine” • The DNA replication machine is probably stationary during the replication process • Recent studies support a model in which DNA polymerase molecules “reel in” parental DNA and “extrude” newly made daughter DNA molecules Animation: DNA Replication Review Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Proofreading and Repairing DNA • DNA polymerases proofread newly made DNA, replacing any incorrect nucleotides • In mismatch repair of DNA, repair enzymes correct errors in base pairing • DNA can be damaged by chemicals, radioactive emissions, X-rays, UV light, and certain molecules (in cigarette smoke for example) • In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -18 Nuclease DNA polymerase DNA ligase

Replicating the Ends of DNA Molecules • Limitations of DNA polymerase create problems for the linear DNA of eukaryotic chromosomes • The usual replication machinery provides no way to complete the 5 ends, so repeated rounds of replication produce shorter DNA molecules Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -19 5 Leading strand Lagging strand Ends of parental DNA strands 3 Last fragment Previous fragment RNA primer Lagging strand 5 3 Parental strand 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 3 Further rounds of replication Shorter and shorter daughter molecules

• Eukaryotic chromosomal DNA molecules have at their ends nucleotide sequences called telomeres • Telomeres do not prevent the shortening of DNA molecules, but they do postpone the erosion of genes near the ends of DNA molecules • It has been proposed that the shortening of telomeres is connected to aging Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -20 1 µm

• If chromosomes of germ cells became shorter in every cell cycle, essential genes would eventually be missing from the gametes they produce • An enzyme called telomerase catalyzes the lengthening of telomeres in germ cells Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• The shortening of telomeres might protect cells from cancerous growth by limiting the number of cell divisions • There is evidence of telomerase activity in cancer cells, which may allow cancer cells to persist Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Concept 16. 3 A chromosome consists of a DNA molecule packed together with proteins • The bacterial chromosome is a doublestranded, circular DNA molecule associated with a small amount of protein • Eukaryotic chromosomes have linear DNA molecules associated with a large amount of protein • In a bacterium, the DNA is “supercoiled” and found in a region of the cell called the nucleoid Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• Chromatin is a complex of DNA and protein, and is found in the nucleus of eukaryotic cells • Histones are proteins that are responsible for the first level of DNA packing in chromatin Animation: DNA Packing Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -21 a Nucleosome (10 nm in diameter) DNA double helix (2 nm in diameter) H 1 Histones DNA, the double helix Histones Histone tail Nucleosomes, or “beads on a string” (10 -nm fiber)

Fig. 16 -21 b Chromatid (700 nm) 30 -nm fiber Loops Scaffold 300 -nm fiber Replicated chromosome (1, 400 nm) 30 -nm fiber Looped domains (300 -nm fiber) Metaphase chromosome

• Chromatin is organized into fibers • 10 -nm fiber – DNA winds around histones to form nucleosome “beads” – Nucleosomes are strung together like beads on a string by linker DNA • 30 -nm fiber – Interactions between nucleosomes cause thin fiber to coil or fold into this thicker fiber Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• 300 -nm fiber – The 30 -nm fiber forms looped domains that attach to proteins • Metaphase chromosome – The looped domains coil further – The width of a chromatid is 700 nm Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• Most chromatin is loosely packed in the nucleus during interphase and condenses prior to mitosis • Loosely packed chromatin is called euchromatin • During interphase a few regions of chromatin (centromeres and telomeres) are highly condensed into heterochromatin • Dense packing of the heterochromatin makes it difficult for the cell to express genetic information coded in these regions Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

• Histones can undergo chemical modifications that result in changes in chromatin organization – For example, phosphorylation of a specific amino acid on a histone tail affects chromosomal behavior during meiosis Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

Fig. 16 -22 RESULTS Condensin and DNA (yellow) Outline Condensin of nucleus (green) Normal cell nucleus DNA (red at periphery) Mutant cell nucleus

Fig. 16 -UN 2 G C A T G Sugar-phosphate backbone C A C C Nitrogenous bases G T G Hydrogen bond T A

Fig. 16 -UN 3 DNA pol III synthesizes leading strand continuously Parental DNA 3 5 DNA pol III starts DNA synthesis at 3 end of primer, continues in 5 3 direction 5 3 5 Primase synthesizes a short RNA primer Lagging strand synthesized in short Okazaki fragments, later joined by DNA ligase 3 5

Fig. 16 -UN 4

Fig. 16 -UN 5

You should now be able to: 1. Describe the contributions of the following people: Griffith; Avery, Mc. Cary, and Mac. Leod; Hershey and Chase; Chargaff; Watson and Crick; Franklin; Meselson and Stahl 2. Describe the structure of DNA 3. Describe the process of DNA replication; include the following terms: antiparallel structure, DNA polymerase, leading strand, lagging strand, Okazaki fragments, DNA ligase, primer, primase, helicase, topoisomerase, single-strand binding proteins Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings

4. Describe the function of telomeres 5. Compare a bacterial chromosome and a eukaryotic chromosome Copyright © 2008 Pearson Education Inc. , publishing as Pearson Benjamin Cummings