dna: molecular basis of inheritanceclasses.biology.ucsd.edu/bild1.sp16/lect-15-sv.pdf · dna:...

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DNA: Molecular basis of Inheritance

Lecture 15: 05/24/16

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DNA Double Helix

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DNA: Molecular basis of InheritanceHistorical Overview!

First Isolation of DNA

DNA as genetic material

Transformation

DNA vs Protein

Chargaff’s Rules

Structure of DNA

Franklin’s X-ray Diffraction photograph of DNA

Watson and Crick – The Double Helix

---------------------------------------------------------DNA Replication: Meselson and Stahl’s Semi-conservative model.

A change in phenotype by the acquisition of external DNA

A:T and G:C composition in cells

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First Isolation of DNA

1869: Miescher isolated nuclei

White blood cells

large molecules, acidic, and rich in phosphorus

salmon sperm

1900s: scientist had determined that nucleic acids were made of:

Nuclein

Nucleic acids

5C SugarPhosphatebase

Nucleotide

AdenineGuanineThymineCytosineUracil (Only in RNA)

HO P

O

O-

O-

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DNA as genetic material

By The 1930’s universally accepted:

1928: Griffith discovered the Transformation Phenomenon

Studied the pathogenicity of Streptococcus pneumoniae

Can the genetic trait of pathogenicity be transferred between bacteria?


All cells contained DNA

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DNA as genetic material – Griffith (1928)

Can the genetic trait of pathogenicity be transferred between bacteria?


2 Strains of S. pneumoniae

Smooth (S) – have a capsule - pathogenic

Rough (R) – no capsule – non pathogenic

Inject

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Extracted all molecular material

DNA as genetic material – Avery (1930s-40s)

Isolated the TRANSFORMATION factor

Systematically remove or destroy various componenents

RNaseProteaseDNase

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DNA as genetic material – Hershey-Chase (~1952)

DNA or Protein as the genetic material of bacteriophages?

bacteriophages are viruses that infect bacteria

DNA encapsulated by proteins

Colorized TEM

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Life Cycle of bacteriophage

Inject DNA into bacteria

Hijacks bacteria replication and translation machinery to replicate its phage DNA and make head and tail proteins

Package replicated DNA

Lyse bacteria and release new bacteriophages

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32P labelled DNA

35S labelled protein

(Cys & Met a.a.)

(phosphate)

Grow phages with radioactive materials

Infect bacteriaSeparate Bacteria

and phage by mixing

Centrifuge mixture

pellet

pellet

supernatent

supernatent

DNA or Protein as the genetic material of bacteriophages to produce more phage?

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H


Structure of DNA

Phosphate

Base

5C sugar (deoxyribose)

C1

C2C3

C4

5

C1 is attached to basePurines A and G (2 rings)Pyrimidines C and T (1 ring)

C2 is missing OH C3 is reactive site (3’end or 3’OH)C5 attached to phosphate: reactive (5’end or 5’ phosphate)

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Structure of DNA

DNA is polymer of nucleotides

Condensation reaction: loss of water

H

H

5’3’

5’

3’

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Structure of DNA

Chargaff’s Rule

Diversity of species

1. All had DNA

2. Total amounts differed – BUT……

Pecentage of A and T the same“ “ G and C “ “

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Structure of DNA

Rosalind Franklin’s Famous photograph of DNA

(DNA)

Diffraction

(Collect and focus x-rays)

Diffraction pattern is directly correlated to the 3D shape of specimen

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Structure of DNA

Watson and Crick: DNA Double Helix

Very Stable – WHY?

• Base pair across the helix (H-bonding)

• Phosphate far apart

• Base – which are hydrophobic: stack atop each other (van der waals)

A T : G C

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Structure of DNA Double Helix UniformDiameter

Right-handed helix

Helix has Uniform Diameter

Strands are antiparallel

0.34 nm rise per nucleotide

3.4nm rise per turn (10 nucleotides per turn)

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A, T, G, C

This is basis for amazing genetic diversity among species – HOW?

Central DogmaDNA RNA Protein

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How is DNA replicated?

3 Hypothesis to Test?

1. Conservative Hypothesis

2. Semi Conservative Hypothesis

3. Dispersive Hypothesis

Parent DNA helix remains intact, and a 2nd new copy is made

2 strands of Parent DNA helix separate, each function as a template to make “COMPLEMENTARY” strands.

Daughter DNA helix get a mix of new DNA.

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How is DNA replicated?

Conservative

Semi-Conservative

Dispersive

DNA Replication11-17-11

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DNA Replication

A

C

T

A

G

A

C

T

A

G

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C

A

C

T

A

G

A

C

T

A

G

T

G

A

T

C

T

G

A

T

C

T

G

A

T

C

T

G

A

T

C

Figure 16.9 a–d

Parent Strand: A-T and G-C base pairs

Separation of parent strands

Each parent strand becomes a template for complementary strands

Nucleotides connected by phosphodiester bond2 new DNA molecules

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3) DNA sample centrifuged after 20 minutes (first

replication)

1) Bacteria cultures in medium containing 15N

2) Bacteria transferred to medium containing

14N

Meselson and Stahl’s Experiment

4) DNA sample centrifuged after 20 minutes (first

replication)

Less dense

More dense

: mechanism of DNA Replication

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First replication Second replication

Conservativemodel

Semiconservativemodel

Dispersivemodel

Meselson and Stahl’s Experiment: mechanism of DNA Replication

15N

15N + 14N

14N

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DNA Replication

Bacteria has 4.6 million bases pairs to replicate

Human diploid cell has 3 billion base pairs to replicate

46 (long) DNA molecules

Has to be fast and accurate

> 2000 bases per second are polymerized!!

only 1 mistake per 10 billion bases added!!

Many proteins involved!

(Polymers)

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DNA Replication

Origin of Replication - specialized site where replication begins

Easy to unwind because high number of A/T base pairs

Bacteria has 1

Eukaryotic cells - 100s to 1000s per chromosome

Replication fork - Ends of replication bubble (origin)

Helicase – special protein which unwinds DNA (uses ATP)

Single stranded binding proteins (SSBP’s) - Binds single stranded DNA and keeps it from base pairing

Replication Fork

Replication Fork

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DNA Replication

Initiation

Fusion

Lateral Expansion(Elongation)

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DNA Replication

DNA Polymerase III

catalyses the addition of deoxyribose nucleotides to the 3’end (3’OH)

dATP, dTTP, dGTP, dCTP

looks at opposite (template) strand and chooses correct base

catalyses the phosphodiester bond

energy source: energy released from PPi 2 Pi (14 kcal of energy)

high fidelity (very seldom makes a mistake) : 1 in 10 billion base additions

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DNA Replication

DNA Polymerase III

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DNA Replication

Primase – synthesizes short (5-10 bases in length) RNA primer

supplies the 3’OH needed by DNA Pol III to add bases

DNA Polymerase I - removes RNA primer and replaces it with DNA

DNA ligase – joins (ligates) DNA together to make one continuous strand

(Note: Make sure you know Table 16.1 (Campbell – 7th edition)!)

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DNA Replication

DNA replication only takes place in the 5’ 3’ direction! Why?

Polymerization only takes place at the 3’end (3’OH)

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Overall direction of replication

3

3

3

35

35

35

35

3

5

3

5

3

5

3 5

5

1

1

21

12

5

5

12

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Templatestrand

RNA primer

Okazakifragment

Figure 16.15

Primase joins RNA nucleotides into a primer.

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DNA pol III adds DNA nucleotides to the primer, forming an Okazaki fragment.

2

After reaching the next RNA primer (not shown), DNA pol III falls off.

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After the second fragment is primed. DNA pol III adds DNAnucleotides until it reaches the first primer and falls off.

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DNA pol 1 replaces the RNA with DNA, adding to the 3 end of fragment 2.

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DNA ligase forms a bond between the newest DNAand the adjacent DNA of fragment 1.

6 The lagging strand in this region is nowcomplete.

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DNA Replication : Lagging strand Synthesis11-17-11

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Helicase

SSBP

DNA Replication: Overview11-17-11

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DNA Replication: Telomeres

Dilemma: What about the ends of linear DNA?

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Dilemma: What about the ends of linear DNA?

Telomerase is the answer!

~2500 base pairs at the end of DNA is a repeatTTAGGGAATCCC

Telomerase is a protein with a built-in RNA template

AAUCCCAAU

Adds back Telomere ends

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Telomerase adds back nucleotides at the ends of DNA

Repeat again and again

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