 Max Perutz-Medical Research Council (MRC)

What is the genetic material?

Griffiths- 1928- discovery of transformation
 Two strains of Streptococcus pneumonia: IIIS-smooth coat and IIR- rough coat,
polysaccharides

 How did these strains act?

 Live S strain in the mice: kill the mouse, live S from the bloodstream
 R strain: mouse lives, live R from the bloodstream
 Heat treated S: mouse lives, didn't get anything out of it
 R + heat treated S: mouse dies, live S from the bloodstream
 What is the transforming principle?

Avery, MacLeod & McCarty-1944-Studied Griffith's transforming principle

 Control: R strain + S strain extract-no components destroyed, mouse dies
 Polysacchardies destroyed, lipids destroyed, RNA destroyed, protein destroyed: no
effect
 DNA destroyed: mouse lives
 DNA is the transforming principle
Hershey-Chase waring blender experiment 1952: proof
 Proof that dna is the genetic material
 Waring blender experiment using T2 bacteriophage and bacteria
 Bacteriophage: made of Dna and protein
 Bacteriophage will attach to the surface of a cell, inject Dna into the cell and
take over the cell. Phage Dna replicates; new phage proteins are made. Phage
particles assemble. Cell bursts, releasing new phage.
 Experiment:
 Radioactive labels for 32P for DNA and 35S for Protein
 treat bacterial cells with phage. Let them go for 10 mins so DNA is injected
 Blender and centrifuge: what's in the bacterial cells that end up going on to
produce phage
 Most of the radioactivity for 32P labeled phages ended up inside the bacterial
cells, indicating that the phage DNA entered the cells (in pellet)
 35S-labeled phages, most of the radioactivity ended up in the phage ghosts
indicating that the phage protein never entered the bacterial cell
The function of the genetic material
 Replication-store information & accurately transmit to offspring
 Gene expression-development & adult functioning
 Mutation-must undergo sufficient variation to allow organism to adapt to
environment
What is the Structure of the DNA molecule?
 The information available to Watson and Crick
 Nucleotides with sugar ring and phosphate and cyclic nitrogen containing base
and there were 4 bases
 Purine: adenine and guanine, double-ring structure
 Pyrimidine: cytosine & thymine, single-ring structure
 Ex: naming: deoxyadenoine 5'-monophophate
 Chargaff's rule: every organism has characteristic amount of A=T and G=C &
always T+C=A+G, pyrimidines=purines
 Between organisms ratio is different (characteristic of the organism)
 Within one organism (human) ratio is the same in different tissues
 Rosalind Franklin's X-ray Diffraction picture B-DNA (wet DNA)
 Crick's contribution: he knew that it was a double helix
 Watson's contribution: base pairing, knew that the structures of the bases would be
the keto forms of the bases not the enol forms

What were others thinking?

 Pauling's triple helix of DNA: bases are on the outside

Watson & Crick structure of DNA

  Put the bases on the inside and phosphate groups on the outside
 DNA is a double helix
 Strands are anti-parallel with a sugar phosphate backbone on the outside and pairs of
bases in the middle
 Strands go to 5' to 3', antiparallel
 Two chains are held together by hydrogen bonds between
 AT and GC base pairs
 Highly regular structure: Two strands wrapped around each other every 3.4
angstroms, once every 10 pairs
 The bases are perpendicular so the weak forces are also holding the bases
together
 AT has 2 hydrogen bonds and GC has 3 hydrogen bonds
 It's easier to pull apart AT rich DNA
 Origins of replication are AT rich
 Purine + pyrimidine: thickness compatible with X-ray data
 5' to 3' directionality
 The backbone of each strand has alternating phosphate and deoxyribose sugars
connected by phosphodiester linkages
 A phosphodiester linkage connects the 5' carbon of one deoxyribose to the 3'
carbon of the adjacent deoxyribose
 5' end phosphate group at one end
 Weak wander walls forces b/w the bases
 Very hydrophobic
 Bases are stacked on top of one another and the stacking adds to the stability of
the DNA molecule by excluding water molecues from the spaces b/w the base
pairs. The most stable form results from a double helix with two distinct sizes of
grooves running in spiral
 Major groove and minor groove
 Most DNA-protein associations are in major grooves
 DNA is a right-handed helix
 DNA fulfilled 3 requirements for a hereditary substance
 Genetic material might determine the structure of proteins, genetic code
 Mutations
 Replication
 Ended their paper with a hypothesis of semi-conservative model for DNA replication

Semi conservative model for Dna replication

 Strands are split apart to serve as templates
 Two strands replicated simultaneously
 Original double helix, strands separate, complementary bases align opposite
templates, enzymes link sugar-phosphate elements of aligned nucleotides into a
continuous new strand
Three models for DNA replication: tested by Meselson & Stahl 1958
 Semiconservative replication: DNA is replicated so that the each daughter cell has one
new strand and one old strand
 Conservative replication: two parent strands stay together and two new strands are
the daughter cells
 Dispersive replication: when you make the new strands, old and new
 Experiment
 Idea: allow parental DNA molecules containing nucleotides of one density to
replicate in medium containing nucleotides of different density
 Grew bacteria in 15N heavy nitrogen (blue)
 Diluted into 14N light nitrogen medium (yellow)
 The bases have nitrogen so the bases would be labeled
 At different time points after DNA replication (20 mins for ecoli to replicate
DNA) they chowed the reaction and isolated the DNA and spun the DNA
through a CsCl density gradient and determine its relative density through
generations
 Cesium chloride gradient centriguation: establishes a gradient of ions in the
tube with the highest density at the bottom. DNA centrifuged with the cesium
chloride form sa band at a position identical with its density in the gradient
 Semiconservative
 1st generation: two strands, one will be heavy and one will be light
 2nd generation: one mixed, one light
 Conservative
 1st generation: one will be heavy and one will be light
 Dispersive
 1st generation: each progeny strands will be 50/50
 2nd generation: would get lighter and lighter
Replication forks: Also predicted by Watson & Crick
 Places where two strands are separated and copied
 John Cairns: allowed replicating DNA in bacteria to incorporate tritiated thymidine-3H
thymidine-
 Each daughter molecule should have one radiactive/hot and one nonradioactive/cold
strand
 Isolated DNA that was radioactive and did a film on it, developed a picture of the
location of 3H in the cell material
 Chromosome after one round of replication: circle
 Chromosome during second round of replication: new strand is pulled out, two forks
of DNA replication
 The newly replicated double helix that crosses the circle consists of two
radioactive strands

Arthur Kornberg: RNA Pol I



 He purified DNA polymerase from E.coli and reconstituted DNA replication in vitro
 Took liters of E.coli
 Made an extract, removed DNA bc he hypothesized that a protein could do this
 Protein purification
 Assay
 Buffer
 The enzyme needs magnesium
 Deoxy triphosphates are needed to make DNA
 Need a DNA template
 Need a DNA primer
 Then put in the enzyme
 Make radioactive polymer

What replicates DNA- an enzyme named DNA polymerase

 DNA polymerase: adds deoxy ribonucleotides to the 3' end of a growing chain
 Substrates: dATP, dGTP, dCTP, and dTTP.
 The addition of each base to the growing polymer is accompanied by the removal of
2/3 phosphates in the form of pyrophosphate Ppi.
 The energy produced by cleaving this high energy bond and the hydrolysis of
pyrophosphate helps drive the endergonic process of building a DNA polymer
 DNA polymerase reads the strand 3' to 5'
  3 critical activities of DNA polymerase
 5' to 3' polymerase works on both strands, leading and lagging
 5' to 3' exonuclease activity, removes RNA primers
 3' to 5' exonuclease activity, proofreads
Details of Dna replication
 If replication only occurs 5' to 3', how are both strands replicated in same direction?
 One strand is made continuously: leading strand
 Lagging strand: made of pieces ligated together
 Need a 3' hydroxyl to start Dna replication

 The replication fork


Helicase Unwrap the helix, uses energy of ATP
Single stranded stabilize the unwound single stranded DNA helix, and keep the
binding proteins helix open for dna polymerase
Primase Dna dependent RNA polymerase, can make the 3' hydroxyl, a
distributive enzyme (falls off quickly)
DNA polymerase III Makes the strand (leading strand is continuous)
DNA polymerase I Removes the RNA primers with its 5' to 3' exonuclease activity
and fills in the gaps with its 5' to 3' polymerase activity
 RNA primer is more likely than DNA to contain errors bc
primase doesn't have proofreading funcition so RNA must
be removed!!
 Only after RNA primer is gone does DNA pol I catalyze DNA
synthesis to replace the primer
DNA ligase Joins the 3' end of the gap-filling DNA to the 5' end of the
downstream Okazaki fragment.
 Formation of a phosphodiester bond b/w the 5' phosphate
end of one fragment and the adjacent 3'-OH group of
another fragment


 Dna replisome: 2 subunits of DNA polymerase and the lagging strand is moved around
 Multiple different polymerases at the replication fork

Okazaki 1968
 Proved 1 strand made in smaller fragments that then become sewn together
 Smaller pieces of lagging strand called Okazaki fragments
 Slowing down and look at size of DNA, very high in molecular weight
 Label dna and stop the reaction really fast
 Always one high strand of DNA: leading strand
 Then smaller pieces
 Pulse chase experiment: proved lagging strand was made in pieces
 Mutation in DNA ligase: lagging strand will always be in pieces, he saw that you have
one set of small pieces and the other leading strand

Scientific concerns about Kornberg's Dna polymerase being the replicative enzyme (NZ)
 Very abundant (if only required for DNA replication, why do you need so much of it?)
 slow (20 nucleotides/sec)
 not processive (continuous enzymatic synthesis without dropping substrate)
 Drops off DNA after 20-50 nucleotides (not a very efficient enzyme)