Chapter 16

The Molecular Basis of Inheritance

PowerPoint Lectures for Biology, Seventh Edition
Neil Campbell and Jane Reece

Lectures by Chris Romero

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Overview: Lifes 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
Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Concept 16.1: DNA is the genetic material

Early in the 20th century, the identification of the molecules of inheritance loomed as a major challenge to biologists

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The Search for the Genetic Material: Scientific Inquiry

When Morgans group showed that genes are located on chromosomes, the two components of chromosomesDNA and proteinbecame 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 2005 Pearson Education, Inc. publishing as 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, a pathogenic S strain and a harmless R strain 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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-2

Living S cells (control)

Living R cells (control)

Heat-killed S cells (control)

Mixture of heat-killed S cells and living R cells

RESULTS
Mouse dies Mouse healthy

Mouse healthy

Mouse dies

Living S cells are found in blood sample

 In 1944, Oswald Avery, Maclyn McCarty, and Colin MacLeod 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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Evidence That Viral DNA Can Program Cells

More evidence for DNA as the genetic material came from studies of a virus that infects bacteria Such viruses, called bacteriophages (or phages), are widely used in molecular genetics research

Animation: Phage T2 Reproductive Cycle

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LE 16-3

Phage head

Tail

Tail fiber

DNA 100 nm

Bacterial cell

 In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2 To determine the source of genetic material in the phage, they designed an experiment showing that only one of the two components of T2 (DNA or protein) enters an E. coli cell during infection They concluded that the injected DNA of the phage provides the genetic information
Animation: Hershey-Chase Experiment
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LE 16-4

Phage Bacterial cell Batch 1: Sulfur (35S)

Radioactive protein

Empty protein shell

Radioactivity (phage protein) in liquid

DNA Phage DNA Centrifuge Pellet (bacterial cells and contents)

Radioactive DNA

Batch 2: Phosphorus (32P)

Centrifuge Pellet Radioactivity (phage DNA) in pellet

Additional Evidence That DNA Is the Genetic Material

In 1947, Erwin Chargaff reported that DNA composition varies from one species to the next This evidence of diversity made DNA a more credible candidate for the genetic material

By the 1950s, it was already known that DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a phosphate group

Animation: DNA and RNA Structure

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-5

Sugarphosphate backbone 5 end

Nitrogenous bases

Thymine (T)

Adenine (A)

Cytosine (C)

Phosphate Sugar (deoxyribose) 3 end

DNA nucleotide

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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-6

Rosalind Franklin

Franklins X-ray diffraction photograph of DNA

 Franklins 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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-7

5 end Hydrogen bond 3 end

1 nm 3.4 nm

3 end 0.34 nm Key features of DNA structure Partial chemical structure 5 end 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 molecules interior 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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-UN298

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 paired only with thymine, and guanine paired only with cytosine

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-8

Sugar Sugar Thymine (T)

Adenine (A)

Sugar Sugar

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 2005 Pearson Education, Inc. publishing as 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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-9_1

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.

LE 16-9_2

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.

LE 16-9_3

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.

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 backbones of the new strands. Each daughter DNA molecule consists of one parental strand and one new strand.

 Watson and Cricks 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 and the dispersive model

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LE 16-10
Parent cell Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix. First replication Second replication

Semiconservative model. The two strands of the parental molecule separate, and each functions as a template for synthesis of a new, complementary strand.

Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA.

 Experiments by Meselson and 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 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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-11
Bacteria cultured in medium containing 15N Bacteria transferred to medium containing 14N

DNA sample centrifuged after 20 min (after first replication)

DNA sample centrifuged after 40 min (after second replication)

Less dense

More dense

First replication Conservative model

Second replication

Semiconservative model

Dispersive model

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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Getting Started: Origins of Replication

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

At the end of each replication bubble is a replication fork, a Y-shaped region where new DNA strands are elongating
Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-12

Parental (template) strand Origin of replication Daughter (new) strand 0.25 m

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

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Elongating a New DNA Strand

Enzymes called DNA polymerases catalyze the elongation of new DNA at a replication fork Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate The rate of elongation is about 500 nucleotides per second in bacteria and 50 per second in human cells

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-13

New strand 5 end

Template strand 3 end

5 end

3 end

Sugar Phosphate Base

3 end

DNA polymerase

Pyrophosphate Nucleoside triphosphate

3 end

5 end

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 3end of a growing strand; therefore, a new DNA strand can elongate only in the 5to3direction

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

 Along one template strand of DNA, called the leading strand, DNA polymerase can synthesize a complementary strand continuously, moving toward the replication fork 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
Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-14
Parental DNA 5 3

3
5 Leading strand

Okazaki fragments Lagging strand 3 5 DNA pol III

Template strand

Leading strand Lagging strand

Template strand

DNA ligase

Overall direction of replication

Animation: Leading Strand

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Priming DNA Synthesis

DNA polymerases cannot initiate synthesis of a polynucleotide; they can only add nucleotides to the 3 end The initial nucleotide strand is a short one called an RNA or DNA primer An enzyme called primase can start an RNA chain from scratch

Only one primer is needed to synthesize the leading strand, but for the lagging strand each Okazaki fragment must be primed separately
Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-15_1
3 Primase joins RNA nucleotides into a primer. 5 5 Template strand 3

Overall direction of replication

LE 16-15_2
3 Primase joins RNA nucleotides into a primer. 5 5 Template strand 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 5

RNA primer 5

Overall direction of replication

LE 16-15_3
3 Primase joins RNA nucleotides into a primer. 5 5 Template strand 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 5

RNA primer 5

After reaching the next RNA primer (not shown), DNA pol III falls off.

Okazaki fragment

3 5

Overall direction of replication

LE 16-15_4
3 Primase joins RNA nucleotides into a primer. 5 5 Template strand 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 5

RNA primer 5

After reaching the next RNA primer (not shown), DNA pol III falls off.

Okazaki fragment

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
3 Primase joins RNA nucleotides into a primer. 5 5 Template strand 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 5

RNA primer 5

After reaching the next RNA primer (not shown), DNA pol III falls off.

Okazaki fragment

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

5 3

DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2.

3 5

Overall direction of replication

LE 16-15_6
3 Primase joins RNA nucleotides into a primer. 5 5 Template strand 3 DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment. 5

RNA primer 5

After reaching the next RNA primer (not shown), DNA pol III falls off.

Okazaki fragment

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

5 3

DNA pol I replaces the RNA with DNA, adding to the 3 end of fragment 2.

3 5

DNA ligase forms a bond between the newest DNA and the adjacent DNA of fragment 1. 5 3

The lagging strand in the region is now complete. 3 5

Overall direction of replication

Animation: Lagging Strand

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

Other Proteins That Assist DNA Replication

Helicase untwists the double helix and separates the template DNA strands at the replication fork 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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

 Primase synthesizes an RNA primer at the 5 ends of the leading strand and the Okazaki fragments

DNA pol III continuously synthesizes the leading strand and elongates Okazaki fragments
DNA pol I removes primer from the 5 ends of the leading strand and Okazaki fragments, replacing primer with DNA and adding to adjacent 3 ends

DNA ligase joins the 3 end of the DNA that replaces the primer to the rest of the leading strand and also joins the lagging strand fragments
Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-16

Overall direction of replication

Lagging Leading Origin of replication strand strand

DNA pol III

Lagging strand Leading strand

OVERVIEW

Leading strand

Replication fork Primase Primer DNA pol I DNA pol III Lagging strand

DNA ligase

3 Parental DNA

3 5

Animation: DNA Replication Review

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

The DNA Replication Machine as a Stationary 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

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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

In nucleotide excision repair, enzymes cut out and replace damaged stretches of DNA

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-17
A thymine dimer distorts the DNA molecule.

A nuclease enzyme cuts the damaged DNA strand at two points and the damaged section is removed. Nuclease

DNA polymerase

Repair synthesis by a DNA polymerase fills in the missing nucleotides.

DNA ligase

DNA ligase seals the free end of the new DNA to the old DNA, making the strand complete.

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 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-18
5

End of parental DNA strands

3

Leading strand Lagging strand

Last fragment
RNA primer Lagging strand 5 3 Primer removed but cannot be replaced with DNA because no 3 end available for DNA polymerase 3

Previous fragment

Removal of primers and replacement with DNA where a 3 end is available 5 Second round of replication 5

New leading strand 3

New leading 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

Copyright 2005 Pearson Education, Inc. publishing as Benjamin Cummings

LE 16-19

1 m

 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 2005 Pearson Education, Inc. publishing as Benjamin Cummings