The molecular basis of inheritance

The Molecular Basis of Inheritance

Information: It must contain the information necessary to make an entire organism

Transmission: It must be passed from parent to offspring

Replication: Able to replicate accurately

Variation: Capable of change to allow evolution

Stable enough to store information for long periods

THE GENETIC MATERIAL

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

© 2011 Pearson Education, Inc.

DNA is the genetic material

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


The Search for the Genetic Material: Scientific Inquiry

• When T. H. Morgan’s group 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


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• 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


Living S cells(control)

Living R cells(control)

Heat-killedS cells(control)

Mixture ofheat-killedS cells andliving R cells

Mouse dies Mouse diesMouse healthy Mouse healthy

Living S cells

EXPERIMENT

RESULTS

Figure 16.2

• 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


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


Figure 16.3

Phagehead

Tailsheath

Tail fiber

DNA

Bacterialcell

100

nm

• In 1952, Alfred Hershey and Martha Chase performed experiments showing that DNA is the genetic material of a phage known as T2

• To determine this, 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


Figure 16.4-3

Bacterial cell

Phage

Batch 1:Radioactivesulfur(35S)

Radioactiveprotein

DNA

Batch 2:Radioactivephosphorus(32P)

RadioactiveDNA

Emptyproteinshell

PhageDNA

Centrifuge

Centrifuge

Radioactivity(phage protein)in liquid

Pellet (bacterialcells and contents)

PelletRadioactivity(phage DNA)in pellet

EXPERIMENT

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

• This evidence of diversity made DNA a more credible candidate for the genetic material


The Discovery of RNA as Viral Genetic Material1. All known cellular organisms have DNA as their

genetic material. Some viruses, however, use RNA instead.

2. Tobacco mosaic virus (TMV) is composed of RNA and protein; it contains no DNA. In 1956 Gierer and Schramm showed that when purified RNA from TMV is applied directly to tobacco leaves, they develop mosaic disease. Pretreating the purified RNA with RNase destroys its ability to cause TMV lesions

3. In 1957 Fraenkel-Conrat and Singer showed that in TMV infections with viruses containing RNA from one strain and protein from another, the progeny viruses were always of the type specified by the RNA, not by the protein.

Typical tobacco mosaic virus (TMV) particle

Demonstration that RNA is the genetic material in tobacco mosaic virus (TMV)

The Composition and Structure of DNA and RNA

1. DNA and RNA are polymers composed of monomers called nucleotides.

2. Each nucleotide has three parts:a. A pentose (5-carbon) sugar.b. A nitrogenous base.c. A phosphate group.

3. The pentose sugar in RNA is ribose, and in DNA it’s deoxyribose. The only difference is at the 2’ position, where RNA has a hydroxyl (OH) group, while DNA has only a hydrogen.

Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Structures of deoxyribose and ribose in DNA and RNA

The Composition and Structure of DNA and RNA

4.There are two classes of nitrogenous bases:a. Purines (double-ring, nine-membered

structures) include adenine (A) and guanine (G).

b. Pyrimidines (one-ring, six-membered structures) include cytosine (C), thymine (T) in DNA and uracil (U) in RNA.

Structures of the nitrogenous bases in DNA and RNA

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Base + sugar nucleoside Example

Adenine + ribose = Adenosine Adenine + deoxyribose = Deoxyadenosine

Base + sugar + phosphate(s) nucleotide Example

Adenosine monophosphate (AMP) Adenosine diphosphate (ADP) Adenosine triphosphate (ATP)

22


Base always attached here

Phosphates are attached here

AdenosineAdenosine monophosphate

Adenosine diphosphate

Adenine

Phosphate groups

Phosphoester bond

Ribose

H

OP CH2

O–OO P

O–

O O O–O P

O–H

OHHO

O

H2′3

1′4′5′

Adenosine triphosphate

NH2

NH

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

H

N

NN

23

• dNTPs• NTPs


Nucleotides are covalently linked together by phosphodiester bonds A phosphate connects the 5’ carbon of one nucleotide

to the 3’ carbon of another Therefore the strand has directionality

5’ to 3’ In a strand, all sugar molecules are oriented in the

same direction

The phosphates and sugar molecules form the backbone of the nucleic acid strand The bases project from the backbone

25

Figure 9.10

NH2

N O

N

O

N O

N

Adenine (A)

Guanine (G)

Thymine (T)

BasesBackbone

Cytosine (C)

O

HH

H

H

HH

OOO

O–

P CH2

O–

HH

H

H

H

HH

OOO

O

P CH2

O–

NH2

N

NH

N

N

HH

H

HH

OOO

O

P CH2

O–

NH2

HN

N

NH

N

HH

HOH

HH

OOO

O

P CH2

O–Singlenucleotide

Phosphodiesterlinkage

Sugar (deoxyribose)

Phosphate

3′

5′

5′4′ 1′

2′3′

5′4′ 1′

2′3′

5′4′ 1′

2′3′

5′4′ 1′

2′3′

CH3


26

Chapter 2 slide 27Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Chemical structures of DNA and RNA

The Discovery of the DNA Double Helix

Erwin Chargaff’s ratios obtained for DNA derived from a variety of sources showed that the amount of purine always equals the amount of pyrimidine, and further, that the amount of G equals C, and the amount of A equals T.

Chargaff’s rules–The base composition of DNA varies between species–But, in any species the number of A and T bases are equal and the number of G and C bases are equal•The basis for these rules was not understood until the discovery of the double helix


Building a Structural Model of DNA: Scientific Inquiry

• After DNA was accepted as the genetic material, the challenge was to determine how its structure accounts for its role in heredity

• 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


Figure 16.6

(a) Rosalind Franklin (b) Franklin’s X-ray diffractionphotograph 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 pattern in the photo suggested that the DNA molecule was made up of two strands, forming a double helix


• 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 outer sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior

• Watson built a model in which the backbones were antiparallel (their subunits run in opposite directions)


Figure 16.5 Sugar–phosphatebackbone

Nitrogenous bases

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

Nitrogenous base

Phosphate

DNA nucleotide

Sugar(deoxyribose)

3 end

5 end

Figure 16.7

3.4 nm

1 nm

0.34 nm

Hydrogen bond

(a) Key features ofDNA structure

Space-fillingmodel

(c)(b) Partial chemical structure

3 end

5 end

3 end

5 end

T

T

A

A

G

G

C

C

C

C

CC

C

C

CC

C

G

G

G

G

G

G

G

GG

T

T

T

T

T

T

A

A

A

A

A

A

• 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 data


Figure 16.UN01

Purine purine: too wide

Pyrimidine pyrimidine: too narrow

Purine pyrimidine: widthconsistent 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


Sugar

Sugar

Sugar

Sugar

Adenine (A) Thymine (T)

Guanine (G) Cytosine (C)

Main features of Watson and Crick’s three-dimensional model

a. It is two polynucleotide chains wound around each other in a right-handed helix.

b. The two chains are antiparallel

c. The sugar-phosphate backbones are on the outside of the helix, and the bases are on the inside, stacked perpendicularly to the long axis like the steps of a spiral staircase.

d. The bases of the two strands are held together by hydrogen bonds between complementary bases (two for A-T pairs and three for G-C pairs). Individual H-bonds are relatively weak and so the strands can be separated (by heating, for example). Complementary base pairing means that the sequence of one strand dictates the sequence of the other strand.

e. The base pairs are 0.34 nm apart, and one full turn of the DNA helix takes 3.4 nm, so there are 10 bp in a complete turn. The diameter of a dsDNA helix is 2 nm.

f. Because of the way the bases H-bond with each other, the opposite sugar-phosphate backbones are not equally spaced, resulting in a major and minor groove. This feature of DNA structure is important for protein binding.

3. The 1962 Nobel Prize in Physiology or Medicine was awarded to Francis Crick, James Watson and Maurice Wilkins (the head of the lab in which Franklin worked). Franklin had already died, and so was not eligible.

Different DNA Structures

X ray diffraction studies show that DNA can exist in different forms

a. A-DNA is the dehydrated form, and so it is not usually found in cells. It is a right-handed helix with 10.9 bp/turn, with the bases inclined 13° from the helix axis. A-DNA has a deep and narrow major groove, and a wide and shallow minor groove.

b. B-DNA is the hydrated form of DNA, the kind normally found in cells. It is also a right-handed helix, with only 10.0 bp/turn, and the bases inclined only 2° from the helix axis. B-DNA has a wide major groove and a narrow minor groove, and its major and minor grooves are of about the same depth.

c. Z-DNA is a left-handed helix with a zigzag sugar-phosphate backbone that gives it its name. It has 12.0 bp/turn, with the bases inclined 8.8° from the helix axis. Z-DNA has a deep minor groove, and a very shallow major groove. Its existence in living cells has not been proven.

Space-filling models of different forms of DNA. a) A-DNA b) B-DNA c) Z-DNA

DNA in the Cell

• All known cellular DNA is in the B form. • A-DNA would not be expected because

it is dehydrated and cells are aqueous. • Z-DNA has never been found in living

cells, although many organisms have been shown to contain proteins that will bind to Z-DNA.

RNA Structure

1. RNA structure is very similar to that of DNA.a. It is a polymer of ribonucleotides (the sugar is ribose

rather than deoxyribose).b. Three of its bases are the same (A, G, and C) while it

contains U rather than T.2. RNA is single-stranded, but internal base pairing

can produce secondary structure in the molecule.3. Some viruses use RNA for their genomes. In

some it is dsRNA, while in others it is ssRNA. Double-stranded RNA is structurally very similar to dsDNA.


The primary structure of an RNA strand is much like that of a DNA strand

RNA strands are typically several hundred to several thousand nucleotides in length

In RNA synthesis, only one of the two strands of DNA is used as a template

RNA Structure

47

Adenine (A)

Guanine(G)

Uracil (U)

BasesBackbone

Cytosine (C)

O

HH HH

OOO

O–

P CH2

O–

HH HH

OOO

O

P CH2

O–

NH2

H

N

HH HH

OOO

O

P CH2

O–

H

H

HH

OH

HH

OOO

O

P CH2

O–

Sugar (ribose)

Phosphate

5′4′ 1′

2′3′

5′4′ 1′

2′3′

5′4′ 1′

2′3′

5′4′ 1′

2′3′OH

OH

OH

OH

RNAnucleotide

Phosphodiesterlinkage

3′

5′

NH2

OH

H

H

O

NH O

N


NN

N

N

N

N

N N

N

NH2

H

H

48


Although usually single-stranded, RNA molecules can form short double-stranded regions This secondary structure is due to complementary

base-pairing A to U and C to G

This allows short regions to form a double helix RNA double helices typically

Are right-handed Have the A form with 11 to 12 base pairs per turn

Different types of RNA secondary structures are possible

49

A U

A U

U AG C

C GC G

A UU A

U A

C GC G

C GC G

C G

A

A

U

U

G

G

C

C

C

(a) Bulge loop (b) Internal loop (c) Multibranched junction (d) Stem-loop

GC

CG

UA

AU

GC

GC

CG

AU

A UA U

G C

A UU AG C

C G

C G

GC

GC

CG

AU

AU

GC

CG

CG

AU

AU

A

U

U

G

G

C

C

C A UA U

G CC G

A

AAU

U

U

U

G

G

C

C



Also called hair-pin

Complementary regions

Non-complementary regions

Held together by hydrogen bonds

Have bases projecting away from double stranded regions

50

Many factors contribute to the tertiary structure of RNA For example

Base-pairing and base stacking within the RNA itself

Interactions with ions, small molecules and large proteins


Figure A: depicts the tertiary structure of tRNAphe

The transfer RNA that carries phenylalanine

Molecule contains single- and double-stranded regions

These spontaneously fold and interact to

produce this 3-D structure

Figure A(a) Ribbon model

3′ end(acceptorsite)5′ end

Double helix

Double helix

Anticodon

51

The Organization of DNA in Chromosomes• Cellular DNA is organized into chromosomes.

A genome is the chromosome or set of chromosomes that contains all the DNA of an organism.

• In prokaryotes the genome is usually a single circular chromsome. In eukaryotes, the genome is one complete haploid set of nuclear chromosomes; mitochondrial and chloroplast DNA are not included.

Viral Chromosomes

A virus is nucleic acid surrounded by a protein coat. The nucleic acid may be dsDNA, ssDNA, dsRNA or ssRNA, and it may be linear or circular, a single molecule or several segments.

Bacteriophages are viruses that infect bacteria. Three different types that infect E. coli are good examples of the variety of chromosome structure found in viruses.

台大農藝系遺傳學 601 20000


chromosome structure varies at stages of lytic infection of E. coli

Prokaryotic Chromosomes1. The typical prokaryotic genome is one circular

dsDNA chromosome, but some prokaryotes are more exotic, with a main chromosome and one or more smaller ones. When a minor chromosome is dispensable to the life of the cell, it is called a plasmid. Some examples:a. Borrelia burgdorferi (Lyme disease in humans) has a 0.91-

Mb linear chromosome, plus an additional 0.53-Mb of DNA in 17 different linear and circular molecules.

b. Agrobacterium tumefaciens (crown gall disease of plants) has a 3.0-Mb circular chromosome and a 2.1-Mb linear one.


Chapter 2 slide 56

2. Archaebacteria also vary in chromosomal organization, but only circular forms have been found. Examples:a. Methanococcus jannaschii has three chromosomes of 1.66-

Mb, 58-kb and 16-kb.b. Archaeoglobus fulgidus has one 2.2-Mb circular

chromosome.3. Both Eubacteria and Archaebacteria lack a

membrane-bounded nucleus, hence their classification as prokaryotes. Their DNA is densely arranged in a cytoplasmic region called the nucleoid.

4. In an experiment where E. coli is gently lysed, it releases one 4.6-Mb circular chromosome, highly supercoiled (Figures 2.19 and 2.20). A 4.6-Mb double helix is about 1mm in length, about 103 times longer than an E. coli cell. DNA supercoiling helps it fit into the cell.



Illustration of DNA supercoiling

5. A molecule of B-DNA, with 10bp/turn of the helix, is in relaxed conformation. If turns of the helix are removed and the molecule circularized, the DNA will form superhelical turns to compensate for the added tension.

6. A nick in supercoiled DNA will allow it to return to a relaxed DNA circle

7. Either overwinding or underwinding DNA will create a structure where 10bp/turn of the helix is not the most energetically favored conformation, and supercoils will be induced. Both positive and negative supercoils will condense the DNA.

8. All organisms contain topoisomerase enzymes to supercoil their DNA.

9. Prokaryotes also organize their DNA into looped domains, with the ends of the domains held so that each is supercoiled independently

10. The compaction factor for looped domains is about 10-fold. In E. coli there are about 100 domains of about 40kb each.

Model for the structure of a bacterial chromosome

Eukaryotic Chromosomes1. The genome of most prokaryotes consists of

one chromosome, while most eukaryotes have a diploid number of chromosomes.

2. A genome is the information in one complete haploid chromosome set. The total amount of DNA in the haploid genome of a species is its C value. The structural complexity and the C value of an organism are not related, creating the C value paradox.

Chapter 2 slide 61

C value paradox

O3. The form of eukaryotic chromosomes changes

through the cell cycle:a. In G1, each chromosome is a single structureb. In S, chromosomes duplicate into sister chromatids but

remain joined at centromeres through G2c. At M phase, sister chromatids separate into daughter

chromosomes

4. In G1 eukaryotic chromosomes are linear dsDNA, and contain about twice as much protein as DNA by weight. The DNA-protein complex is called chromatin, and it is highly conserved in all eukaryotes.

• Chromatin, a complex of DNA and protein, is found in the nucleus of eukaryotic cells

• Chromosomes fit into the nucleus through an elaborate, multilevel system of packing


• Chromatin undergoes changes in packing during the cell cycle

• At interphase, some chromatin is organized into a 10-nm fiber, but much is compacted into a 30-nm fiber, through folding and looping

• Though interphase chromosomes are not highly condensed, they still occupy specific restricted regions in the nucleus


Figure 16.22a

DNA double helix(2 nm in diameter)

DNA, the double helix

Nucleosome(10 nm in diameter)

Histones

Histones

Histonetail

H1

Nucleosomes, or “beads ona string” (10-nm fiber)

Figure 16.22b

30-nm fiber

30-nm fiber

Loops Scaffold

300-nm fiber

Chromatid(700 nm)

Replicatedchromosome(1,400 nm)

Looped domains(300-nm fiber) Metaphase

chromosome

• 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

• Histones can undergo chemical modifications that result in changes in chromatin organization


• Eukaryotic chromosomal DNA molecules have special nucleotide sequences at their ends called telomeres

• Telomeres are needed for chromosomal replication and stability. Generally composed of heterochromatin, they interact with both the nuclear envelope and each other. All telomeres in a species have the same sequence.

• 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


Telomeres

• 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

• 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



Chapter 2 slide 70

Unique-Sequence and Repetitive-Sequence DNA

1. Sequences vary widely in how often they occur within a genome. The categories are:a. Unique-sequence DNA, present in one or a few

copies.b. Moderately repetitive DNA, present in a few to 105

copies.c. Highly repetitive DNA, present in about 105–107

copies.2. Prokaryotes have mostly unique-sequence DNA,

with repeats only of sequences like rRNAs and tRNAs. Eukaryotes have a mix of unique and repetitive sequences.

3. Unique-sequence DNA includes most of the genes that encode proteins, as well as other chromosomal regions. Human DNA contains about 65% unique sequences.


Chapter 2 slide 71

4. Repetitive-sequence DNA includes the moderately and highly repeated sequences. They may be dispersed throughout the genome, or clustered in tandem repeats.

5. Dispersed repetitive sequences occur in families that have a characteristic sequence. Often the same few sequences are highly repeated, and comprise most of the dispersed repeats in the genome. Little is known of their function, or indeed whether they actually serve a function. There are two types of interspersion patterns found in all eukaryotic organisms:a. SINEs (short interspersed repeated sequences) with 100–500

bp sequences. An example is the Alu repeats found in some primates, including humans, where these 200–300 bp repeats make up 9% of the genome.

b. LINEs (long interspersed repeated sequences) with sequences of 5 kb or more. The common example in mammals is LINE-1, with sequences up to 7 kb in length.

6. Tandemly repetitive sequences are common in eukaryotic genomes, ranging from very short (1–10 bp) sequences to genes and even longer sequences. This group includes centromere and telomere sequences, and rRNA and tRNA genes.

DNA Replication

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


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


Figure 16.9-3

(a) Parent molecule (b) Separation ofstrands

(c)“Daughter” DNA molecules,each consisting of oneparental strand and onenew strand

A

A

A

A

A

A

A

A

A

A

A

A

T

T

T

T

T

T

T

T

T

T

T

T

C

C

C

C

C

C

C

C

G

G

G

G

G

G

G

G

• 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)


Figure 16.10

(a) Conservativemodel

(b) Semiconservativemodel

(c) Dispersive model

Parentcell

Firstreplication

Secondreplication

• 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

• 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


Figure 16.11

Bacteriacultured inmedium with15N (heavyisotope)

Bacteriatransferred tomedium with14N (lighterisotope)

DNA samplecentrifugedafter firstreplication

DNA samplecentrifugedafter secondreplication

Less dense

More dense

Predictions: First replication Second replication

Conservativemodel

Semiconservativemodel

Dispersivemodel

21

3 4

EXPERIMENT

RESULTS

CONCLUSION

Getting Started

• Replication begins at particular sites called origins of replication, where the two DNA strands are separated, opening up a replication “bubble”

• A bacterial chromosome has 1 origin of replication

• 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


Figure 16.12(a) Origin of replication in an E. coli cell (b) Origins of replication in a eukaryotic cell

Origin ofreplication Parental (template) strand

Double-strandedDNA molecule

Daughter (new)strand

Replicationfork

Replicationbubble

Two daughterDNA molecules

Origin of replicationDouble-strandedDNA molecule

Parental (template)strand

Daughter (new)strand

Bubble Replication fork

Two daughter DNA molecules

0.5

m

0.25

m

• 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 proteins bind to and stabilize single-stranded DNA

• Topoisomerase corrects “overwinding” ahead of replication forks by breaking, swiveling, and rejoining DNA strands


• 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• 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


Figure 16.13

Topoisomerase

Primase

RNAprimer

Helicase

Single-strand bindingproteins

5

3

5

53

3

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


• Each nucleotide that is added to a growing DNA strand is a nucleoside triphosphate

• dATP supplies adenine to DNA and is similar to the ATP of energy metabolism

• The difference is in their sugars: dATP has deoxyribose while ATP has ribose

• As each monomer of dATP, dGTP, dCTP, or dTTP joins the DNA strand, it loses two phosphate groups as a molecule of pyrophosphate


Figure 16.14

New strand Template strand

Sugar

Phosphate Base

Nucleosidetriphosphate

DNApolymerase

Pyrophosphate

5

5

5

5

3

3

3

3

OH

OH

OH

P P i

2 P i

PP

P

A

A

A

A

T T

TT

C

C

C

C

C

C

G

G

G

G

Antiparallel Elongation

• The antiparallel structure of the double helix 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

• Along one template strand of DNA, the DNA polymerase synthesizes a leading strand continuously, moving toward the replication fork


Figure 16.15

Leadingstrand

Laggingstrand

Overview

Origin of replication Laggingstrand

Leadingstrand

Primer

Overall directionsof replication

Origin of replication

RNA primer

Sliding clamp

DNA pol IIIParental DNA

35

5

33

5

3

5

3

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


Figure 16.16a

Origin of replication

Overview

Leadingstrand

Leadingstrand

Laggingstrand

Lagging strand

Overall directionsof replication

12

LE 16-15_653

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

33

5

5

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

33

5

5

DNA ligase forms abond between the newestDNA and the adjacent DNAof fragment 1.

The lagging strand in the regionis now complete.

Figure 16.17

OverviewLeadingstrand

Origin of replication Lagging

strand

LeadingstrandLagging

strand Overall directionsof replicationLeading strand

DNA pol III

DNA pol III Lagging strandDNA pol I DNA ligase

PrimerPrimase

ParentalDNA

5

5

5

5

5

33

3

333 2 1

4

The DNA Replication Complex

• The proteins that participate in DNA replication form a large complex, a “DNA replication machine”

• The DNA replication machine may be 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

• Primase may act as a molecular brake, coordinating primer placement and the rates of replication on the leading and lagging strands


Figure 16.18

Parental DNADNA pol III

Leading strand

Connectingprotein

Helicase

Lagging strandDNA pol III

Laggingstrandtemplate

5

5

5

5

5

5

3 3

33

3

3

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 exposure to harmful chemical or physical agents such as cigarette smoke and X-rays; it can also undergo spontaneous changes

• In nucleotide excision repair, a nuclease cuts out and replaces damaged stretches of DNA


Figure 16.19

Nuclease

DNA polymerase

DNA ligase

5

5

5

5

5

5

5

5

3

3

3

3

3

3

3

3

A thymine dimerdistorts the DNA molecule.

A nuclease enzyme cutsthe damaged DNA strandat two points and the damaged section isremoved.

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

DNA ligase seals thefree end of the new DNAto the old DNA, making thestrand complete.

Evolutionary Significance of Altered DNA Nucleotides

• Error rate after proofreading repair is low but not zero

• Sequence changes may become permanent and can be passed on to the next generation

• These changes (mutations) are the source of the genetic variation upon which natural selection operates


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 with uneven ends

• This is not a problem for prokaryotes, most of which have circular chromosomes


LE 16-18

End of parentalDNA strands

5

3

Lagging strand 5

3

Last fragment

RNA primer

Leading strandLagging strand

Previous fragment

Primer removed butcannot be replacedwith DNA becauseno 3 end available

for DNA polymerase5

3

Removal of primers andreplacement with DNAwhere a 3 end is available

Second roundof replication

5

3

5

3Further roundsof replication

New leading strand

New leading strand

Shorter and shorterdaughter molecules

• Eukaryotic chromosomal DNA molecules have special nucleotide sequences at their ends 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


Figure 16.21

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

• 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


Internet

The molecular basis of inheritance