DNA and Its Role in Heredity

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13DNA and Its Role in Heredity


Chapter 13 DNA and Its Role in Heredity

Key Concepts

13.1 Experiments Revealed the Function of DNA

as Genetic Material

13.2 DNA Has a Structure That Fits Its Function

13.3 DNA Is Replicated Semiconservatively

13.4 Errors in DNA Can Be Repaired

13.5 The Polymerase Chain Reaction Amplifies

DNA


Chapter 13 DNA and Its Role in Heredity (IL 1)

Investigating LIFE introduction

Targeting DNA Replication in Cancer Therapy

• Testicular cancer is highly curable due to the drug

cisplatin, which irreversibly cross links DNA strands and

prevents replication.

• Without DNA replication, cells cannot divide, and

apoptosis follows.

Q&A: What do we need to know about DNA

replication to describe the mechanism of a drug

that blocks it? (See slides 34–35 and 69–71.)


Concept 13.1 Experiments Revealed the Function of DNA as

Genetic Material (1)

In the 1920s, it was known that chromosomes

consisted of DNA and proteins.

A new DNA stain provided evidence that DNA is

the genetic material.

• It was in the right place

• It varied among species

• It was present in the right amounts




Experimental evidence came from work on two

strains of Streptococcus pneumoniae.

A substance from cells of one strain (even when

dead) could produce a heritable change in the

other strain.

If DNA was destroyed, the transforming activity

was lost. But not with the destruction of proteins

or RNA.

Figure 13.1 Genetic Transformation (Experiment)

Figure 13.2 Genetic Transformation by DNA (Experiment)




Hershey-Chase experiment: bacteriophage T2

virus was used to determine whether DNA or

protein is the genetic material.

Part of the virus enters E. coli cells and converts

the cell into a virus replication machine.

Figure 13.3 Bacteriophage T2: Reproduction Cycle




Bacteriophage were grown with either 35S to label

the proteins, or with 32P to label the DNA.

After infection, bacterial cells and viral remains

were separated—the bacterial cells were labeled

with 32P, indicating that DNA had entered the

cells.

Figure 13.4 The Hershey–Chase Experiment (Experiment)




Eukaryotic cells can also be transformed

(transfection).

A genetic marker (gene that confers an

observable phenotype, such as antibiotic

resistance) is used to demonstrate transfection.

Any cell can be transfected, even an egg cell,

resulting in a transgenic organism.


Concept 13.2 DNA Has a Structure That Suits Its Function (1)

The structure of DNA was determined using many

lines of evidence.

Chemical composition:

Biochemists knew that DNA is a polymer of

nucleotides, which differed only in the bases:

• Purines: adenine (A), guanine (G)

• Pyrimidines: cytosine (C), thymine (T)



Chargaff’s rule:

In the 1950s, Chargaff and others found that in all

DNA, the amount of purines is always equal to

the amount of pyrimidines.

But the relative abundances of A + T versus G + C

varies among species.



Evidence from X-ray diffraction:

A purified substance can be made to form crystals.

When X-rays are passed through it, the position

of atoms is inferred from the pattern of diffraction.

Figure 13.5 X-Ray Diffraction Helped Reveal the Structure of DNA (Part 1)



Rosalind Franklin prepared crystallographs from

DNA samples.

Her images suggested a double-stranded helix

with 10 nucleotides in each full turn.

The diameter of 2 nm suggested that the sugar-

phosphate backbone of each strand must be on

the outside.

Figure 13.5 X-Ray Diffraction Helped Reveal the Structure of DNA (Part 2)



In 1953, Francis Crick and James Watson built a

model of DNA using the physical and chemical

evidence, published in 1953.

To satisfy Chargaff’s rule, the model paired

purines on one strand with pyrimidines on

the other, resulting in uniform width.

Figure 13.6 DNA Is a Double Helix (Part 1)



The X-ray diffraction data indicated that

• The bases are on the inside of each strand.

• The sugar-phosphate groups are on the outside

of each strand.

• The chains run in opposite directions—

antiparallel.

Figure 13.6 DNA Is a Double Helix (Part 2)



Key features of DNA structure:

• It is a double-stranded helix

• It is right-handed helix

• It is antiparallel

• The strands are held together by

complementary base pairing

• The outer edges of the bases are exposed in

major and minor grooves



The sugar–phosphate backbones form a coil

around the outside of the helix; the nitrogenous

bases point toward the center.

Hydrogen bonds between complementary base

pairs hold the two strands of the DNA helix

together.

van der Waals forces occur between adjacent

bases on the same strand.



Antiparallel strands: direction is determined by

sugar–phosphate bonds.

Phosphate groups connect the 3′ C of one sugar

with the 5′ C of the next.

One strand has a free 5′ phosphate group—the 5′

end.

The other chain has a free 3′ hydroxyl group—the

3′ end.

Figure 13.7 Each DNA Strand Consists of a Sugar–Phosphate Backbone



The backbones are closer together on one side

(forming the minor groove) than on the other

(forming the major groove).

The outer edges of the base pairs are accessible

for hydrogen bonding.

Binding of proteins to specific base pair sequences

is the key to protein-DNA interactions.

Figure 13.8 Base Pairs in DNA Can Interact with Other Molecules



The double-helix structure is essential to DNA

function:

• With millions of nucleotides, the base

sequences store a huge amount of genetic

information.

• Susceptible to mutations, simple changes in the

linear sequence of base pairs.



• Precise replication in cell division is possible by

complementary base pairing.

• Genetic information is expressed as the

phenotype—nucleotide sequences determine

sequences of amino acids in proteins; proteins

determine phenotypes.


Concept 13.3 DNA Is Replicated Semiconservatively (1)

Researchers found that DNA could be replicated in

a test tube using

• Nucleoside monomers of DNA

• DNA molecules to serve as templates

• DNA polymerase

• Salts and pH buffer

This confirmed that DNA contains the information

needed for its own replication.



Possible replication patterns:

• Semiconservative: each parent strand is a

template; new molecules have one old and one

new strand

• Conservative: original molecule serves as a

template only

• Dispersive: DNA fragments are templates; old

and new pieces are assembled into new

molecules

Figure 13.9 Three Models for DNA Replication



Investigating LIFE: The Meselson-Stahl

Experiment

Meselson and Stahl showed that

semiconservative replication was the correct

model:

• E. coli were grown with 15N (a heavy isotope

that makes DNA more dense), then transferred

to a medium with 14N.

• DNA densities could only be explained by the

semiconservative model.

Investigating Life: The Meselson–Stahl Experiment, Experiment



Three steps in DNA replication:

• Initiation: double helix is unwound, making two

template strands

• Elongation: addition of complementary base

pairs linked by phosphodiester bonds

• Termination: DNA synthesis ends when all DNA

regions have been replicated



Nucleotides are deoxyribonucleoside

monophosphates (dNMPs)—they contain

deoxyribose and one phosphate group.

Monomers added during elongation are

deoxyribonucleoside triphosphates (dNTPs)—

they have 3 phosphate groups attached to the 5′

C on the sugar.



Nucleotides are added to the new strand at the 3′

end.

In formation of the phosphodiester bond, two

phosphates of an incoming dNTP are removed.

Energy is released, which drives the reaction, a

transesterification.

Figure 13.10 Each New DNA Strand Grows from Its 5′ End to Its 3′ End



DNA replication starts when a large protein

complex (pre-replication complex) binds to a

region called origin of replication (ori).

In E. coli, DNA is unwound and replication

proceeds in both directions, forming two

replication forks.



Eukaryote chromosomes are much longer and

have multiple origins of replication.

Otherwise, it would take weeks to replicate

chromosomes, which have up to a billion base

pairs.

Figure 13.11 The Origin of DNA Replication



DNA polymerase requires a primer, a short starter

strand—usually RNA.

The primer is complementary to the DNA template

and is synthesized by a primase.

DNA polymerase then adds nucleotides to the 3′

end until that section is complete, and the primer

is degraded.

Figure 13.12 DNA Synthesis Requires a Primer



DNA polymerases are much larger than the dNTPs

and template DNA.

The enzyme is shaped like an open right hand—

the “palm” brings the active site and the

substrates into contact.

The “fingers” recognize the nucleotide bases. They

bind to bases by hydrogen bonding and rotate

inward.

Figure 13.13 DNA Polymerase Binds to the Template Strand



Other proteins have roles in replication:

• DNA helicase uses energy from ATP

hydrolysis to unwind the DNA.

• Single-strand binding proteins keep the

strands from getting back together.

Figure 13.14 Many Proteins Collaborate in the Pre-Replication Complex



At the replication fork, DNA opens up like a zipper

in one direction.

The leading strand grows at the 3′ end as the fork

opens.

In the lagging strand, the exposed 3′ end gets

farther from the fork, and an unreplicated gap

forms.

Figure 13.15 The Two New Strands Form in Different Ways



Synthesis of the lagging strand occurs in small,

discontinuous stretches called Okazaki

fragments.

Each fragment requires its own primer.

DNA polymerase III adds nucleotides to the 3′ end,

until reaching the primer of the previous

fragment.

Figure 13.16 The Lagging Strand Story



DNA polymerase I then replaces the primer with

DNA.

The final phosphodiester linkage between

fragments is catalyzed by DNA ligase.



DNA polymerases work fast because

• They are processive: they catalyze many

linkages each time they bind to DNA, rather

than just one.

• The polymerase-DNA complex is stabilized by a

sliding DNA clamp, a protein that keeps the

enzyme and DNA in close contact.

Figure 13.17 A Sliding DNA Clamp Increases the Efficiency of DNA Polymerization



The enzymes used in replication don’t move

separately, but form a large replication complex.

The replication complex is stationary, while the

DNA moves through it.

DNA enters the complex as a double stranded

molecule and emerges as two double-stranded

molecules.



Eukaryote chromosomes have repetitive

sequences at the ends called telomeres.

In humans, the sequence is TTAGGG-3′, repeated

about 2,500 times.

The repeats bind proteins that prevent the DNA

repair system from recognizing chromosome

ends as breaks.



On lagging strands, when the terminal Okazaki

primer is removed, no DNA can be synthesized

to replace it (no 3′ end).

The short piece of single stranded DNA is

removed, and the chromosome becomes shorter

with each replication.

After many divisions, genes may be lost and the

cell dies.

Figure 13.18 Telomeres and Telomerase



Continuously dividing cells, such as bone marrow

stem cells, have telomerase, which catalyzes

addition of lost telomeres.

Telomerase is expressed in most cancer cells and

is important in their ability to keep dividing. It is a

target for anti-cancer drugs.


Concept 13.4 Errors in DNA Can Be Repaired (1)

DNA polymerases make mistakes and DNA can

be damaged by chemicals, UV radiation, and

other threats.

Cells have three repair mechanisms:

1. Proofreading: DNA polymerase recognizes

mismatched pairs and removes incorrectly

paired bases. Catches 99% or more

mismatches.


Concept 13.4 Errors in DNA Can Be Repaired (2)

2. Mismatch repair: Newly replicated DNA is

scanned for mistakes by other proteins and

mismatches can be corrected.

3. Excision repair: Enzymes scan DNA for

damaged bases—they are excised and DNA

polymerase I adds the correct ones.

Figure 13.19 DNA Repair Mechanisms


Concept 13.5 The Polymerase Chain Reaction Amplifies DNA (1)

The principles of DNA replication were used to

develop the polymerase chain reaction (PCR)

technique.

An automated process makes multiple copies of

short DNA sequences for genetic manipulation

and research (DNA amplification).



A PCR mixture contains:

• A sample of double-stranded DNA (the

template)

• Two artificially synthesized primers

• Four dNTPs

• DNA polymerase that can tolerate high

temperatures

• Salts and pH buffer



In PCR amplification:

• DNA strands are separated (denatured) by

heating

• Reaction is cooled to allow primers to bind

(anneal) to template strands

• Reaction is warmed; DNA polymerase

catalyzes new strands

• The sequence is repeated many times

Figure 13.20 The Polymerase Chain Reaction



Base sequences at the 3ʹ ends of the DNA strands

must be known, so that primers can be made.

The specificity of the primers is a key to the power

of PCR.

The DNA polymerase that does not denature at

high temperatures (90°C) was taken from a hot

springs bacterium, Thermus aquaticus.



Investigating LIFE conclusion (1 of 2)



that blocks it?

• Cisplatin has a platinum atom bonded to 2

chlorines and 2 amino groups.

• The chlorines can be displaced by nitrogen on

guanine bases to form covalent bonds.



Investigating LIFE conclusion (2 of 2)



that blocks it?

• This results in irreparable cross-linking of the

DNA strands; replication cannot occur and the

cells die.

• Preventing replication is why cisplatin is

effective at halting growth of certain tumors.

Figure 13.21 Cisplatin: A Small but Lethal Molecule

DNA and Its Role in Heredity

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