2006-2007

DNAAND ITS ROLE IN HEREDITY

DNA is the genetic material:a short history

- DNA was found in the nucleus by Miescher (1868)

• Early in the 20th century, the search for genetic material led to DNA– T. H. Morgan’s group (1908): genes are on chromosomes– Frederick Griffith (1928): experiments on S. pneumoniae– Oswald Avery (1944): confirmed Griffith’s experiments– Hershey and Chase (1952): DNA is the genetic material– Erwin Chargaff (1947): The amount of thymine = adenine – Watson and Crick (1953): Structure of DNA– Rosalind Franklin (1951): X-Ray Structure of DNA– Meselson and Stahl (1958): DNA Replication

DNA was found in chromosomes using dyes that bind specifically to DNA.

Oswald Avery Maclyn McCarty Colin MacLeod

Avery, McCarty & MacLeod

• Conclusion– first experimental evidence that DNA was the genetic material

injected protein into bacteria- no effect

injected DNA into bacteria- transformed harmless bacteria into virulent bacteria

Evidence That Viral DNA Can Program Cells

• Bacteriophages (or phages), are viruses that infect bacteria

T2 Phage

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

DNA Structure Reflects Its Role as the Genetic Material

• After identifying DNA as the genetic material, scientists hoped to answer two questions about the structure:

1. How is DNA replicated between cell divisions?

2. How does it direct the synthesis of specific proteins?

Structure of DNA• How does the structure of DNA account for its role

in genetic inheritance?• Maurice Wilkins and Rosalind Franklin – used X-ray

crystallography to study molecular structure

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

DNA STRUCTURE

Erwin Chargaff reported (1947) that DNA composition varies from one species to the next.

Chargaff’s rules state that in any species there is an equal number of A and T bases, and an equal number of G and C bases

Hydrogen bond 3 end

5 end

3.4 nm

0.34 nm3 end

5 end

1 nm

Watson and Crick

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width consistent with X-ray data

Watson and Crick reasoned that the pairing was specific, dictated by the base structures

DNA in the Nucleus and in the Cell Cycle

DNA Is a Double Helix

Base Pairs in DNA Can

Interact with Other

Molecules

Cytosine (C)

Adenine (A) Thymine (T)

Guanine (G)

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

Models of DNA Replication

• Alternative models– become experimental predictions

conservative semiconservative dispersive

1

2

P

DNA Replication

• Semiconservative replication• Each half of the double helix

acquires a new mate• Each new DNA molecule,

then, is really half old and half new

DNA has directionality

• 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

OPO

C

O–O

CH2

1

2

4

5

1

2

3

3

4

5

5

Each New DNA Strand Grows by the Addition of Nucleotides to Its 3 End ′

DNA Replication • Large team of enzymes coordinates replication

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

Fig. 16-13

Topoisomerase

Helicase

PrimaseSingle-strand binding proteins

RNA primer

55

5 3

3

3

Replication: 1st step

Fig. 16-13

Topoisomerase

Helicase

PrimaseSingle-strand binding proteins

RNA primer

55

5 3

3

3

Replication: 2nd step

Primase starts an RNA chain from scratch - adds RNA nucleotides one at a time using the parental DNA as a template- 3 end serves as the starting point for the new DNA strand

DNAPolymerase III

Replication: 2nd step Build daughter DNA strand

add new complementary bases to the 3’ end of the RNA primer

DNA polymerase III

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

DNA polymerase

What is driving polymerization?

Fig. 16-16a

OverviewOrigin of replication

Leading strand

Leading strand

Lagging strand

Lagging strand

Overall directions of replication

12

DNA Replication Animation

Fig. 16.16

• The lagging strand is copied away from the fork in short segments, each requiring a new primer.

• To summarize, at the replication fork, the leading stand is copied continuously into the fork from a single primer.

Template strand

5

53

3

The Lagging Strand: A Closer Look

Template strand

5

53

3

RNA primer 3 5

5

3

1

DNA Pol III works in the direction away from the replication fork

Template strand

5

53

3

RNA primer 3 5

5

3

1

13

35

5

Okazaki fragment

Okazaki

Template strand

5

53

3

RNA primer 3 5

5

3

1

13

35

5

Okazaki fragment

12

3

3

5

5

Okazaki

Template strand

5

53

3

RNA primer 3 5

5

3

1

13

35

5

Okazaki fragment

12

3

3

5

5

12

3

3

5

5

Okazaki

Template strand

5

53

3

RNA primer 3 5

5

3

1

13

35

5

Okazaki fragment

12

3

3

5

5

12

3

3

5

5

12

5

5

3

3

Overall direction of replication

Okazaki

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

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

Fig. 16-20

1 µm

Correcting Mistakesmore than 130 DNA repair enzymes have been identified in

humans

• An enzyme detects something wrong in one strand of the DNA and removes it

• Then DNA polymerase copies the information in the intact second strand and creates a new stretch of DNA

• DNA ligase seals the gap

Nobel Prize in Chemistry 2015 Interview

Sunburn Damages DNA

Nucleotide excision repair

Nuclease – a DNA cuttingenzyme

• In mismatch repair, repair enzymes fix incorrectly paired nucleotides.– A hereditary defect in

one of these enzymesis associated with a form of colon cancer.

Ghosts of Lectures Past

Frederick Griffith The “Transforming Principle”

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.

Semiconservative replication• Meselson & Stahl

– label “parent” nucleotides in DNA strands with heavy nitrogen = 15N

– label new nucleotides with lighter isotope = 14N

“The Most Beautiful Experiment in Biology”

1958

parent replication

15N parent strands

15N/15N

conservative semiconservative dispersive

1

2

P

1

P

2

DNA: Count the Carbons!

3’

5’

3’5’

3’

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 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