The Molecular Basis

of Inheritance

(Ch. 13)

Many people contributed to our understanding of DNA T.H. Morgan (1908) Frederick Griffith (1928) Avery, McCarty & MacLeod (1944) Erwin Chargaff (1947) Hershey & Chase (1952) Watson & Crick (1953) Meselson & Stahl (1958)

T.H. Morgan working with Drosophila

associated phenotype with

specific chromosome white-eyed male had specific X

chromosome

Morgan’s conclusions genes are on chromosomes

but is it the protein or the DNA of the chromosomes that are the genes? initially proteins were

thought to be genetic material… Why?

What’s so impressive about proteins?!

Frederick Griffith Streptococcus pneumonia

bacteria 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 “Transforming Principle”

Avery, McCarty & MacLeod Purified DNA & proteins from

Streptococcus pneumonia bacteria

injected protein into bacteria no effect

injected DNA into bacteria transformed harmless bacteria

into virulent bacteria

1944

What’s the conclusion?

mice die

Oswald Avery Maclyn McCarty Colin MacLeod

Conclusion first experimental evidence that DNA

was the genetic material

Hershey & Chase classic “blender” experiment

worked with bacteriophage viruses that infect bacteria

grew phages in 2 media, radioactively labeled with either 35S in their proteins

32P in their DNA

infected bacteria phages Why use Sulfur

vs. Phosphorus?

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!

Alfred Hershey Martha Chase

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%

That’s interesting! What do you notice?

Rules A = T C = G

Watson & Crick developed double helix model of DNA

other leading scientists working on question: Rosalind Franklin

Maurice Wilkins

Linus Pauling

Franklin Wilkins Pauling

Watson and Crick 1953 article in Nature

Crick Watson

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

Alternative models become experimental predictions

conservative semiconservative

Can you design a nifty experiment

to verify?

dispersive

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2

P

Meselson & Stahl label “parent” nucleotides in DNA

strands with heavy nitrogen = 15N

label new nucleotides with lighter isotope = 14N

Franklin Stahl

Matthew Meselson

March to understanding that DNA is the genetic material T.H. Morgan (1908): genes are on chromosomes Frederick Griffith (1928): a transforming factor can

change phenotype Avery, McCarty & MacLeod (1944): transforming factor

is DNA Erwin Chargaff (1947): Chargaff rules: A = T, C = G Hershey & Chase (1952): confirmation that DNA is

genetic material Watson & Crick (1953): determined double helix

structure of DNA Meselson & Stahl (1958): semi-conservative replication

protein RNA

Flow of genetic information in a cell

DNA

transcription translation

replication

You need to number the carbons! it matters!

OH

CH2

O

4

5

3 2

1

PO4

N base

sugar

nucleotide

This will be

IMPORTANT!!

Putting the DNA backbone together refer to the 3 and 5

ends of the DNA the last trailing carbon

OH

O

3

PO4

base

CH2

O

base

O

P

O

C

O –O

CH2

1

2

4

5

1

2

3

3

4

5

5

Sounds trivial, but…

this will be IMPORTANT!!

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

….strong or weak bonds?

How do the bonds fit the mechanism for copying DNA?

3

5 3

5

covalent

phosphodiester

bonds

hydrogen

bonds

Purines adenine (A) guanine (G)

Pyrimidines thymine (T) cytosine (C)

Pairing A : T

2 bonds C : G

3 bonds

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

Large team of enzymes coordinates replication

Let’s meet the team…

Unwind DNA helicase enzyme

unwinds part of DNA helix

stabilized by single-stranded binding proteins

single-stranded binding proteins replication fork

helicase

I’d love to be helicase & unzip

your genes…

DNA

Polymerase III

But… We’re missing

something! What?

Where’s the ENERGY

for the bonding!

Build daughter DNA

strand

add new

complementary bases

DNA polymerase III

energy

ATP GTP TTP CTP

Where does energy for bonding usually come from?

ADP AMP GMP TMP CMP

modified nucleotide

energy

We come with our own

energy!

And we leave behind a nucleotide!

You remember

ATP! Are there other ways

to get energy out of it?

Are there other energy nucleotides?

You bet!

The nucleotides arrive as nucleosides DNA bases with P–P–P

P-P-P = energy for bonding DNA bases arrive with their own energy source

for bonding bonded by enzyme: DNA polymerase III

ATP GTP TTP CTP

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

DNA

Polymerase III

DNA

Polymerase III

DNA

Polymerase III

DNA

Polymerase III

energy

energy

energy

energy

3

3

5 B.Y.O. ENERGY! The energy rules

the process

5

Limits of DNA polymerase III

can only build onto 3 end of

an existing DNA strand

5

5

5

5

3

3

3

5

3 5

3 3

Leading strand

Lagging strand 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

5

3 5

3

leading strand

lagging strand

leading strand

lagging strand leading strand

5

3

3

5

5

3

5

3

5

3 5

3

growing replication fork

growing replication fork

5

5

5

5

5

3

3

5

5 lagging 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

5

5

5

3

3

3

5

3 5

3 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

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

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 5

5

5

5

3

3

3

3

growing replication fork

TTAAGGG TTAAGGG

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 polymerase III 1000 bases/second! main DNA builder

DNA polymerase I 20 bases/second editing, repair &

primer removal

DNA polymerase III enzyme

Arthur Kornberg 1959

Thomas Kornberg ??

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

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 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

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