dna structure & replication bio 12 e. mcintyre 1

DNA Structure & Replication

Bio 12E. McIntyre

1

Historical Events

1869 Friedrich Miescher identified DNA, which he called nuclein, from pus cells

1889 Richard Altman renamed nuclein nucleic acid

1928 Griffith discovered that genetic information could be passed from one

bacteria to another; known as the transforming principle

1944 Avery showed that the transforming material was pure DNA not protein,

lipid or carbohydrate.

1952 Hershey and Chase used bacteriophage (virus) and E. coli to show that only viral DNA entered the host

1953 Watson and Crick discovered the structure of DNA was a double helix 2

Friedrich Miescher-1869

• Swiss chemist• Isolated the nuclei from pus cells.• Discovered a chemical that didn`t act like

protein. He named it “nuclein”

• Many chemists continued his work• Found that nuclein was rich in

phosphorus and contained no sulfur (unlike proteins)

• Found that nuclein contained an acidic substance they termed “nucleic acid”

• Found 2 types of nucleic acids: DNA & RNA

3

Walter Sutton-1902

• Suggested that “genes” are located on chromosomes.

• Noticed that “genes” are inherited in the same fashion as chromosomes.

Thomas Hunt Morgan-1910• Stated that each “gene” had a locus on a

particular chromosome. • Used Drosophila melanogaster (fruit fly) for

his studies. 4

Hit the PAUSE button…

• At this point, the idea of “genes” was an accepted notion. It was a very abstract idea: gene = trait that can be passed on.

• DNA had been discovered. Chromosomes had been discovered.

• Nobody had yet made the connection between genes & DNA.

5

BIG questions remained…

• What IS the genetic material? What molecular substance is a “gene” made of?

• Scientists agreed that—no matter what substance genes were made of—this substance must be:

1. able to store information

2. stable so that it can be copied and passed on

3. able to undergo rare changes called mutations in order for evolution to occur

6

Frederick Griffith-1931• Giffith was trying to make a vaccine to prevent pneumonia infections in the

"Spanish flu" influenza pandemic by using two strains of the Streptococcus pneumoniae bacterium.

• The smooth strain (S strain) had a polysaccharide capsule and was virulent .• The rough strain (R strain) had a no polysaccharide capsule and was avirulent

• The capsule is a slimy layer on the cells' surface that allows the bacteria to resist the human immune system. The rough strain (R strain) did not cause pneumonia when injected into mice (it was avirulent), since it lacked a capsule.

7

Oswald Avery & associates-1944• Griffith’s experiment led Avery to design

his own experiments to identify the “transforming agent.”

• Avery’s evidence showed:1. DNA from S strain bacteria caused

R strain bacteria to be transformed.2. Enzymes that degrade proteins and

RNA did not prevent transformation.3. Enzymes that digest DNA did prevent transformation.4. The DNA segment that transformed

the bacteria contained about 1600 nucleotides—enough for genetic variability to be possible.

8

So Avery had proven that the genetic material was DNA, right?

• Nope. Some folks still didn’t buy it.

• The skeptics replied that maybe DNA was just used to activate protein-based genes.

• In the 1950s, bacteriophages were beginning to be used as scientific tools.

• Scientists of the day were still asking: What does the virus inject into the bacteria? That’s where we’ll find the answer to our question:

• Is protein or DNA the genetic material??

9

Alfred Hershey/Martha Chase-1952

Note: DNA contains phosphorus but no sulphurProtein contains sulphur but no phosphorus 10

Hershey & Chase experiment

http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter14/animations.html

Hershey & Chase’s conclusion?

DNA—not protein—is the genetic material! DNA transmits all the genetic

information needed to produce new viruses.

11

Case Closed! Well, not quite…

• After almost 100 years, scientists had finally proven that DNA is the material that genes are made of.

• But they only had a foggy idea of what DNA really was.

• What was its structure?

12

Since Friedrich Mieschner discovered “nuclein” in 1869, chemists had been working to figure out its chemical composition

= 5 carbon sugar

Chemists knew DNA was a polymer made of nucleotide monomers.

They also knew that each nucleotide consisted of:

1.a phosphate group

2. a 5-carbon sugar

3.One of 4 nitrogen-containing bases

13

Nucleotides are classified into 2 groups based on structure:

1. Purinescontain these bases…– Adenine– Guanine

2. Pyrimidinescontain these bases… – Cytosine– Thymine– Uracil (RNA only)

14

Study tip: small name = BIG BASE STRUCTURE (2 RINGS)

Purine Nucleotides

15

Study tip: BIG NAME = small base structure (one ring)

Pyrimidines Nucleotides

16

Erwin Chargaff-1940s• Remember each nucleotide

has one of four possible bases, A, T, C & G

• Chargaff analyzed DNA from various species.

• Conclusions of his experiments came to be known as “Chargaff’s Rules”

1. What can you say about the relative amounts of A, T, C & G in different species

2. What can you say about the relative amounts of A, T, C & G in a given species

17

Erwin Chargaff-1940s• Remember each nucleotide

has one of four possible bases, A, T, C & G

• Chargaff analyzed DNA from various species.

• Conclusions of his experiments came to be known as “Chargaff’s Rules”

1. What can you say about the relative amounts of A, T, C & G in different speciesThe amount of A, T, G and C in DNA varies from species to species

2. What can you say about the relative amounts of A, T, C & G in a given speciesIn each species, the amount of A = T and the amount of G = C are the same 18

Suppose you were Chargaff who observed the ratios of A, T, C & G we just discussed. Use deductive reasoning to determine how the bases pair up.

Stop & Think

19

•A pairs with T and G pairs with C.

•These rules were presented before scientists knew the structure of DNA. The rules would only be useful in the next decade, when the structure was more certain.

Stop & Think

Suppose you were Chargaff who observed the ratios of A, T, C & G we just discussed. Use deductive reasoning to determine how the bases pair up.

20

Chargaff’s Rules

• DNA from any cell of all organisms should have a 1:1 ratio of pyrimidine and purine bases.

• The amount of guanine is equal to cytosine & the amount of adenine is equal to thymine. This pattern is found in both strands of the DNA.

Rosalind Franklin-1953• An expert in X-ray crystallography. Used this technique to

discover the physical shape of DNA!

• This was the first indication that DNA was composed of a

double helix structure which had a constant diameter of 2nm.

22

James Watson & Francis Crick-1953

• Constructed a model for DNA structure

• Awarded the Nobel Prize in 1962• Rosalind Franklin’s work was

essential to the Watson and Crick model. She died of cancer due to overexposure to x-rays. Her lab partner, Maurice Wilkins was also awarded the Nobel Prize along with Watson & Crick because of his contributions to Ms. Franklin’s work.

23

Watson & Crick’s model of DNA structure

• The rules of complementary base pairing (or nucleotide pairing) are:• Purines are always paired with

pyrimidines but only in certain combinations…

• A with T: the purine adenine (A) always pairs with the pyrimidine thymine (T)

• C with G: the purine guanine (G) always pairs with the pyrimidine cytosine (C)

24


• The 2 strands of DNA are held together by the hydrogen bonds between the complimentary bases.

25

Stop & Think

So why can`t…

A (purine) pair with C (pyrimidine)

or

G (purine) pair with T (pyrimidine)?

26

Stop & Think

So why can`t…

A (purine) pair with C (pyrimidine)

or

G (purine) pair with T (pyrimidine)?

• A & T form double hydrogen bonds while…

• C & G form triple hydrogen bonds

• So… an A-C or G-T pairing won`t work 27

Stop & Think

Why can`t …

A (purine) pair with G (purine)

or

C (pyrimidine) pair with T (pyrimidine)?

28

Stop & Think

Why can`t …

A (purine) pair with G (purine)

or

C (pyrimidine) pair with T (pyrimidine)?

There is not enough space for two

purines to fit within the helix and too

much space for two pyrimidines to get

close enough to each other to form

hydrogen bonds between them.

29


Space-filling model

30

What`s this?

31

What`s this?

32

Schematic axial view of DNA

Prokaryotic DNA

• Where is the DNA found in prokaryotes?

• In the cytoplasm!

• What shape is the DNA found in?• A circle. (ends are connected together.)

33

Eukaryotic DNA

• Where is the DNA found in a eukaryote?

• In the nucleus.

• Human Cell: Nucleus

has ~1 meter of DNA!

34

DNA Structure• DNA– Double Helix

• Histones– Proteins

• Nucleosome– DNA coils around histones.

• Coils• Supercoils• Chromosome

35

DNA Packaging

36

Linked Nucleotides in DNA

5

• Nucleotides are linked by phosphodiester bonds.

• Hydroxyl groups from the 3rd carbon and a phosphate group (off 5th carbon) of two nucleotides react to form water.

• A phosphodiester bond is a type of condensation reaction

Strands of DNA are Antiparallel

• The strands in a DNA molecule run antiparallel to each other.

• The two strands in a DNA molecule run antiparallel to each other (the two strands have opposite orientations; the 5' end of one strand aligns with the 3' end of the other strand.

• 3’ and 5’ pertain to the 3rd and 5th carbons in the deoxyribose molecules.

That wraps up one mystery!

But in science, new information almost always raises new questions… 39

Now we know “what”… but how?

• What is the genetic material? –DNA!

• How is it passed on to the next generation? How is it copied?

40

DNA Replication & Repair

41

DNA Replication

• When does a cell replicate its DNA?

42

DNA Replication

• When does a cell replicate its DNA?

• During the S-Phase of Interphase.

43

DNA Replication-The Basics1. ‘parent’ DNA

molecule separates into 2 strands. (Unzips by breaking hydrogen bonds)

2. Produces 2 new complementary strands following base-pairing rules.

3. Backbone formation in daughter strands

44

DNA Replication

• Watson & Crick’s model of DNA suggested how DNA could replicate; by breaking hydrogen bonds allowing each strand to act as a template to form new complimentary strands.

45

Meselson and Stahl (1958)

46Meselson & Stahl Experiment

Meselson and Stahl conducted an experiment to determine the mode of DNA replication…


47

Meselson & Stahl Experiment

14N & 15N are isotopes of nitrogen (light & heavy respectively)

What do you think is the mode of DNA replication? • semiconservative, • conservative or • dispersive?


48

Meselson & Stahl Experiment

14N & 15N are isotopes of nitrogen (light & heavy respectively)


DNA Replication • Since DNA replication is semiconservative, therefore the helix must be

unwound.

• John Cairns (1963) showed that initial unwinding is localized to a region of the bacterial circular genome, called an “origin” or “ori” for short.

• DNA replication is semiconservative. Each strand of both replication forks is being copied.

• DNA replication is bidirectional. Bidirectional replication involves two replication forks, which move in opposite directions

Summary of DNA Replication

• The topoisomerases introduce negative supercoils and relieve strains in the double helix at either end of the bubble.

• The helicase unwinds and unzips the DNA helix by breaking the Hydrogen bonds between the base pairs, thus allowing the two strands to separate.

• The SSB proteins (Single Strands Binding) stabilize the single strands thus preventing them to zip back together and to form hairpin loops.

• The primase (RNA polymerase) added to other proteins (forming a Primeosome) makes short pieces of RNA (RNA primers) that are recognised by DNA polymerase III to initiate replication.

• Following the DNA pol III, the DNA pol I removes the RNA primers, and fills in with DNA. Finally, the DNA ligases repair the gaps between DNA Okazaki fragments.

•

50

• Strand SeparationStrand Separation:1.1. TopoisomeraseTopoisomerase: enzymes which relieves relieves stressstress on the DNA moleculeDNA molecule by allowing free rotation around a single strand. Gyrase is a type of topoisomerase.

Enzyme

DNA

Enzyme

DNA Replication: Exposing the TemplatesDNA Replication: Exposing the Templates

DNA Replication Fork

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter11/animation_quiz_2.html

DNA Replication: Exposing the TemplatesDNA Replication: Exposing the Templates• Strand SeparationStrand Separation:

2.2. HelicaseHelicase: enzyme catalyze the unwindingunwinding and separationseparation (breaking H-Bonds) of the parental double helix. 3.3. Single-Strand Binding Proteins (SSBs)Single-Strand Binding Proteins (SSBs): proteins

attach and help keep the separated strands apart.



Replication Forks & BubblesReplication Forks & BubblesReplication ForksReplication Forks:

hundredshundreds of Y-shapedY-shaped regions of replicating replicating DNA moleculesDNA molecules where new strands are growing.



Replication Forks & BubblesReplication Forks & BubblesReplication BubblesReplication Bubbles:

a.HundredsHundreds of replicating bubbles (Eukaryotes)(Eukaryotes).b.SingleSingle replication fork (bacteria).(bacteria).



DNA Replication: Building the New StrandsDNA Replication: Building the New Strands• Priming:Priming:• RNA primersRNA primers: before new DNA strands can form,

there must be small pre-existing primers (RNA)primers (RNA) (typically with twenty or fewer bases) present to start the addition of new nucleotides (DNA Polymerase)(DNA Polymerase).

• PrimasePrimase: enzyme that polymerizes (synthesizes) the RNA Primer.



• Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:

1.1. DNA Polymerase IIIDNA Polymerase III: with an RNA primerRNA primer in place, DNA Polymerase III (enzyme) catalyzes the synthesis of a new DNA strand in the synthesis of a new DNA strand in the 5’ to 3’ 5’ to 3’ directiondirection. Nucleotides are added to the parent strand based on the rules of complementary base pairs; A with T, C with G.

RNARNAPrimerPrimerDNA PolymeraseDNA Polymerase

NucleotideNucleotide

5’

5’ 3’

DNA Replication: Building the New StrandsDNA Replication: Building the New Strands



Stop & Think

• Based on what you have learned about the directionality of DNA replication what problems do you see in replicating one of the strands?

57DNA Replication Fork


DNA replication is semi-discontinuous

Continuous synthesis

Discontinuous synthesisDNA Replication Fork


DNA ReplicationDNA Replication


2.2. Leading StrandLeading Strand: synthesized as a single polymersingle polymer in the 5’ to 3’ direction5’ to 3’ direction.

RNARNAPrimerPrimerDNA PolymeraseDNA PolymeraseNucleotidesNucleotides

3’5’

5’



A 3’ hydroxylgroup is necessaryfor addition of nucleotides



3.3. Lagging StrandLagging Strand: also synthesized in the 5’ to 5’ to 3’ direction3’ direction, but discontinuouslydiscontinuously against overall direction of replication.

RNA PrimerRNA Primer

Leading StrandLeading Strand

DNA PolymeraseDNA Polymerase

5’

5’

3’

3’

Lagging StrandLagging Strand

5’

5’

3’

3’


• Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:4.4. Okazaki FragmentsOkazaki Fragments: series of short segments on the lagginglagging strand strand. . Assembly of each Okazaki fragment is preceded by the addition of an RNA primer.

Okazaki FragmentOkazaki Fragment


Lagging Strand

RNARNAPrimerPrimer

DNADNAPolymerasePolymerase

3’

3’

5’

5’


• Removal of RNA primers:Removal of RNA primers:5.5. DNA Polymerase I DNA Polymerase I removes RNA primers and replaces them with the appropriate DNA nucleotides.


Lagging Strand

RNA Primer has been RNA Primer has been removed and replaced removed and replaced with nucleotides by with nucleotides by by DNA Polymerase Iby DNA Polymerase I

3’

3’

5’

5’

Replication of the Lagging Strand


DNA ReplicationDNA Replication• Synthesis of the new DNA Strands:Synthesis of the new DNA Strands:

6.6. DNA ligaseDNA ligase: a linking enzyme that catalyzes the formation of a covalent bond (a phosphodiester bond to be precise) from the 3’ to 5’ end 3’ to 5’ end of joining stands.

Example: joining two Okazaki fragments together.Example: joining two Okazaki fragments together.

Lagging Strand

Okazaki Fragment 2Okazaki Fragment 2

DNA ligaseDNA ligase

Okazaki Fragment 1Okazaki Fragment 1

5’

5’

3’

3’


Semi-discontinuous Replication

67DNA Replication Fork

Evidence points to bidirectional replication

Label at both replication forksDNA Replication Fork



6.6. ProofreadingProofreading: initial base-pairing errors are usually corrected by DNA polymeraseDNA polymerase.

DNA RepairDNA Repair

• Excision repair:Excision repair:1. Damaged segment is excisedexcised by a repair repair enzymeenzyme (there are over 50 repair enzymes).

2. DNA polymerase DNA polymerase and DNA ligase DNA ligase replace and bond the new nucleotides together.

Question:

• What would be the complementary DNA strand for the following DNA sequence?

DNA 5’-GCGTATG-3’DNA 5’-GCGTATG-3’

Answer:Answer:

DNA 5’-GCGTATG-3’DNA 5’-GCGTATG-3’DNA 3’-CGCATAC-5’DNA 3’-CGCATAC-5’

ALWAYS SHOW DIRECTIONALITYALWAYS SHOW DIRECTIONALITY

Multiple Origins in Eukaryotes

• Eukaryotes replicate their DNA only in S-phase• Eukaryotes have larger chromosomes• Replication speed 2,600 npm. • Largest Drosophila chromosome is 6.5 x 107 nucl.,

but it can replicate in 3-4 min. From a single origin, bidirectional replication would take 8.5 days. ==> The chromosome must have some 7,000 origins of replication.

73

The Central Dogma of Molecular Biology

74

DNA Replication is Uncommonly Accurate

DNA REPAIR

Thymine Dymers: Formation and Repair76

Thymine Dimers: Formation & repair

• UV radiation from sunlight can cause damage to DNA. UV radiation can cause covalent bonds to form between adjacent bonds of thymines on the same strand of DNA no replication or transcription possible. This can be repaired in two ways:

1. light repair: Photolase can break the bonds between the thymine dimers (light is required)

2. Excision repair/dark repair: An excision enzyme removes the damaged section Excised nuceotides are then replaced with nucleotides by DNA polymerase. DNA ligase forms the phosphodiester bond.

DNA REPAIR

77

Mutation by Base SubstitutionRemember: Nucleotide sequence in DNA determines Nucleotide

sequence in mRNA which determines the amino acids in proteins

• Purines and Pyrimidines occur in two structural forms.

• Most common forms result in A-T and C-G pairings.

• A tautomeric shift (change in position of a hydrogen) in a base can cause altered hydrogen bonding properties.

• A tautomeric shift in A allows it to bond to C.

• A tautomeric shift in T allows it to bond to G.

• A change in a single nucleotide is passed on to mRNA. Therefore, protein synthesis is affected.

Mutation by Base Substitution

DNA REPAIR

78

Addition & Deletion Mutation (Frameshift mutations)

• A mutation can result in an alteration to the the amino acid sequence of a protein.

• spontaneously or as the result of a mutagen

• Addition or deletion of one or more nucleotides during DNA replication. This shifts the codons in the mRNA

• This affects all codons past the site of the mutation

Addition & Deletion Mutation

79

Bacterial DNA Replication

Transcription

Protein Synthesis

DNA ligase seals nicks left by lagging strand replication

DNA Helicase Separates Strands

dna structure & replication bio 12 e. mcintyre 1

Documents

genes dna

dna rna

structure of dna

pure dna

viral dna

dna segment

r strain bacteria

s strain bacteria