DNA (Deoxyribonucleic Acid)

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)

Transformation of Bacteria

What carries hereditary information?

By the 1940s, scientists knew that chromosomes carried genes.

They also knew that chromosomes were made of DNA and protein.

They did NOT know which of these molecules actually carried the genes.

Since protein has 20 types of amino acids that make it up, and DNA only has 4 types of building blocks, it was a logical conclusion.

Most Scientists thought protein carried genes

Chromosomes are made of DNA and protein

Transformation of Bacteria

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

Avery’s Experiment1. Avery repeated Griffith’s experiments with an additional step to see what type of molecule caused transformation.

3. When Avery added enzymes that destroy

DNA, no transformation occurred.

2. Avery used enzymes to destroy the sugars and transformation still occurred—Sugar did not cause transformation. Avery used enzymes to destroy lipids, RNA, and protein one by one. Every time transformation still occurred—none of these had anything to do with the transformation.

So…he knew that DNA carried hereditary

information!

Oswald Avery Maclyn McCarty Colin MacLeod

Avery, McCarty & MacLeod Conclusion

– first experimental evidence that DNA was the genetic material

1944 | ??!!

The experiment involved viruses to see if DNA or protein was injected into the bacteria in order to make new viruses.

One group of viruses was infected with radioactive protein and another group with radioactive DNA.

Then the viruses attack the bacteria.

Radioactive DNA shows up in the bacteria, but no radioactive protein.

Hershey-Chase Experiment

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

Rosalind Franklin Took X-ray

pictures of DNA. The photos

revealed the basic helix, spiral shape of DNA.

Maurice Wilkins Worked with

Rosalind Franklin. Took her x-ray

photos and information to Watson and Crick

Watson and Crick

Used Franklin’s pictures to build a series of large models.

Stated that DNA is a double-stranded molecule in the shape of a double helix, or twisted ladder.

Won the Nobel Prize for their work in 1962.

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)

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

Basic DNA Structure

S

P

A

CS

P

S

P

T

A nucleotide is the monomer of DNA A nucleotide is made of

– a sugar called deoxyribose– a phosphate– and a base (ATCG)

Directionality of DNA You need to

number the carbons!– it matters!

OH

CH2

O

4

5

3 2

1

PO4

N base

ribose

nucleotide

This will beIMPORTANT!!

Deoxyribose

Simple sugar molecule like glucose that has 5 carbons

The five carbons are numbered clockwise starting from the first one after the oxygen

Phosphate The negatively charged phosphate

bonds to the 5’ Carbon of the deoxyribose.

Bases

The base bonds to the 1’ Carbon.

Base

Bases There are two main types of bases

purines and pyrimidines. – Purines have two rings in their structure.

• Adenine and guanine are purines.

– Pyrimidines only have one ring.• Thymine and Cytosine are pyrimidines.

Pyrimidines Purines

Basic DNA Structure

S

P

A

CS

P

S

P

T

To form one strand of DNA, the phosphate of one nucleotide covalently bonds to the 3’ Carbon of the deoxyribose from another nucleotide.

S

P

A

CS

P

S

P

T

G S

P

S

P

A

T S

P

The two strands of DNA are held together by hydrogen bonds

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

Base Pairs The nucleotides that bond together by

their bases are called base pairs.– Adenine only bonds to Thymine– Guanine only bonds to Cytosine

Does each of your cells have the same DNA?

YES

DNA Replication

Before a cell divides, DNA must make a copy of itself so that each new cell has a complete set of DNA.

Step 1-Unzip DNA An enzyme called helicase untwists the

ladder and breaks the hydrogen bonds between the bases and “unzips” DNA down the middle.

Helicase Enzyme

Step 2-Prime the DNA

An enzyme called DNA primase put a few nucleotides of RNA on the DNA.

This is only to create a starting place and these will later be removed.

Step 3-Elongation The two strands of the

Parent DNA become templates for the new strands.

New nucleotides are added by an enzyme called DNA polymerase.

Step 3-Elongation

DNA polymerase only adds nucleotides in the 5’ to 3’ direction on both strands beginning at the RNA primer.

Step 4 – Fine tuning RNA primer is removed and any gaps

are sealed by an enzyme called ligase. DNA polymerase proof reads the new

copy and fixes any mistakes.

Helicase unwinds and unzips DNA

S

P

A

CS

P

S

P

T

T

G S

S

P

P

S

P

A

DNA Polymerase Adds New Nucleotides

T

G S

S

P

P

S

P

A

S

P

A

CS

P

S

P

T

T S

P

G S

P

S

P

A

S

P

A

CS

P

S

P

T

Are the two copies of DNA the same?

Why would it be important for the two copies of DNA to be the same?

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 strand

Okazaki 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 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

Length of DNA

The length of the DNA from one cell is– 3 meters

"Unravel your DNA and it would stretch from here to the moon"

DNA PackingDNAdoublehelix(2-nmdiameter

Histones“Beads ona string”

Nucleosome(10-nm diameter)

Tight helical fiber(30-nm diameter) Supercoil

(200-nm diameter)

Metaphase chromosome

700nm

Nucleosomes “Beads on a string”

– 1st level of DNA packing– histone proteins

• 8 protein molecules• positively charged amino acids • bind tightly to negatively charged DNA

8 histone molecules

DNA packing as gene control Degree of packing of DNA regulates transcription

– tightly wrapped around histones • no transcription

• genes turned off heterochromatindarker DNA (H) = tightly packed

euchromatinlighter DNA (E) = loosely packed

H E

The End!