Chapter 11DNA: The Carrier of Genetic

Information

Experiments in DNA

• ???Protein as the genetic material• 20 AA – many different combinations = unique

properties• Genes control protein synthesis• DNA and RNA – only 4 nucleotides = dull

Experiments in DNA• Frederick Griffith – 1928

– Bacteria – pneumococcus – 2 strains– (S) smooth strain – virulent (lethal)

• Mice – pneumonia - death

– (R) rough strain – avirulent• Mice survive

– Heat killed (S) strain• Mice survive

– Heat killed (S) + live (R)• Mice died• Found living (S) in dead mice

• Griffith continued– transformation - type of permanent genetic

change where the properties of 1 strain of dead cells are conferred on a different strain of living cells

– “transforming principle” was transferred from dead to living cells

Fig. 16-2

Living S cells (control)

Living R cells (control)

Heat-killed S cells (control)

Mixture of heat-killed S cells and living R cells

Mouse diesMouse dies Mouse healthy Mouse healthy

Living S cells

RESULTS

EXPERIMENT

• Avery, MacLeod, McCarty - 1944– Identified Griffith’s transforming principle as DNA– Live (R) + purified DNA from (S) R cells

transformed– R + (S) DNA die– R = (S) protein live

– DNA responsible for transformation– Really?

• Hershey and Chase – 1952– Bacteriophages– Radioactive labels

• Viral protein – sulfur• Viral DNA - phosphorus

– infect bacteria, agitate in blender, centrifuge– Found

• Sulfur sample – all radioactivity in supernatant (not cells)• Phosphorus sample – radioactivity in pellet (inside cells)

– SO – bacteriophages inject DNA into bacteria, leaving protein on outside

– DNA = hereditary material

Fig. 16-3

Bacterial cell

Phage head

Tail sheath

Tail fiber

DNA

100

nm

Fig. 16-4-3

EXPERIMENT

Phage

DNA

Bacterial cell

Radioactive protein

Radioactive DNA

Batch 1: radioactive sulfur (35S)

Batch 2: radioactive phosphorus (32P)

Empty protein shell

Phage DNA

Centrifuge

Centrifuge

Pellet

Pellet (bacterial cells and contents)

Radioactivity (phage protein) in liquid

Radioactivity (phage DNA) in pellet

• Rosalind Franklin (in lab of Wilkins)– X-ray diffraction on crystals of purified DNA– (X-ray crystallography)– Determine distance between atoms of molecules

arranged in a regular, repeating crystalline structure

• Helix structure• Nucleotide bases like rungs on ladder

Fig. 16-6

(a) Rosalind Franklin (b) Franklin’s X-ray diffraction photograph of DNA

• James Watson and Francis Crick – 1953– Model for DNA structure = double helix– DNA now widely accepted as genetic material– Took all available info on DNA and put together– Showed –

• DNA can carry info for proteins• Serve as own template for replication

Structure of DNA

• Nucleotides– Deoxyribose– Phosphate– Nitrogenous base (ATCG)

• Purines – adenine, guanine – 2 rings• Pyrimidines – thymine, cytosine – 1 ring

– covalent bonds link = sugar-phosphate backbone• 3’ C of sugar bonded to 5’ phosphate = phophodiester

linkage• 5’ end – 5’ C attached to phosphate• 3’ end – 3’ C attached to hydroxyl

Chargaff - 1950

• # purines = # pyrimidines– #A = #T– #C = # G

• Each cross rung of ladder– 1 purine + 1 pyrimidine

Fig. 16-UN1

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width consistent with X-ray data

• Hydrogen bonding between N bases• A-T = 2 H bonds• G-C = 3 H bonds• Complementary base pairs

• # possible sequences virtually unlimited• many genes, much info

Fig. 16-8

Cytosine (C)

Adenine (A) Thymine (T)

Guanine (G)

Fig. 16-5 Sugar–phosphate backbone

5 end

Nitrogenous

bases

Thymine (T)

Adenine (A)

Cytosine (C)

Guanine (G)

DNA nucleotide

Sugar (deoxyribose)

3 end

Phosphate

Fig. 16-7a

Hydrogen bond 3 end

5 end

3.4 nm

0.34 nm3 end

5 end

(b) Partial chemical structure(a) Key features of DNA structure

1 nm

DNA Replication

• Semiconservative – each strand of DNA is template to make opposite new strand

• Meselson and Stahl– E. coli and isotopes of N– 15N – heavy/dense; 14N “normal”– Bacteria with 15N in DNA replicated with medium

having 14N– Centrifuge – Supports semiconservative model

• Explains how mutagens can be passed on

Fig. 16-11a

EXPERIMENT

RESULTS

1

3

2

4

Bacteria cultured in medium containing 15N

Bacteria transferred to medium containing 14N

DNA sample centrifuged after 20 min (after first application)

DNA sample centrifuged after 20 min (after second replication)

Less dense

More dense

Fig. 16-9-3

A T

GC

T A

TA

G C

(a) Parent molecule

A T

GC

T A

TAG C

(c) “Daughter” DNA molecules, each consisting of one parental strand and one new strand

(b) Separation of strands

A T

GC

T A

TA

G C

A T

GC

T A

TAG C

Fig. 16-10Parent cell

First replication

Second replication

(a) Conservative model

(b) Semiconserva- tive model

(c) Dispersive model

Steps of DNA Replication

• 1. DNA helicase – • 2. Helix-destabilizing proteins – • 3. Topoisomerases – • 4. RNA primer – • 5. DNA polymerase – • 6. Origin of replication –

– Leading strand– Lagging strand

• 7. DNA Ligase

Leading Strand

Fig. 16-12b

0.25 µm

Origin of replication Double-stranded DNA molecule

Parental (template) strandDaughter (new) strand

Bubble Replication fork

Two daughter DNA molecules

(b) Origins of replication in eukaryotes

Fig. 16-14

A

C

T

G

G

G

GC

C C

C

C

A

A

AT

T

T

New strand 5 end

Template strand 3 end 5 end 3 end

3 end

5 end5 end

3 end

BaseSugar

Phosphate

Nucleoside triphosphate

Pyrophosphate

DNA polymerase

Fig. 16-15a

Overview

Leading strand

Leading strandLagging strand

Lagging strandOrigin of replication

Primer

Overall directions of replication

Fig. 16-17

OverviewOrigin of replicationLeading strand

Leading strand

Lagging strand

Lagging strandOverall

directions of

replicationLeading strand

Lagging strand

Helicase

Parental DNA

DNA pol IIIPrimerPrimase

DNA ligase

DNA pol IIIDNA pol I

Single-strand

binding protein

53

5

55

5

3

3

3313 2

4

Telomeres

• Telomeres – caps end of chromosome; short non-coding sequences repeated many times

• Cell can divide many times before losing crucial info

• Lagging strand is discontinuous, so DNA polymerase unable to complete replication , leaving small part unreplicated small part lost with each cycle

Fig. 16-19Ends of parental DNA strands

Leading strandLagging strand

Lagging strand

Last fragment Previous fragment

Parental strand

RNA primer

Removal of primers and replacement with DNA where a 3 end is available

Second round of replication

New leading strand

New lagging strand

Further rounds of replication

Shorter and shorter daughter molecules

5

3

3

3

3

3

5

5

5

5