ch16 lecture (08-09).pptnleaders.org/Download/1st_year/biology/powerpoint_presentations/C… ·...

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

Chapter 16

The molecular basis of

inheritance


The search for the genetic material• T.H. Morgan’s group showed that genes are

located on chromosomes

• The two chemical components of chromosomes are:

– Protein

– DNA

• The BIG QUESTION: Which one is the genetic material??

– until the 1940s, protein was the favored candidate


The search for the genetic material• The key is in choosing the right experimental

organism!

– Mendel – peas

– Morgan – fruit flies

– Griffith, Avery, Hershey & Chase and others –bacteria & viruses


The search for the genetic material• Why protein?

– Great heterogeneity & specificity of function

• Why not DNA?

– Little known about nucleic acids

– Physical & chemical properties seemed far too uniform


Evidence: the case for DNA• Frederick Griffith was studying the bacterium

that causes pneumonia in mammals (Streptococcus pneumoniae)

• He worked with two strains:

– pathogenic S strain

• S = smooth(slime capsule; protected from mouse’s defenses)

– nonpathogenic R strain

• R = rough (no slime capsule; harmless)


Evidence: the case for DNA• Griffith mixed heat-killed pathogenic S strain

with living nonpathogenic R strain

• Found that some of living R cells became pathogenic Heat-killed S

(control)Mixture of

heat-killed S& living R

Living S(control)

Living R(control)

Living S cellsfound in

blood sample

dies healthy healthy dies


Evidence: the case for DNA• Conclusion: molecules from dead S cells had

genetically transformed living R into S

• Griffith called this transformation

– change in genotype & phenotype due to assimilation of external DNA by a cell

• Next question: what is the transforming substance?


Evidence: the case for DNA• Griffith’s work set the stage for the work of

Oswald Avery, Maclyn McCarty & Colin McLeod (1944)

– Purified various chemicals from heat-killed S bacteria

– Tried to transform R bacteria with each

– Only DNA worked

– DNA is transforming agent!


Evidence: the case for DNA• Additional evidence for DNA as the genetic

material came from studies using bacteriophages, viruses that infect bacteria

– widely used as tools by researchers in molecular genetics

Phagehead

Tail

Tail fiber

DNA

Bacterialcell

100

nm


• Next question: which part of a phage is the transforming agent – protein or DNA?

• Alfred Hershey and Martha Chaseperformed experiments using a phage called T2

• Labeled batches of phages with radioisotopes:

– 35S – incorporated into which part? Why?

– 32P – incorporated into which part? Why?

– Why not use 15N?

Evidence: the case for DNA


Evidence: the case for DNA• Used a blender to knock off phages

• Used a centrifuge to separate bacterial cells (pellet) from phages (supernatant)

• Measured radioactivityin pellet & supernatantto determine which partwas incorporated intobacterial cells


• The Hershey and Chase “blender” experiment

Radioactivityin pellet(=phage DNA)

Radioactivityin supernatant(=phage protein)

PhageBacterial cell

Radio-activeprotein

Emptyprotein shell

PhageDNA

DNA

CentrifugePellet (bacterialcells and contents)

Radio-activeDNA

Centrifuge

Pellet

Batch 1: Phagesgrown with 35S –incorporated into phage protein (pink)

Batch 2: Phagesgrown with 32P –was incorporated into phage DNA (blue)

1 2 3 4Blender dislodges phages frombacterial cells

Radioactive phages infect bacterial cells

Centrifuge separates phage shells from bacterial cells

Measureradioactivityin pellet &supernatant


Evidence: the case for DNA• Prior to the 1950s, it was already known that

DNA:

– is a polymer of nucleotides

– each consists of three components

• nitrogenous base

• sugar (deoxyribose)

• phosphate group

Sugar-phosphatebackbone

Nitrogenousbases

5 endO–

O P O CH25

4O–H

HO

H

H

H3

1H O

CH3

N

O

NH

Thymine (T)

O

O P O

O–CH2

HH

OH

HH

HN

N

N

H

NH

H

Adenine (A)

O

O P O

O–CH2

HH

OH

HH

HH H

HN

NN

OCytosine (C)

O

O P O CH25

4O–

H

O

H

H3

1

OH2

H

N

NN H

ON

N HH

H H

Sugar (deoxyribose)3 end

Phosphate

Guanine (G)

DNA nucleotide

2

N


• Erwin Chargaff analyzed the DNA base compositionfrom a number of different organisms

• In 1947, Chargaff reported the following:

– DNA composition varies from one species to the next

• Made DNA a more credible candidate for the genetic material

– Number of adenine roughly equals number of thymine and number of cytosine roughly equals number of guanine

• Chargaff had no explanation for this


SAS: Building a Structural Model of DNA

• 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 (i.e. its function)

• the race was on!


• Maurice Wilkins and Rosalind Franklin

– were using X-ray crystallography to study molecular structure

• Rosalind Franklin produced a picture of the DNA molecule using this technique

(a) Rosalind Franklin Franklin’s X-ray diffractionPhotograph of DNA (“Photo 51”)

(b)


• James Watson and Francis Crick constructed 3-D models to fit known data (no original research)


• Observed Franklin’s X-ray crystallographic images of DNA and deduced that DNA was a double helix

– reported their double helix model in Nature in April 1953

– structure suggested mechanism for replication!

C

T

A

A

T

CG

GC

A

C G

AT

AT

A T

TA

C

TA0.34 nm

3.4 nm

(a) Key features of DNA structure

G

1 nm

G

(c) Space-filling model

T


• What Franklin’s X-ray data revealed about DNA structure:

– Overall shape is helix

– Width of helix = 2 nm

• indicates 2-stranded helix (not 3-stranded)

– Spacing of bases = .34 nm; 1 turn = 3.4 nm

• indicates 10 bases per turn

• hydrophobic bases must be interior

– Double helix has uniform diameter

• purines (A,G) must pair with pyrimidines (C,T)


O

–O O

OH

O

–O O

O

H2C

O

–OO

O

H2C

O

–O O

O

OH

O

O

OT A

C

GC

A T

O

O

O

CH2

O O–

OO

CH2

CH2

CH2

5 end

Hydrogen bond3 end

3 end

G

P

P

P

P

O

OH

O–

OO

O

P

P

O–

OO

O

P

O–

OO

O

P

(b) Partial chemical structure

H2C

5 end

O


• 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


N H O CH3

N

N

O

N

N

N

N H

Sugar

Sugar

Adenine (A) Thymine (T)

N

N

N

N

Sugar

O H N

H

NH

N OH

H

N

Sugar

Guanine (G) Cytosine (C)

H


DNA replication and repair

• The relationship between structure and function is seen in the double helix


Base Pairing to a Template Strand

• the two strands of DNA are complementary

– each strand acts as a template for building a new strand in replication

– the parent molecule unwinds, and two new daughter strands are built based on base-pairing rules

(a) The parent molecule has twocomplementary strands of DNA.Each base is paired by hydrogenbonding with its specific partner,A with T and G with C.

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

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

(d) The nucleotides are connectedto form the sugar-phosphatebackbones of the new strands. Each “daughter” DNA molecule consists of one parentalstrand and one new strand.

ACTAG

ACTAG

ACTAG

ACTAG

TGATC

TGATC

ACTAG

ACTA

G

TGATC

TGATC

TGATC

TG

ATC


Conservativemodel. The twoparental strandsreassociate after acting astemplates fornew strands,thus restoringthe parentaldouble helix.

Semiconservativemodel. The two strands of the parental moleculeseparate, and each functionsas a templatefor synthesis ofa new, comple-mentary strand.

Dispersivemodel. Eachstrand of bothdaughter mol-ecules containsa mixture ofold and newlysynthesizedDNA.

Parent cellFirstreplication

Secondreplication

• 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 (a)

(b)

(c)


• Experiments performed by Matthew Meselson and Franklin Stahl supported the semiconservative model of DNA replication

• cultured several generations of E. Coli with nucleotide precursors labeled with 15N (heavynitrogen) allowing bacteria to incorporate 15N into their DNA

• then transferred bacteria to medium with only 14N(light nitrogen); any newly synthesized DNA wouldbe lighter than the parental DNA

• then separated DNA of different densities by centrifuging DNA extracted from the bacteria.

EXPERIMENT


RESULTS

Bacteriacultured inmediumcontaining15N

Bacteriatransferred tomediumcontaining14N

DNA samplecentrifugedafter 20 min(after firstreplication)

DNA samplecentrifugedafter 40 min(after secondreplication)

less dense

more dense

1 band (heavy nitrogen only) 2 bands (light & heavy nitrogen)


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

Conservativemodel

Semiconservativemodel

Dispersivemodel


Details of DNA Replication• DNA replication is remarkably fast & accurate

• More than a dozen enzymes & other proteins participate in DNA replication


Origin of Replication• specific DNA sequence recognized by DNA-

replicating proteins

• initiates replication

• location where the two DNA strands are first separated


Origin of Replication• bacterial chromosomes have 1 replication

origin

• eukaryotic chromosomes have hundreds or thousands of origins of replication.

• Why?

– Faster! Much more DNA to copy

• 6 billion bps in eukaryotes vs. 6 million bps in bacteria


Origin of Replication• replication fork

– Y-shaped region where parental strands separate & new strands form

– forms a replication bubble

– replication bubbles expand laterally, as DNA replication proceeds in both directions

– replication bubbles eventually fuse, completing synthesis of the daughter strands


• In eukaryotes, DNA replication begins at many sites along the giant DNA molecule of each chromosome.

Origin of replication

Bubble

Parental (template) strandDaughter (new) strand

Replication fork

Two daughter DNA molecules

3 replication bubbles are visible along the DNA of a cultured Chinese hamster cell (TEM)

(b)

0.25 µm


New strand Template strand5 end 3 end

Sugar A TBase

C

G

G

C

A

COH

P P

5 end 3 end

5 end 5 end

A T

C

G

G

C

A

C

T

3 endPyrophosphate

2 P

OH

Phosphate

Elongating a New DNA Strand• elongation of new DNA at a replication fork

is catalyzed by enzymes called DNA polymerases

– add nucleotides to the 3 end of a growing strand

Nucleosidetriphosphate


Rate of Elongation• In bacteria: 500 nucleotides/sec

• In human cells: 50 nucleotides/sec


Polymerization requires energy!• Energy is provided by nucleoside

triphosphates (the monomers themselves)

– Nucleoside = 5C sugar + N base

• nucleotide = nucleoside + phosphate

– Chemically reactive (similar to ATP!)

• Only difference is deoxyribose (ATP contains ribose)


Polymerization requires energy!• As each monomer joins DNA, it loses a

phosphate dimer (pyrophosphate) which breaks down, releasing energy (exergonic)

New strand Template strand5 end 3 end

Sugar A TBase

C

G

G

C

A

COH

P P

5 end 3 end

5 end 5 end

A T

C

G

G

C

A

C

T

3 endPyrophosphate

2 P

OH

Phosphate


Antiparallel Elongation• How does the antiparallel structure of the

double helix affect replication?

– DNA strand is polar

– PO4 is attached to 5’ C

– DNA polymerases can only add to free 3’ C!

– DNA grows 5’ 3’ only!(DNA polymerase reads 3’ 5’)


Leading & Lagging Strands• Leading strand:

– Able to form continuously towardsreplication fork along 3’ template strand

• DNA polymerases can only add to 3end!

• Lagging strand:

– Must form in sections away from replication fork along 5’ template strand

• Enough parent DNA must “unzip” first

• Like “backstitching”


• Okazaki fragments

– sections of DNA forming lagging strand

– ~100-200 nucleotides long in eukaryotes

• DNA ligase

– enzyme that joins Okazaki fragments at sugar-phosphate backbone


Parental DNA

DNA polymerase

Okazakifragments

DNA polymeraseTemplate strand

Lagging strand32

Overall direction of replication!

DNA ligase

35

53

35

21

Leading strand

1

• Synthesis of leading & lagging strands during DNA replication

Leading strand

Lagging strand

Template strand


Priming DNA Synthesis• DNA polymerases cannot initiate the

synthesis of a polynucleotide

– can only add nucleotides to the 3 end

• Primer:

– short section (~10 nucleotides) of RNAattached to parent DNA strand

– provides starting point for polymerase (provides 3’ carbon)


Priming DNA Synthesis• Primase

– Enzyme that makes RNA primers

• Like all RNA synthesizing enzymes, primase can start RNA from scratch

– Another DNA polymerase later replaces it with DNA


• Only one primer is needed for synthesis of the leading strand

• Many primers need for synthesis of the lagging strand

– Each Okazaki fragment needs a primer!


Overall direction of replication

3

3

3

35

35

35

35

35

35

35

3 5

5

1

1

21

12

5

5

12

35

Templatestrand

RNA primer

Okazakifragment

Primase joins RNA nucleotides into a primer.

1

DNA polymerase adds DNA nucleotides to primer, forming Okazaki fragment.

2

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

3

After 2nd fragment is primed, DNA polym adds DNA nucleotides until it reaches the first primer and falls off.

4

A different DNA polym replaces RNA with DNA, adding to the 3 end of fragment 2.

5

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

6

The lagging strand in this region is nowcomplete.

7


Other Proteins Assisting DNA Replication• Helicase

– Enzyme that untwists double helix & separates parental strands

• Single-strand binding proteins

– Line up along unpaired strands to hold them apart


Overall direction of replicationLeadingstrand

Laggingstrand

Laggingstrand

LeadingstrandOVERVIEW

Leadingstrand

Replication fork

DNA pol III

PrimasePrimer

DNA pol III Laggingstrand

DNA pol I

Parental DNA

53

43

2

Origin of replication

DNA ligase

1

5

3

Helicase unwinds theparental double helix.

1

single-strand binding proteinsStabilize unwoundtemplate strands

2

leading strand issynthesized continuously in 5 3 direction by DNA pol III.

3

Primase begins synthesis of RNA primer for 5th Okazaki fragment

4

summary of DNA replication


Summary of main proteins of DNA replication

• Initiation of replication:

• Untwisting – helicases

• Stabilizing – single-strand binding proteins

• Strand synthesis:

• Priming – primase (leading & lagging)

• Elongation – DNA polymerase III (leading & lagging)

• Primer replacement – DNA polymerase I (leading & lagging)

• Joining of fragments – ligase (lagging only)


DNA Replication Complex

• The various proteins that participate in DNA replication form a single large complex

• = DNA replication “machine”

• probably stationary during the replication process (DNA feeds through)

• replication may be more like DNA fabric moving through a sewing machine rather than a train riding on a DNA track


Proofreading and Repairing DNA• DNA polymerase

– proofreads newly made DNA

– compares new strand against template

• older strand can be distinguished by chemical accumulations (ex. methyl groups)

– removes any incorrect nucleotides (then resumes synthesis)


Proofreading and Repairing DNA• mismatch repair of DNA

– occurs after DNA molecule completed

• corrects mismatches that were missed by DNA polymerase or that occurred after synthesis

– repair enzymes correct errors in base pairing

• must be important - 130 repair enzymes identified so far in humans!

– defective enzyme linked to colon cancer

• Nuclease – enzyme that removes damaged section of DNA


Nuclease

DNApolymerase

DNAligase

A thymine dimerdistorts the DNA molecule.1

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

2

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

3

DNA ligase seals theFree end of the new DNATo the old DNA, making thestrand complete.

4

• nucleotide excision repair

– enzymes cut out and replace damaged stretches of DNA


Replicating the Ends of DNA Molecules• The ends of eukaryotic chromosomal DNA get

shorter with each round of replicationEnd of parentalDNA strands

Leading strandLagging strand

Last fragment Previous fragment

RNA primer

Lagging strandRemoval of primers and replacement with DNA where a 3 end is availablePrimer removed but

cannot be replacedwith DNA becauseno 3 end available

for DNA polymerase Second roundof replicationNew leading strand

New lagging strand 5Further roundsof replication

Shorter and shorterdaughter molecules

53

5

3

53

53

3


telomeres• Eukaryotic chromosomal DNA molecules

have at their ends nucleotide sequences, called telomeres, that postpone the erosion of genes near the ends of DNA molecules

1 µm


telomeres• In humans, telomeres consist of the

sequence TTAGGG repeated 100-1,000 times

1 µm


telomerase• If the chromosomes of germ cells became

shorter in every cell cycle

– essential genes would eventually be missing from the gametes they produce

– Prokaryotes don’t have this problem – circular chromosomes!

• An enzyme called telomerase

– catalyzes the lengthening of telomeres in germ cells

– provides short RNA molecule that serves as template for lengthening DNA molecule


Any questions?

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