Chapter 16~ The Molecular Basis of Inheritance

Scientific History • The march to understanding that DNA is

the genetic material– T.H. Morgan (1908)– Frederick Griffith (1928)– Avery, McCarty & MacLeod (1944)– Erwin Chargaff (1947)– Hershey & Chase (1952)– Watson & Crick (1953)– Meselson & Stahl (1958)

The “Transforming Principle” 1928

• Frederick Griffith – Streptococcus pneumonia bacteria

• was working to find cure for pneumonia

– harmless live bacteria (“rough”) mixed with heat-killed pathogenic bacteria (“smooth”) causes fatal disease in mice

– a substance passed from dead bacteria to live bacteria to change their phenotype

• “Transforming Principle”

The “Transforming Principle”

Transformation = change in phenotypesomething in heat-killed bacteria could still transmit disease-causing properties

live pathogenicstrain of bacteria

live non-pathogenicstrain of bacteria

mice die mice live

heat-killed pathogenic bacteria

mix heat-killed pathogenic & non-pathogenicbacteria

mice live mice die

A. B. C. D.

DNA is the “Transforming Principle”

• Avery, McCarty & MacLeod– purified both DNA & proteins separately from

Streptococcus pneumonia bacteria• which will transform non-pathogenic bacteria?

– injected protein into bacteria• no effect

– injected DNA into bacteria• transformed harmless bacteria into

virulent bacteria

1944

What’s theconclusion?

mice die

Oswald Avery Maclyn McCarty Colin MacLeod

Avery, McCarty & MacLeod• Conclusion

– first experimental evidence that DNA was the genetic material

1944 | ??!!

Confirmation of DNA• Hershey & Chase

– classic “blender” experiment– worked with bacteriophage

• viruses that infect bacteria– grew phage viruses in 2 media,

radioactively labeled with either • 35S in their proteins• 32P in their DNA

– infected bacteria with labeled phages

1952 | 1969Hershey

Why useSulfurvs.Phosphorus?

Protein coat labeledwith 35S DNA labeled with 32P

bacteriophages infectbacterial cells

T2 bacteriophagesare labeled withradioactive isotopesS vs. P

bacterial cells are agitatedto remove viral protein coats

35S radioactivityfound in the medium

32P radioactivity foundin the bacterial cells

Which radioactive marker is found inside the cell?

Which molecule carries viral genetic info?

Hershey &

Chase

Blender experiment• Radioactive phage & bacteria in blender

– 35S phage• radioactive proteins stayed in supernatant• therefore viral protein did NOT enter bacteria

– 32P phage• radioactive DNA stayed in pellet• therefore viral DNA did enter bacteria

– Confirmed DNA is “transforming factor”

Taaa-Daaa!

Hershey & Chase

Alfred HersheyMartha Chase

1952 | 1969Hershey

Chargaff

• DNA composition: “Chargaff’s rules”– varies from species to species– all 4 bases not in equal quantity– bases present in characteristic ratio

• humans:A = 30.9%

T = 29.4% G = 19.9% C = 19.8%

1947

That’s interesting!What do you notice?

RulesA = TC = G

Structure of DNA• Watson & Crick

– developed double helix model of DNA• other leading scientists working on question:

– Rosalind Franklin– Maurice Wilkins– Linus Pauling

1953 | 1962

Franklin Wilkins Pauling

Watson and Crick 1953 article in Nature

CrickWatson

Rosalind Franklin (1920-1958)

Double helix structure of DNA

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” Watson & Crick

Directionality of DNA• You need to

number the carbons!– it matters!

OH

CH2O

4

5

3 2

1

PO4

N base

ribose

nucleotide

This will beIMPORTANT!!

The DNA backbone• Putting the DNA

backbone together– refer to the 3 and 5

ends of the DNA• the last trailing carbon

OH

O

3

PO4

base

CH2O

base

OPO

C

O–O

CH2

1

2

4

5

1

2

3

3

4

5

5

Sounds trivial, but…this will beIMPORTANT!!

Anti-parallel strands• Nucleotides in DNA

backbone are bonded from phosphate to sugar between 3 & 5 carbons– DNA molecule has “direction”– complementary strand runs in

opposite direction

3

5

5

3

Bonding in DNA

….strong or weak bonds?How do the bonds fit the mechanism for copying DNA?

3

5 3

5

covalentphosphodiesterbonds

hydrogenbonds

Base pairing in DNA• Purines

– adenine (A)– guanine (G)

• Pyrimidines– thymine (T)– cytosine (C)

• Pairing– A : T

• 2 bonds– C : G

• 3 bonds

But how is DNA copied?• Replication of DNA

– base pairing suggests that it will allow each side to serve as a template for a new strand

“It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” — Watson & Crick

Copying DNA• Replication of DNA

– base pairing allows each strand to serve as a template for a new strand

– new strand is 1/2 parent template & 1/2 new DNA

• semi-conservative copy process

Semiconservative replication, • when a double helix replicates each of the daughter molecules will

have one old strand and one newly made strand.• Experiments in the late 1950s by Matthew Meselson and Franklin

Stahl supported the semiconservative model, proposed by Watson and Crick, over the other two models. (Conservative & dispersive)

DNA Replication • Large team of enzymes coordinates replication

Let’s meetthe team…

Replication: 1st step• Unwind DNA

– helicase enzyme• unwinds part of DNA helix• stabilized by single-stranded binding proteins

single-stranded binding proteins replication fork

helicase

DNAPolymerase III

Replication: 2nd step Build daughter DNA

strand add new

complementary bases DNA polymerase III

• Adding bases – can only add

nucleotides to 3 end of a growing DNA strand• need a “starter”

nucleotide to bond to

– strand only grows 53

DNAPolymerase III

DNAPolymerase III

DNAPolymerase III

DNAPolymerase III

energy

energy

energy

Replicationenergy

3

3

5

5

Limits of DNA polymerase III can only build onto 3 end of an

existing DNA strand

Leading & Lagging strands

5

5

5

5

3

3

3

53

53 3

Leading strand

Lagging strandOkazaki fragments

ligase

Okazaki

Leading strand continuous synthesis

Lagging strand Okazaki fragments joined by ligase

“spot welder” enzyme

DNA polymerase III

3

5

growing replication fork

DNA polymerase III

Replication fork / Replication bubble

5

3 5

3

leading strand

lagging strand

leading strand

lagging strandleading strand

5

3

3

5

5

3

5

3

5

3 5

3

growing replication fork

growing replication fork

5

5

5

5

53

3

5

5lagging strand

5 3

DNA polymerase III

RNA primer built by primase serves as starter sequence for DNA

polymerase III

Limits of DNA polymerase III can only build onto 3 end of an

existing DNA strand

Starting DNA synthesis: RNA primers

5

5

5

3

3

3

5

3 53 5 3

growing replication fork primase

RNA

DNA polymerase I removes sections of RNA primer and

replaces with DNA nucleotides

But DNA polymerase I still can only build onto 3 end of an existing DNA strand

Replacing RNA primers with DNA

5

5

5

5

3

3

3

3

growing replication fork

DNA polymerase I

RNA

ligase

Loss of bases at 5 ends in every replication

chromosomes get shorter with each replication limit to number of cell divisions?

DNA polymerase III

All DNA polymerases can only add to 3 end of an existing DNA strand

Chromosome erosion

5

5

5

5

3

3

3

3

growing replication fork

DNA polymerase I

RNA

Houston, we have a problem!

Repeating, non-coding sequences at the end of chromosomes = protective cap

limit to ~50 cell divisions

Telomerase enzyme extends telomeres can add DNA bases at 5 end different level of activity in different cells

high in stem cells & cancers -- Why?

telomerase

Telomeres

5

5

5

5

3

3

3

3

growing replication fork

TTAAGGGTTAAGGGTTAAGGG

Replication fork

3’

5’3’

5’

5’

3’3’ 5’

helicase

direction of replication

SSB = single-stranded binding proteins

primase

DNA polymerase III

DNA polymerase III

DNA polymerase I

ligase

Okazaki fragments

leading strand

lagging strand

SSB

DNA polymerases• DNA polymerase III

– 1000 bases/second!– main DNA builder

• DNA polymerase I– 20 bases/second– editing, repair & primer removal

DNA polymerase III enzyme

Arthur Kornberg1959

Roger Kornberg2006

Editing & proofreading DNA• 1000 bases/second =

lots of typos!

• DNA polymerase I – proofreads & corrects typos – repairs mismatched bases– removes abnormal bases

• repairs damage throughout life

– reduces error rate from 1 in 10,000 to 1 in 100 million bases

Fast & accurate!• It takes E. coli <1 hour to copy

5 million base pairs in its single chromosome – divide to form 2 identical daughter cells

• Human cell copies its 6 billion bases & divide into daughter cells in only few hours– remarkably accurate– only ~1 error per 100 million bases– ~30 errors per cell cycle

1

2

3

4

What does it really look like?

2007-2008

Any Questions??