For quite some time scientists have been interested in chromosomes
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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?

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For quite some time scientists have been interested in chromosomes

For quite some time, scientists have been interested in chromosomes

  • WHY???


Chromosomes

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

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

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

Mixture of heat-killed

S cells and living

R cells

Heat-killed

S cells (control)

Living R cells

(control)

Living S cells

(control)

LE 16-2

RESULTS

Mouse dies

Mouse healthy

Mouse healthy

Mouse dies

Living S cells

are found in

blood sample


Le 16 3

Phage

head

LE 16-3

Tail

Tail fiber

DNA

100 nm

Bacterial

cell


Figure 16 2b the hershey chase experiment

Figure 16.2b The Hershey-Chase experiment


The race to discover the structure of dna

The Race to discover the structure of DNA

  • Watson and Crick

  • Chargaff

  • Pauling

  • Wilkins and Franklin


Figure 16 01

Figure 16-01


Le 16 6

LE 16-6

Franklin’s X-ray diffraction

photograph of DNA

Rosalind Franklin


X ray diffraction insights

X-ray diffraction insights

  • Double helix with a uniform width of 2nm

  • Purine and pyrimidine bases stacked .34 nm apart

  • Helix makes a turn every 3.4 nm

  • 10 layers of nitrogen bases every turn of the helix


Le 16 un298

Purine + purine: too wide

LE 16-UN298

Pyrimidine + pyrimidine: too narrow

Purine + pyrimidine: width

consistent with X-ray data


The birth of genetics and genetic engineering

The Birth of Genetics and Genetic Engineering


The double helix paper

The “Double Helix” paper

  • A copy is posted on Angel-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 5

Sugar–phosphate

backbone

Nitrogenous

bases

5 end

Thymine (T)

LE 16-5

Adenine (A)

Cytosine (C)

Phosphate

DNA nucleotide

Sugar (deoxyribose)

3 end

Guanine (G)


Le 16 8a

LE 16-8a

Sugar

Sugar

Thymine (T)

Adenine (A)


Le 16 8b

LE 16-8b

Sugar

Sugar

Cytosine (C)

Guanine (G)


Le 16 7b

5 end

Hydrogen bond

3 end

LE 16-7b

3 end

5 end

Partial chemical structure


Le 16 7a

1 nm

3.4 nm

LE 16-7a

0.34 nm

Key features of DNA structure


Le 16 7c

LE 16-7c

Space-filling model


Structure answers a question of function

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

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

LE 16-9_2

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

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 3

LE 16-9_3

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.

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.


Experimental evidence for semi conservative replication

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

Second

replication

First

replication

Parent cell

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

LE 16-10

Semiconservative model. The two strands of the parental

molecule

separate, 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.


Le 16 10a

Second

replication

First

replication

Parent cell

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

LE 16-10a


Le 16 10b

Second

replication

First

replication

Parent cell

Semiconservative model. The two strands of the parental

molecule

separate, and

each functions

as a template for synthesis of a

new, comple-mentary strand.

LE 16-10b


Le 16 10c

Second

replication

First

replication

Parent cell

Dispersive model. Each strand of both daughter molecules contains

a mixture of

old and newly synthesized

DNA.

LE 16-10c


Figure 16 9 the meselson stahl experiment tested three models of dna replication layer 4

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

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

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

Consideration #1-DNA must unwind prior to replication

  • DNA helicase (unwindase)

  • Topoisomerase (relieves twisting)

  • Single strand binding proteins


Consideration 2 speed of replication

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

Template strand

5¢ end

3¢ end

5¢ end

3¢ end

Sugar

Base

LE 16-13

Phosphate

DNA polymerase

3¢ end

3¢ end

Pyrophosphate

Nucleoside

triphosphate

5¢ end

5¢ end


Le 16 12

Parental (template) strand

0.25 µm

Origin of replication

Daughter (new) strand

LE 16-12

Replication fork

Bubble

Two daughter DNA molecules

In this micrograph, three replication

bubbles are visible along the DNA

of a cultured Chinese hamster cell

(TEM).

In eukaryotes, DNA replication begins at may sites

along the giant DNA molecule of each chromosome.


Consideration 3 accuracy

Consideration #3-Accuracy

  • DNA polymerase has “proofreading capabilities”-mismatch repair


Consideration 4 limitations of dna polymerase

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

Primase joins RNA

nucleotides into a primer.

3¢

Template

strand

LE 16-15_1

Overall direction of replication


Le 16 15 2

Primase joins RNA

nucleotides into a primer.

3¢

Template

strand

DNA pol III adds

DNA nucleotides to

the primer, forming

an Okazaki fragment.

RNA primer

LE 16-15_2

Overall direction of replication


Consideration 4 limitations of dna polymerase continued

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

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

Leading strand

Okazaki

fragments

Lagging strand

LE 16-14

DNA pol III

Template

strand

Leading strand

Lagging strand

Template

strand

DNA ligase

Overall direction of replication


Le 16 15 11

Primase joins RNA

nucleotides into a primer.

3¢

Template

strand

LE 16-15_1

Overall direction of replication


Le 16 15 21

Primase joins RNA

nucleotides into a primer.

3¢

Template

strand

DNA pol III adds

DNA nucleotides to

the primer, forming

an Okazaki fragment.

RNA primer

LE 16-15_2

Overall direction of replication


Le 16 15 3

Primase joins RNA

nucleotides into a primer.

3¢

Template

strand

DNA pol III adds

DNA nucleotides to

the primer, forming

an Okazaki fragment.

RNA primer

After reaching the

next RNA primer (not

shown), DNA pol III

falls off.

LE 16-15_3

Okazaki

fragment

Overall direction of replication


Le 16 15 4

Primase joins RNA

nucleotides into a primer.

3¢

Template

strand

DNA pol III adds

DNA nucleotides to

the primer, forming

an Okazaki fragment.

RNA primer

After reaching the

next RNA primer (not

shown), DNA pol III

falls off.

LE 16-15_4

Okazaki

fragment

After the second fragment is

primed, DNA pol III adds DNA

nucleotides until it reaches the

first primer and falls off.

Overall direction of replication


Le 16 15 5

Primase joins RNA

nucleotides into a primer.

3¢

Template

strand

DNA pol III adds

DNA nucleotides to

the primer, forming

an Okazaki fragment.

RNA primer

After reaching the

next RNA primer (not

shown), DNA pol III

falls off.

LE 16-15_5

Okazaki

fragment

After the second fragment is

primed, DNA pol III adds DNA

nucleotides until it reaches the

first primer and falls off.

DNA pol I replaces

the RNA with DNA,

adding to the 3¢ end

of fragment 2.

Overall direction of replication


Le 16 15 6

Primase joins RNA

nucleotides into a primer.

3¢

Template

strand

DNA pol III adds

DNA nucleotides to

the primer, forming

an Okazaki fragment.

RNA primer

After reaching the

next RNA primer (not

shown), DNA pol III

falls off.

LE 16-15_6

Okazaki

fragment

After the second fragment is

primed, DNA pol III adds DNA

nucleotides until it reaches the

first primer and falls off.

DNA pol I replaces

the RNA with DNA,

adding to the 3¢ end

of fragment 2.

DNA ligase forms a

bond between the newest

DNA and the adjacent DNA

of fragment 1.

The lagging

strand in the region

is now complete.

Overall direction of replication


Le 16 16

Overall direction of replication

Lagging

strand

Leading

strand

Origin of replication

LE 16-16

Leading

strand

Lagging

strand

OVERVIEW

DNA pol III

Leading

strand

DNA ligase

Replication fork

DNA pol I

Primase

Lagging

strand

Parental DNA

DNA pol III

Primer


Figure 16 15 the main proteins of dna replication and their functions

Figure 16.15 The main proteins of DNA replication and their functions


Repair of damaged dna

Repair of Damaged DNA

  • Environmental factors including UV radiation can damage DNA

  • DNA polymerase can repair damage (excision repair)


Le 16 17

A thymine dimer

distorts the DNA molecule.

A nuclease enzyme cuts

the damaged DNA strand

at two points and the

damaged section is

removed.

LE 16-17

Nuclease

Repair synthesis by

a DNA polymerase

fills in the missing

nucleotides.

DNA

polymerase

DNA

ligase

DNA ligase seals the

free end of the new DNA

to the old DNA, making the

strand complete.


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