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Chapter 13. The Molecular Basis of Inheritance Central Dogma Song Sing along Hip Hip Hooray for DNA Blame it on the DNA . DNA, the substance of inheritance is the most celebrated molecule of our time Hereditary information

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chapter 13

Chapter 13

The Molecular Basis of Inheritance

Central Dogma Song Sing along

Hip Hip Hooray for DNA

Blame it on the DNA

slide2

DNA, the substance of inheritance

    • is the most celebrated molecule of our time
  • Hereditary information
    • is encoded in the chemical language of DNA and reproduced in all the cells of your body
  • It is the DNA program
    • that directs the development of many different types of traits
slide3

Early in the 20th century

    • the identification of the molecules of inheritance loomed as a major challenge to biologists
  • The role of DNA in heredity
    • was first worked out by studying bacteria and the viruses that infect them
evidence that dna can transform bacteria

Frederick Griffith was studying Streptococcus pneumoniae

    • A bacterium that causes pneumonia in mammals
  • He worked with two strains of the bacterium
    • A pathogenic strain (capsulated- smooth) and a nonpathogenic strain (uncapsulated - rough)
Evidence That DNA Can Transform Bacteria
slide5

Griffith found that when he mixed heat-killed remains of the pathogenic strain (smooth) with living cells of the nonpathogenic strain (rough), some of these living cells became pathogenic

slide6

EXPERIMENT

RESULTS

CONCLUSION

Bacteria of the “S” (smooth) strain of Streptococcus pneumoniae are pathogenic because they

have a capsule that protects them from an animal’s defense system. Bacteria of the “R” (rough) strain lack a capsule

and are nonpathogenic. Frederick Griffith injected mice with the two strains as shown below:

Heat-killed

(control) S cells

Living R

(control) cells

Mixture of heat-killed S cells

and living R cells

Living S

(control) cells

Mouse dies

Mouse healthy

Mouse healthy

Mouse dies

Living S cells

are found in

blood sample.

Figure 16.2

Griffith concluded that the living R bacteria had been transformed into pathogenic S bacteria by an unknown, heritable substance from the dead S cells.

slide7

Griffith called the phenomenon transformation

    • Now defined as a change in genotype and phenotype due to the assimilation of external DNA by a cell
evidence that viral dna can program cells

Phage

head

Tail

Tail fiber

DNA

100 nm

Bacterial

cell

Figure 16.3

  • Additional evidence for DNA as the genetic material
    • Came from studies of a virus that infects bacteria
  • Viruses that infect bacteria,bacteriophages
    • Are widely used as tools by researchers in molecular genetics
Evidence That Viral DNA Can Program Cells
slide9

Alfred Hershey and Martha Chase

    • Performed experiments showing that DNA is the genetic material of a phage known as T2
slide10

In their famous 1952 experiment, Alfred Hershey and Martha Chase used radioactive sulfur

and phosphorus to trace the fates of the protein and DNA, respectively, of T2 phages that infected bacterial cells.

EXPERIMENT

CONCLUSION

Agitated in a blender to

separate phages outside

the bacteria from the

bacterial cells.

Centrifuged the mixture

so that bacteria formed

a pellet at the bottom of

the test tube.

Mixed radioactively

labeled phages with

bacteria. The phages

infected the bacterial cells.

3

2

4

1

Measured the

radioactivity in

the pellet and

the liquid

Radioactivity

(phage protein)

in liquid

Radioactive

protein

Empty

protein shell

Phage

Bacterial cell

RESULTS

Batch 1: Phages were

grown with radioactive

sulfur (35S), which was

incorporated into phage

protein (pink).

DNA

Phage

DNA

Centrifuge

Radioactive

DNA

Pellet (bacterial

cells and contents)

Batch 2: Phages were

grown with radioactive

phosphorus (32P), which

was incorporated into

phage DNA (blue).

Centrifuge

Radioactivity (phage DNA) in pellet

Pellet

Phage proteins remained outside the bacterial cells during infection, while phage DNA entered the cells.

When cultured, bacterial cells with radioactive phage DNA released new phages with some radioactive phosphorus.

Hershey and Chase concluded that DNA, not protein, functions as the T2 phage’s genetic material.

Figure 16.4

The Hershey and Chase experiment

slide11

P 32

S 35

P 32 ends up in DNA of new virons

additional evidence that dna is the genetic materia

Sugar-phosphate

backbone

Nitrogenous

bases

5 end

CH3

O–

5

O

H

CH2

O

P

O

O

1

4

N

O–

N

H

H

H

H

H

O

2

3

H

Thymine (T)

O

H

H

CH2

O

O

P

N

O

N

H

O–

H

N

H

H

H

N

N

H

H

Adenine (A)

H

H

O

H

N

CH2

O

O

P

H

O

O–

N

H

N

H

H

H

O

H

Cytosine (C)

O

5

H

CH2

O

P

N

O

O

O

1

4

O–

H

N

H

Phosphate

H

H

N

2

H

3

DNA nucleotide

N

H

OH

N

H

Sugar (deoxyribose)

3 end

H

Figure 16.5

Guanine (G)

  • Prior to the 1950s, it was already known that DNA
    • Is a polymer of nucleotides, each consisting of three components: a nitrogenous base, a Pentose sugar, and a phosphate group
Additional Evidence That DNA Is the Genetic Materia
slide13

Erwin Chargaff analyzed the base composition of DNA

    • from a number of different organisms
  • In 1947, Chargaff reported
    • that DNA composition varies from one species to the next
    • The % of Cytosine = the % of Guanine & the % of Thymine = the % of Adenine – Chargaff’s rule
  • This evidence of molecular diversity among species
    • made DNA a more credible candidate for the genetic material
building a structural model of dna scientific inquiry

Once most biologists were convinced that DNA was the genetic material

    • The challenge was to determine how the structure of DNA could account for its role in inheritance
Building a Structural Model of DNA: Scientific Inquiry
slide15

Franklin’s X-ray diffraction

Photograph of DNA

(b)

(a) Rosalind Franklin

Figure 16.6 a, b

  • Maurice Wilkins and Rosalind Franklin
    • were using a technique called X-ray crystallography to study molecular structure
  • Rosalind Franklin produced a picture (Photo 51) of the DNA molecule using this technique
secret of photo 51

Franklin's Photo 51 shows the mysterious "X" shape that inspired Watson and Crick to visualize the double helix structure of DNA. She was able to get this remarkable image—the clearest image of DNA ever created up until that time—with her advanced techniques of X-ray diffraction. Using Franklin's image as physical evidence, Watson and Crick then went on to publish their Nobel Prize-winning theoretical structure of DNA in Nature in 1953

Secret of Photo 51
slide17

Figure 16.1

  • In 1953, James Watson and Francis Crick shook the world (thanks to Franklin’s photo)
    • with sheet metal & clamps double-helical model for the structure of deoxyribonucleic acid, or DNA
slide18

G

C

A

T

T

A

1 nm

C

G

3.4 nm

C

G

A

T

G

C

T

A

T

A

A

T

T

A

G

C

0.34 nm

A

T

Figure 16.7a, c

(a) Key features of DNA structure

(c) Space-filling model

  • Watson and Crick deduced that DNA was a double helix
    • through observations of Franklin’s X-ray crystallographic images of DNA
slide19

Franklin had concluded that DNA

    • was composed of two antiparallel sugar-phosphate backbones, with the nitrogenous bases paired in the molecule’s interior
  • The nitrogenous bases
    • are paired in specific combinations: adenine with thymine, and cytosine with guanine
structure of dna

5 end

3 end

O

OH

Hydrogen bond

P

–O

OH

O

O

A

T

O

CH2

O

O

P

O

–O

O–

O

P

O

H2C

O

O

G

C

O

O

CH2

O

P

O

O

O

–O

O–

O–

O–

O

P

P

P

O

O

O

H2C

O

O

O

O

C

G

O

O

CH2

O

P

–O

O

H2C

A

T

O

O

CH2

OH

3 end

(b) Partial chemical structure

5 end

Figure 16.7b

Structure of DNA
slide21

Watson and Crick reasoned that there must be additional specificity of pairing dictated by the structure of the bases

  • Each base pair forms a different number of hydrogen bonds
    • Adenine and thymine form two bonds, cytosine and guanine form three bonds
    • BioRap
purines vs pyrimidines

H

N

O

H

CH3

N

N

N

N

H

Sugar

N

N

O

Sugar

Adenine (A)

Thymine (T)

H

O

N

H

N

N

N

H

N

Sugar

N

N

N

O

H

Sugar

H

Figure 16.8

Cytosine (C)

Guanine (G)

PurinesvsPyrimidines
the basic principle base pairing to a template strand

The relationship between structure and function

    • is manifest in thedouble helix
  • Since the two strands of DNA are complementary
    • each strand acts as a template for building a new strand in replication
The Basic Principle: Base Pairing to a Template Strand
slide24

DNA replication is semiconservative

    • Each of the two new daughter molecules will have one old strand, derived from the parent molecule, and one newly made strand
slide25

EXPERIMENT

Matthew Meselson and Franklin Stahl cultured E. coli bacteria for several generations

on a medium containing nucleotide precursors labeled with a heavy isotope of nitrogen, 15N. The bacteria

incorporated the heavy nitrogen into their DNA. The scientists then transferred the bacteria to a medium with

only 14N, the lighter, more common isotope of nitrogen.Any new DNA that the bacteria synthesized would be

lighter than the parental DNA made in the 15N medium. Meselson and Stahl could distinguish DNA of different

densities by centrifuging DNA extracted from the bacteria.

3

2

4

1

Bacteria

cultured in

medium

containing

15N

Bacteria

transferred to

medium

containing

14N

RESULTS

Less

dense

DNA sample

centrifuged

after 40 min

(after second

replication)

DNA sample

centrifuged

after 20 min

(after first

replication)

More

dense

The bands in these two centrifuge tubes represent the results of centrifuging two DNA samples from the flask

in step 2, one sample taken after 20 minutes and one after 40 minutes.

Figure 16.11

  • Experiments performed by Meselson & Stahl
    • Supported the semiconservative model of DNA replication
slide26

CONCLUSION

Meselson and Stahl concluded that DNA replication follows the semiconservative

model by comparing their result to the results predicted by each of the three models in Figure 16.10.

The first replication in the 14N medium produced a band of hybrid (15N–14N) DNA. This result eliminated

the conservative model. A second replication produced both light and hybrid DNA, a result that eliminated

the dispersive model and supported the semiconservative model.

First replication

Second replication

Conservative

model

Semiconservative

model

Dispersive

model

slide28

T

A

A

A

A

T

A

T

T

T

T

A

G

C

G

C

C

C

C

G

G

G

G

C

A

A

T

T

T

T

A

A

A

A

T

T

T

A

A

A

A

T

A

T

T

T

T

A

C

G

G

G

G

C

G

C

C

C

C

G

(a) 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.

(c) Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

(d) 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.

(b) The first step in replication is separation of the two DNA strands.

Figure 16.9 a–d

  • In DNA replication
    • The parent molecule unwinds, and two new daughter strands are built based on base-pairing rules
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
    • Topoisomerase - Relieves strain ahead of the replication fork due to untwisting of the double helix. Flattens out helix
    • Helicase - Untwists the double helix at replication forks to make two parental strands available as template strands. Unzips helix
    • Single stranded binding protein – stabilizes the open strands following helicase- keeps replication fork open
    • DNA Polymerase I or III - Elongates a new DNA strand at a replication fork. Adds nucleotides one by one to the new and growing DNA strand. Binds nucleotides to exposed bases
    • DNA ligase - Joins sugar-phosphates of Okazaki fragments. Okazaki fragments are found on the lagging strand. Molecular glue
    • Primase - Starts an RNA chain from scratch that will eventually be replaced by DNA nucleotides (remember nucleotides come from DNA polymerase). Produces temporary RNA fragments
DNA Replication: A Closer Look
getting started origins of replication

The replication of a DNA molecule begins at special sites called origins of replication, where the two strands are separated by DNA helicase

Getting Started: Origins of Replication
slide32

Origin of replication

Parental (template) strand

0.25 µm

Daughter (new) strand

Replication begins at specific sites

where the two parental strands

separate and form replication

bubbles.

1

Bubble

Replication fork

2

The bubbles expand laterally, as

DNA replication proceeds in both

directions.

Eventually, the replication

bubbles fuse, and synthesis of

the daughter strands is

complete.

3

Two daughter DNA molecules

In this micrograph, three replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM).

In eukaryotes, DNA replication begins at many sites along the giant

DNA molecule of each chromosome.

(a)

(b)

Figure 16.12 a, b

  • A eukaryotic chromosome
    • may have hundreds or even thousands of replication origins
elongating a new dna strand hhmi animation

New strand

Template strand

3 end

5 end

3 end

5 end

Sugar

A

T

A

T

Base

Phosphate

C

G

C

G

G

G

C

C

A

T

A

T

OH

P

P

P

P

3 end

P

Pyrophosphate

C

C

OH

2 P

5 end

5 end

Figure 16.13

  • Elongation of new DNA at a replication fork
    • Is catalyzed by enzymes called DNA polymerases, which add nucleotides to the 3 end of a growing strand or leading strand

DNA Polymerase

Elongating a New DNA Strand HHMI animation

Nucleoside

triphosphate

how does the antiparallel structure of the double helix affect replication

DNA polymerases add nucleotides

    • only to the free 3end (sugar first then phosphate group) of a growing strand
  • Along one template strand of DNA, the leading strand
    • DNA polymerase III can synthesize a complementary strand continuously, moving toward the replication fork
    • DNA strands are replicated from the 3’ end to the 5’ end only so the new strands formed goes in the 5’ to 3’ direction
How does the antiparallel structure of the double helix affect replication?
slide35

To elongate the other new strand of DNA, the lagging strand of 5` side,

    • DNA polymerase III must work in the direction away from the replication fork (5’ to 3’)
  • The lagging strand
    • is synthesized as a series of segments called Okazaki fragments, which are then joined together by DNA ligase
slide36

4

2

3

1

DNA pol Ill elongates

DNA strands only in the

5 3 direction.

3

One new strand, the leading strand,

can elongate continuously 5 3 (DNA)as the replication fork progresses.

5

Parental DNA

5

3

Okazaki

fragments

The other new strand, the

lagging strand must grow in an overall

3 5 direction by addition of short

segments, Okazaki fragments, that grow

5 3 (numbered here in the order

they were made).

2

3

1

5

DNA pol III

Template

strand

DNA ligase joins Okazaki

fragments by forming a bond between

their free ends. This results in a

continuous strand.

Leading strand

Lagging strand

3

1

2

Template

strand

DNA ligase

Figure 16.14

Overall direction of replication

Synthesis of leading & lagging strands during DNA replication

priming dna synthesis

DNA polymerases cannot initiate the synthesis of a polynucleotide

    • They can only add nucleotides to the 3 end
  • The initial nucleotide strand
    • Is an RNA or DNA primer to set down a starting point for the DNA fragment to be added on in the 3’ to 5’ direction
Priming DNA Synthesis
slide38

Only one primer is needed for synthesis of the leading strand

    • But for synthesis of the lagging strand, each Okazaki fragment must be primed separately
slide39

7

2

3

6

5

1

4

3

5

3

5

Templatestrand

DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment.

Primasejoins RNA nucleotides into aprimer.

RNA primer

3

5

3

1

5

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

Okazakifragment

3

3

5

1

5

After the second fragment is primed. DNA pol III adds DNAnucleotides until it reaches the first primer and falls off.

5

3

3

2

5

1

DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2.

5

3

3

5

2

1

DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1.

The lagging strand in this region is nowcomplete.

5

3

3

2

5

1

Figure 16.15

Overall direction of replication

other proteins that assist dna replication

Table 16.1

  • Helicase, topoisomerase, single-strand binding protein
    • Are all proteins that assist DNA replication
Other Proteins That Assist DNA Replication
dna replication song and a good animation

Overall direction of replication

Lagging

strand

Leading

strand

Helicase unwinds the

parental double helix.

Origin of replication

1

Molecules of single-

strand binding protein

stabilize the unwound

template strands.

2

The leading strand is

synthesized continuously in the

5 3 direction by DNA pol III.

3

Leading

strand

Lagging

strand

OVERVIEW

DNA pol III

Leading

strand

5

Replication fork

DNA ligase

DNA pol I

3

Primase

2

Parental DNA

Lagging

strand

DNA pol III

1

Primer

3

Primase begins synthesis

of RNA primer for fifth

Okazaki fragment.

4

3

5

4

DNA pol III is completing synthesis of

the fourth fragment, when it reaches the

RNA primer on the third fragment, it will

dissociate, move to the replication fork,

and add DNA nucleotides to the 3 endof the fifth fragment primer.

DNA pol I removes the primer from the 5 end

of the second fragment, replacing it with DNA

nucleotides that it adds one by one to the 3 end

of the third fragment. The replacement of the

last RNA nucleotide with DNA leaves the sugar-

phosphate backbone with a free 3 end.

DNA ligase bonds

the 3 end of the

second fragment to

the 5 end of the first

fragment.

5

6

7

Figure 16.16

  • A summary of DNA replication
DNA Replication Song and a good animation
the dna replication machine as a stationary complex

The various proteins that participate in DNA replication

    • Form a single large complex, a DNA replication “machine”
  • The DNA replication machine
    • Is probably stationary during the replication process
The DNA Replication Machine as a Stationary Complex
proofreading and repairing dna

DNA replication inserts an incorrect base on average only once in every 104 to 106bases.

  • DNA polymerases proofread newly made DNA replacing any incorrect nucleotides
  • In mismatch repair of DNA,
    • repair enzymes correct errors in base pairing to 1/100,000,000 bases (our entire genome is ~ 3.2 billion base pairs)
Proofreading and Repairing DNA
slide44

1

2

4

A thymine dimer

distorts the DNA molecule.

  • In nucleotide excision repair
    • Enzymes cut out and replace damaged stretches of DNA
    • Animation

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.

3

DNA

ligase

DNA ligase seals the

Free end of the new DNA

To the old DNA, making the

strand complete.

Figure 16.17

replicating the ends of dna molecules

5

End of parental

DNA strands

Leading strand

Lagging strand

3

Last fragment

Previous fragment

RNA primer

Lagging strand

5

3

Primer removed but

cannot be replaced

with DNA because

no 3 end available

for DNA polymerase

Removal of primers and

replacement with DNA

where a 3 end is available

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

Figure 16.18

  • The ends of eukaryotic chromosomal DNA
    • get shorter with each round of replication
    • Explains the process of aging
Replicating the Ends of DNA Molecules
slide46

1 µm

Figure 16.19

  • Eukaryotic chromosomal DNA molecules
    • have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DNA molecules just like the tips on your shoelaces
slide47

If the 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
    • Animation