for quite some time, scientists have been interested in chromosomes why???

For quite some time, scientists have been interested in chromosomes

• WHY???

Chromosomes

• They replicate prior to both mitosis and meiosis? How?

• They carry information for genetic traits (genotype determines phenotype). How?

• These are questions of function-to address these questions it seemed logical to look at the structure of chromosomes

Pre-1953-What did we know about chromosomes

• What is significant about 1953?

• Chromosomes made of DNA and protein

• Which of these molecules stored the genetic information?

• Most researchers favored protein. Why?

History-DNA or protein is the genetic material?

• Griffith-1928

• Avery, McCloud, McCarty-1944

• Hershey and Chase-1952

• Conclusion-DNA was the genetic information in the chromosome

• To understand questions of function regarding genes-we had to know the structure of DNA

LE 16-2

Living S cells(control)

Living R cells(control)

Heat-killedS cells (control)

Mixture of heat-killedS cells and livingR cells

Mouse dies

Living S cellsare found in blood sample

Mouse healthy Mouse healthy Mouse dies

RESULTS

LE 16-3

Bacterialcell

Phagehead

Tail

Tail fiber

DNA

100

nm

Figure 16.2b The Hershey-Chase experiment

The Race to discover the structure of DNA

• Watson and Crick

• Chargaff

• Pauling

• Wilkins and Franklin

Figure 16-01

LE 16-6

Franklin’s X-ray diffractionphotograph of DNA

Rosalind Franklin

X-ray diffraction insights

1.Double helix with a uniform width of 2nm

2.Purine and pyrimidine bases stacked .34 nm apart

3.Helix makes a turn every 3.4 nm

4.10 layers of nitrogen bases every turn of the helix

LE 16-UN298

Purine + purine: too wide

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: widthconsistent with X-ray data

The Birth of Genetics and Genetic Engineering

The “Double Helix” paper

• A copy is posted on cell web site-please read it

• Major insights:

• A. DNA is a double helix

• B. The two strands are held together by hydrogen bonding between complementary base pairs (A-T) and G-C)

• DNA is antiparallel

LE 16-5Sugar–phosphate

backbone

5 end

Nitrogenousbases

Thymine (T)

Adenine (A)

Cytosine (C)

DNA nucleotidePhosphate

3 endGuanine (G)

Sugar (deoxyribose)

LE 16-8a

Adenine (A) Thymine (T)

Sugar

Sugar

LE 16-8b

Guanine (G) Cytosine (C)

Sugar

Sugar

LE 16-7b5 end

3 end

5 end

3 end

Partial chemical structure

Hydrogen bond

LE 16-7a

Key features of DNA structure

0.34 nm

3.4 nm

1 nm

LE 16-7c

Space-filling model

Structure answers a question of function

• Question-How does DNA replicate?

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

• Semi-conservative replication

LE 16-9_1

The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

LE 16-9_2

The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

The first step in replication is separation of the two DNA strands.

LE 16-9_3

The parent molecule has two complementary strands of DNA. Each base is paired by hydrogen bonding with its specific partner, A with T and G with C.

The first step in replication is separation of the two DNA strands.

Each parental strand now serves as a template that determines the order of nucleotides along a new, complementary strand.

Experimental Evidence for Semi-conservative Replication

• Just because something is logical does not mean it is true.

• Three possible mechanisms of DNA replication-

• A. Conservative

• Semi-conservative

• C. Dispersive

• Messelson and Stahl experiment

LE 16-10

Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix.

Semiconservative model. The two strands of the parental moleculeseparate, and each functions as a template for synthesis of a new, comple-mentary strand.

Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA.

Parent cellFirstreplication

Secondreplication

LE 16-10a

Conservative model. The two parental strands reassociate after acting as templates for new strands, thus restoring the parental double helix.

Parent cellFirstreplication

Secondreplication

LE 16-10b

Semiconservative model. The two strands of the parental moleculeseparate, and each functions as a template for synthesis of a new, comple-mentary strand.

Parent cellFirstreplication

Secondreplication

LE 16-10c

Dispersive model. Each strand of both daughter molecules contains a mixture of old and newly synthesized DNA.

Parent cellFirstreplication

Secondreplication

Figure 16.9 The Meselson-Stahl experiment tested three models of DNA replication (Layer 4)

DNA replication-It’s more complicated than Watson and Crick thought

• Considerations-DNA replication• 1. DNA must unwind (it’s a double helix)• 2. It’s fast (mammals-50 nucls/sec;

bacteria-500 nucls/sec).• 3.Accuracy-1 mistake/1 billion nucleotides • 4. DNA polymerase limitations-can’t

synthesize denovo; only works in 5’3’ direction

• 5. DNA is antiparallel

DNA replication proteins

• Several of the replication considerations suggest the involvement of proteins (especially enzymes) in DNA replication

Consideration #1-DNA must unwind prior to replication

• DNA helicase (unwindase)

• Topoisomerase (relieves twisting)

• Single strand binding proteins

Consideration #2-Speed of Replication

• Enzymes involved-DNA polymerase (11 forms in eukaryotes)-III is the major replicative enzyme)

• DNA replication is bi-directional

LE 16-13

New strand

5 end

Phosphate Base

Sugar

Template strand

3 end 5 end 3 end

5 end

3 end

5 end

3 end

Nucleosidetriphosphate

DNA polymerase

Pyrophosphate

LE 16-12

In eukaryotes, DNA replication begins at may sitesalong the giant DNA molecule of each chromosome.

Two daughter DNA molecules

Parental (template) strand

Daughter (new) strand0.25 µm

Replication fork

Origin of replication

Bubble

In this micrograph, three replicationbubbles are visible along the DNAof a cultured Chinese hamster cell(TEM).

Consideration #3-Accuracy

• DNA polymerase has “proofreading capabilities”-mismatch repair

Consideration #4-Limitations of DNA polymerase

• DNA polymerase can’t synthesize a new strand “denovo”-needs a free 3’ OH group to attach the next nucleotide to

• Solution-RNA primase-adds RNA primer (5-10 nucleotides)-later primer removed by a form of DNA polymerase that replaces RNA nucleotides with DNA nucleotides

• Pieces of DNA joined by DNA ligase

LE 16-15_1

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

LE 16-15_2

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Consideration #4-Limitations of DNA polymerase (continued)

• DNA polymerase only works in 5’3’ direction

• Why is this a problem?• Because of consideration #5-DNA is

antiparallel-One strand runs in the 5’3’ direction; the other runs in the 3’5’ direction

• Solution1- Is there a 3’5’ DNApolymerase? (haven’t found one yet)

Solution 2-DNA replication occurs differently on the 2 strands

• Leading strand (continuous replication)

• Lagging strand (discontinuous replication)-involvement of Okasaki fragments (approximately 200 nucleotides in length in eukaryotes).

LE 16-14

Parental DNA

5

3

Leading strand

35

3

5

Okazakifragments

Lagging strand

DNA pol III

Templatestrand

Leading strand

Lagging strand

DNA ligase Templatestrand

Overall direction of replication

LE 16-15_1

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

LE 16-15_2

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

LE 16-15_3

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

LE 16-15_4

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

LE 16-15_5

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

33

5

5

DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.

LE 16-15_6

53

Primase joins RNAnucleotides into a primer.

Templatestrand

5 3

Overall direction of replication

RNA primer3

5

35

DNA pol III addsDNA nucleotides to the primer, formingan Okazaki fragment.

Okazakifragment

3

5

5

3

After reaching thenext RNA primer (not

shown), DNA pol IIIfalls off.

33

5

5

After the second fragment isprimed, DNA pol III adds DNAnucleotides until it reaches thefirst primer and falls off.

33

5

5

DNA pol I replaces the RNA with DNA,adding to the 3 endof fragment 2.

33

5

5

DNA ligase forms abond between the newestDNA and the adjacent DNAof fragment 1.

The lagging strand in the regionis now complete.

LE 16-16

5

3Parental DNA

3

5

Overall direction of replication

DNA pol III

Replication fork

Leadingstrand

DNA ligase

Primase

OVERVIEW

PrimerDNA pol III

DNA pol I

Laggingstrand

Laggingstrand

Leadingstrand

Leadingstrand

LaggingstrandOrigin of replication

Figure 16.15 The main proteins of DNA replication and their functions

Repair of Damaged DNA

• Environmental factors including UV radiation can damage DNA

• DNA polymerase can repair damage (excision repair)

LE 16-17

DNA ligase

DNA polymerase

DNA ligase seals thefree end of the new DNAto the old DNA, making thestrand complete.

Repair synthesis bya DNA polymerasefills in the missingnucleotides.

A nuclease enzyme cutsthe damaged DNA strandat two points and the damaged section isremoved.Nuclease

A thymine dimerdistorts the DNA molecule.