DNA: The Genetic Material Frederick Griffith – 1928 · DNA: The Genetic Material Chapter 14 Frederick Griffith – 1928 •Studied Streptococcus pneumoniae, a ... Hershey & Chase

DNA: The Genetic Material

Chapter 14

Frederick Griffith – 1928

• Studied Streptococcus pneumoniae, a

pathogenic bacterium causing pneumonia

• 2 strains of Streptococcus

– S strain is virulent

– R strain is nonvirulent

• Griffith infected mice with these strains

2

3

Griffith’s Experiments

4

Griffith’s Experiments

5

• Transformation

– Information specifying virulence passed

from the dead S strain cells into the live

R strain cells

• Our modern interpretation is that genetic

material was actually transferred between

the bacterial cells

Griffith’s Results

6

Avery, MacLeod, & McCarty – 1944

• Repeated Griffith’s experiment usingpurified cell extracts as transformingmaterial

• Removal of all protein did not destroy abilityto transform R strain cells

• DNA-digesting enzymes destroyed alltransforming ability

• Supported DNA as the genetic material

7

Hershey & Chase –1952

• Investigated bacteriophages

– Viruses that infect bacteria

• Bacteriophage was composed of only

DNA and protein

• Wanted to determine which of these

molecules is the genetic material

• Only the DNA entered the bacteria and

was used to produce more bacteriophage

• Conclusion: DNA is the genetic material8

Hershey & Chase

9

DNA Structure

• DNA is a nucleic acid

• Composed of nucleotides

– 5-carbon sugar called deoxyribose

– Phosphate group (PO4)

• Attached to 5! carbon of sugar

– Nitrogenous base

• Adenine, thymine, cytosine, guanine

– Free hydroxyl group (—OH)

• Attached at the 3! carbon of sugar

10

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

Pu

rin

es

Pyri

mid

ines

Adenine Guanine

NH2CC

NN

N

C

H

N

C

CH

O

H

H

OC

NC

H

N

C

NH2

H

CH O

O

C

NC

H

N

CH3C

CH

H

O

O

C

NC

H

N

CH

CH

NH2

CC

NN

N

C

H

N

C

CHH

Nitrogenous Base

4´

5´

1´

3´ 2´

2

8

7 6

39

4

5

1

O

P

O–

–O

Phosphate group

Sugar

Nitrogenous base

O CH2

N N

O

N

NH2

OH in RNA

Cytosine

(both DNA and RNA)Thymine

(DNA only)

Uracil

(RNA only)

OH

H in DNA

• Phosphodiesterbond– Bond between

adjacentnucleotides

– Formed betweenthe phosphategroup of onenucleotide and the3! —OH of thenext nucleotide

• Each DNA strandhas a 5! end and a3! end

11


BaseCH2

O

5´

3´

O

P

O

OH

CH2

–O O

C

Base

O

PO4

Phosphodiester

bond

Chargaff’s Rules

• Erwin Chargaff determined that

– Amount of adenine = amount of thymine

– Amount of cytosine = amount of guanine

– Always an equal proportion of purines

(A and G) and pyrimidines (C and T)

12

13

Rosalind Franklin

• Performed X-ray diffraction

studies to identify the 3-D

structure

– Discovered that DNA is helical

– Using Maurice Wilkins’ DNA

fibers, discovered that the

molecule has a diameter of 2

nm and makes a complete

turn of the helix every 3.4 nm

14

James Watson and Francis Crick – 1953

• Deduced the double helix structure of DNA

using evidence from Chargaff, Franklin,

and others

Double helix

• 2 strands are polymers

of nucleotides

• Phosphodiester

backbone – repeating

sugar and phosphate

units joined by

phosphodiester bonds

• Antiparallel strands

15


5´

3´

P

P

P

P

OH

5-carbon sugar

Nitrogenous base

Phosphate group

Phosphodiester bond

O

O

O

O

4´

5´

1´

3´ 2´

4´

5´

1´

3´ 2´

4´

5´

1´

3´ 2´

4´

5´

1´

3´2´

• Complementarity of

bases

• A forms 2 hydrogen

bonds with T

• G forms 3 hydrogen

bonds with C

• Gives consistent

diameter

16


A

H

Sugar

Sugar

Sugar

Sugar

T

G C

N

H

N O

H

CH3

H

HN

N N H N

N

N

H

H

H

N O H

H

H N

N H

N

N HN N

Hydrogen

bond

Hydrogen

bond

17 18

DNA Replication

• 3 possible models

1. Conservative model

2. Semiconservative model

3. Dispersive model

2nd

replication

Parental

DNA

Replicated

DNA

KEY

a. Semiconservative replication

1st

replication

b. Conservative replication c. Dispersive replication

1957 Meselson and Stahl - 3 Replication Models

Meselson-Stahl Experiment

1st replication

15N

medium 14N medium

2nd replication


(cont.)

15N–14N hybrid DNA

14N–14N (light) DNA

15N–15N (heavy) DNA

CsCl forms a density gradient

during centrifugation, with the

highest density at the bottom

of the tube.

DNA molecules move to positions

where their density equals that of the

CsCl solution and form bands. Shown

are the positions of differently labeled

DNA molecules. Experimentally the

bonds are detected by absorbance of

UV light.


(cont.)

15N–14N

hybrid DNA15N–15N

(heavy) DNA

DNA from15N medium

DNA after one

replication in 14N

15N–14N

hybrid DNA

DNA after two

replications in 14N

14N–14N

(light) DNA


Semiconservative

Conservative

Dispersive

The results support the semiconservative model.

Matches

results

Does not

match

results

Does not

match

results

Two replications in 14N15N medium One replication in 14N

Direction of

replication

OldNew

Complementary base

pairing in the DNA double

helix: A pairs with T, G

pairs with C.

The two chains unwind

and separate.

Each “old” strand is a

template for the addition

of bases according to the

base-pairing rules.

The result is two DNA

helices that are exact

copies of the parental

DNA molecule with one

“old” strand and one

“new” strand

25

DNA Replication

• Requires 3 things

– Something to copy

• Parental DNA molecule

– Something to do the copying

• Enzymes

– Building blocks to make copy

• Nucleotide triphosphates

26

Enzymes of DNA Replication

• DNA polymerases assemble nucleotides into a

chain

• Require 3! end (e.g., RNA primer)

• Synthesize in 5!-to-3! direction only

• Helicase unwinds the DNA

• Primase synthesizes RNA primer (starting point

for nucleotide assembly by DNA polymerases)

• Nuclease removes primers

• DNA ligase closes remaining single-chain nicks

Semidiscontinous

• DNA polymerase can synthesize only in 1

direction

• Leading strand synthesized continuously

from an initial primer

• Lagging strand synthesized

discontinuously with multiple priming

events

– Okazaki fragments

28

Assembling Antiparallel Strands Enzyme ActivitiesUnwinding enzyme

(helicase)

Primase

RNA

primers

Replication fork

Overall

direction of replication

1 Helicase unwinds the DNA,

and primases synthesize

short RNA primers.

RNA

Leading strand

Lagging strand

DNA polymerase

DNA polymerase

RNADNA

2 RNA primers are used as

starting points for the

addition of DNA nucleotides

by DNA polymerases.

Primer being extended

by DNA polymerase

DNA polymerase

Newly synthesized primer

DNA unwinds further, and

leading strand synthesis

proceeds continuously, while

a new primer is synthesized

on the lagging strand

template and extended by

DNA polymerase III.

3

DNA

polymerase

Nick Another type of DNA

polymerase (I) removes the

RNA primer, replacing it with

DNA, leaving a nick between

the newly synthesized

segments.

4

DNA ligase Nick is closed

by DNA ligase.5

Lagging strand

Leading strand

Newly

synthesized

primer

DNA polymerase

Primer being extended

by DNA polymerase III

6 DNA continues to unwind,

and the synthesis cycle

repeats as before:

continuous synthesis of

leading strand and synthesis

of a new segment to be

added to the lagging strand.

Prokaryotic Replication

• E. coli model

• Single circular molecule of DNA

• Replication begins at one origin of

replication

• Proceeds in both directions around the

chromosome

33 34

35

• E. coli has 3 DNA polymerases

– DNA polymerase I (pol I)

• Acts on lagging strand to remove primers

and replace them with DNA

– DNA polymerase II (pol II)

• Involved in DNA repair processes

– DNA polymerase III (pol III)

• Main replication enzyme

– All 3 have 3!-to-5! exonuclease activity –

proofreading

– DNA pol I has 5!-to-3! exonuclease activity

36

37

Eukaryotic Replication

• Complicated by

– Larger amount of DNA in multiple

chromosomes

– Linear structure

• Basic enzymology is similar

– Requires new enzymatic activity for

dealing with ends only

Telomeres

• Specialized structures found on the ends

of eukaryotic chromosomes

• Protect ends of chromosomes from

nucleases and maintain the integrity of

linear chromosomes

• Gradual shortening of chromosomes with

each round of cell division

– Unable to replicate last section of lagging

strand38

39 40

• Telomeres composed of short repeatedsequences of DNA

• Telomerase – enzyme makes telomere oflagging strand using an internal RNA template(not the DNA itself)– Leading strand can be replicated to the end

• Telomerase developmentally regulated– Relationship between senescence and telomere

length

• Cancer cells generally show activation oftelomerase

41 42

DNA Repair

• Errors due to replication

– DNA polymerases have proofreading ability

• Mutagens – any agent that increases the

number of mutations above background

level

– Radiation and chemicals

• Importance of DNA repair is indicated by

the multiplicity of repair systems that have

been discovered

Excision repair

• Targets multiple types of lesions

• Damaged region is removed and replaced

by DNA synthesis

• 3 steps

1. Recognition of damage

2. Removal of the damaged region

3. Resynthesis using the information on the

undamaged strand as a template

43 44

DNA: The Genetic Material Frederick Griffith – 1928 · DNA: The Genetic Material Chapter 14 Frederick Griffith – 1928 •Studied Streptococcus pneumoniae, a ... Hershey & Chase

Documents