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Cellular Anatomy
• typical cell: 1. nucleus
2. cell membrane = plasma membrane
3. cytoplasm
-cytosol
-cytoskeleton
4. cytoplasmic organelles
-membranous
-non-membranous
http://www.wisconline.com/objects/index.asp
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The Typical
Cell
TEM of a typical eukaryotic
cell
The Nucleus: Information Central
• nucleus contains most of the cell’s genes and is usually the most
conspicuous organelle
• bound by a double, phospholipid membrane = nuclear membrane
• each membrane is a phospholipid bilayer
• the nuclear envelope connects to the endoplasmic reticulum (protein synthesis)
• nuclear membrane - contains
nuclear pores = channels of
over 100 proteins
• allows entry and exit of
materials
e.g. mRNA
Nucleus
Nucleolus
Chromatin
Nuclear envelope:
Inner membrane
Outer membrane
Nuclear pore
Rough ER
Pore
complex
0.25 m
Ribosome
Pore complexes (TEM)
Close-up
of nuclear
envelope
Chromatin
• Nucleus is comprised of the
following
–1. Nucleoplasm
–2. Nucleolus
–3. Chromatin
1 m
A. Nucleoplasm:
• specialized fluid of the nucleus
• suspends the DNA in its form as chromatin
• contains a network of filaments = nuclear matrix or nuclear lamina
•matrix gives the nucleus its shape
•matrix helps organize the chromatin
•also helps in DNA replication & transcription
Nuclear lamina (TEM)
B. Nucleolus:
• “little nucleus”
• dense body DNA, RNA and protein with no defining
membrane
• site of ribosome production and rRNA synthesis
(via transcription)
•site for the assembly of rRNA with the two protein
subunits of the ribosome
•two ribosome subunits migrate out
through nuclear pores – for assembly
in the cytoplasm
•nucleolus can be very prominent in
cells that produce high amounts of
proteins (e.g. neurons)
C. Chromatin:
•loosely coiled fibers of DNA wrapped around proteins = histones
•found only in eukaryotic cells
Function of Chromatin:
1) to package DNA into a smaller volume
to fit in the cell
2) to strengthen the DNA to allow mitosis
3) to prevent DNA damage
4) to control gene expression and DNA
replication
C. Chromatin:
• described as a “beads on a string” model
histone
- the histone DNA complex is
called a nucleosome
-DNA in between nucleosomes
is called “linker DNA” – 50bp
-the entire complex is known as
the 10nm fiber or euchromatin
Chromatin Organization
-10nm fiber = euchromatin
-10nm fiber is packed further into a 30nm fiber = heterochromatin
-compacted further by forming “loops”
-eventually condenses into a chromosome
DNA
Nucleosome
Euchromatin
Heterochromatin
Further looping and condensing
Chromosome
• organization of chromatin depends on the cell cycle – i.e. the stage of cell
replication
• during interphase of the cell cycle - two forms of chromatin: euchromatin
and heterochromatin (more condensed form of chromatin)
• with cell replication - heterochromatin condenses even more ->
chromosomes
• condensing of chromatin through its various forms – regulated by specific
proteins
– e.g. protein called condensin
• chromatin arrangement is critical to DNA function
–
more condensed the less access the molecular “machinery” of gene expression has
•human karyotype
Histones
• complex of small, basic proteins
– core of 4 proteins: H2A, H2B, H3, H4
– 2 complexes = 8 total proteins per histone
– DNA wraps around this core = 10nm fiber
(euchromatin)
– plus 2 linker histones H1, H5
– linkers interact with the DNA and pack it into a
thicker 30nm fiber (heterochromatin)
• many histone amino acids are positively charged
•
•
•
so DNA-histone interaction is just an attraction between
negatively charged DNA and positively charged histones
not dependent upon DNA sequence
proteins of the histone make ionic bonds to the acidic
sugar-PO4 backbone of the DNA helix
• bonds between histone and DNA can be modified
by enzymes
•
•
•
•
e.g. methylation, phosphorylation
this modification can change the interaction between the
DNA and the histone
can make the DNA either more or less accessible to the
replication machinery
modifications can either make replication easier or harder
The Search for the Genetic Material
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early in the 20th century, the identification of the molecules of inheritance
loomed as a major challenge to biologists
when T. H. Morgan’s group showed that genes are located on chromosomes,
the two components of chromosomes—DNA and protein—became
candidates for the genetic material
key factor in determining the genetic material was choosing appropriate
experimental organisms
role of DNA in heredity was first discovered by studying bacteria and the
viruses that infect them
DNA as the source of genetic material
• 1928
• Frederick Griffith – working on a vaccine for pneumonia (Streptococcus
pneumonia)
– worked with 2 strains – one harmless, one pathnogenic
– killed the pathenogenic bacteria and mixed them with cultures of living,
harmless bacteria
– some of the harmless bacteria became pathenogenic
– their progeny remained pathenogenic
– DNA had to have been transferred from path. to harmless when he mixed the
bacteria
– called this transfer = transformation (assimilation of external by a cell)
EXPERIMENT
Living S cells
(control)
Living R cells
(control)
Heat-killed
S cells
(control)
Mixture of
heat-killed
S cells and
living R cells
RESULTS
Mouse dies
Mouse healthy
Mouse healthy
Mouse dies
Living S cells
DNA as the source of genetic material
• transformation now defined as – change in genotype and phenotype due to
the assimilation of external DNA by a cell
• 1944 -Oswald Avery, Maclyn McCarty, and Colin MacLeod announced that
the transforming substance was DNA
–
–
–
–
–
14 year project!!
possible transforming agents: DNA, RNA or protein
broke open heat-killed bacteria and extracted their contents
treated each sample with chemicals that inactivated either DNA, RNA or protein
tested the modified samples for their ability to transform live, non-pathenogenic
bacteria
• only DNA worked in transforming harmless bacteria into pathogenic
bacteria
– lost this function once they were inactivated with chemicals
• many biologists remained skeptical, mainly because little was known about
DNA
Viral DNA Can Program Cells
•
•
more evidence for DNA as the genetic material - studies of viruses that infect bacteria
• called bacteriophages (or phages)
1952 - Alfred Hershey and Martha Chase performed experiments showing that DNA is
the genetic material of a phage known as T2
•
•
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phage that normally infects E. coli strains
composed of DNA and protein
they designed an experiment showing that only the DNA enters an E. coli cell during
infection
Viral DNA Can Program Cells
– labelled phages with radioactive
sulfur to label proteins (batch #1) or
phosphorus to label DNA (batch #2)
– mixed the labelled phages with
bacteria to cause infection
– separated the bacteria from the
phages and looked for what
radioactive signal was in the isolated
bacteria
– confirmed and measured the
presence of radioactive phosphorus
in the transformed bacteria
Proteins labelled
DNA labelled
Chargaff’s Rules
•
•
•
•
DNA is a polymer of nucleotides, each consisting of a nitrogenous base, a sugar, and a
phosphate group
1950 - Erwin Chargaff reported that DNA composition varies from one species to the
next
BUT he also noticed two other things
these 2 findings became known as Chargaff’s rules
– 1. WHILE the base composition of DNA varies between species
– 2. the number of A and T bases are equal and the number of G and C bases are
equal in any species
Watson & Crick
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1950s – three groups working on the structure of DNA
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1. Linus Pauling – Cal Tech
2. Maurice Wilkins and Rosalind Franklin – King’s College, London, UK
3. American James Watson and Englishman Francis Crick – Cavendish Laboratory, Cambridge, UK
1953- Watson and Crick constructed a model for DNA – two complementary strands that
run anti-parallel to one another in a double helix
Watson & Crick
• based their work on Rosalind Franklin’s X-ray crystallography findings
•
•
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X-rays bent as they passed through the strands of purified DNA
photo 51 – suggested a double helix, with the bases facing inward, making a full turn every 3.4nm,
10 base pairs every full turn
1962 – Nobel prize – Watson, Crick and Wilkins (Franklin dies in 1958)
(a) Rosalind Franklin
(b) Franklin’s X-ray diffraction
photograph of DNA
Nucleic acids
• two types: DNA, RNA
• C,H,O,N,P
• building blocks = nucleotides
• nucleotide:
Sugar-phosphate backbone
5 end
•5 carbon sugar (pentose)
phosphate group (negative
charge) located at the 5’ carbon
organic base located at the
1’carbon
sugar and the base is known as a
nucleoside
bases: 5 types: adenine (A)
cytosine (C)
guanine (G)
thymine (T)
uracil (U)
5C
3C
Nucleoside
Nitrogenous
base
5C
1C
5C
3C
Phosphate 3C
group
Sugar
(pentose)
(b) Nucleotide
3 end
(a) Polynucleotide, or nucleic acid
Naming nucleotides
• nucleotide is conventionally
named “based” on its base
– adenine, guanine etc…
• but the true name is a
combination of the nucleoside
and the number of phosphate
groups
• nucleoside= combination of a
base and a sugar
– e.g. adenine + sugar = adenosine
• so “adenine” is really called
– e.g. RNA - adenosine
monophosphate (AMP)
– e.g. DNA - deoxy-adenosine
monophosphate (dAMP)
 There are two families of
nitrogenous bases
Nitrogenous bases
Pyrimidines
1. Pyrimidines (cytosine,
thymine, and uracil) have a
single six-membered ring
Cytosine
(C)
Thymine
(T, in DNA)
Uracil
(U, in RNA)
2. Purines (adenine and
guanine) have a sixmembered ring fused to a
five-membered ring
Sugars
Purines
Adenine (A)
Guanine (G)
(c) Nucleoside components
Deoxyribose
(in DNA)
Ribose
(in RNA)
DNA is double stranded: The DNA double
helix
• DNA strand is known as a
polynucleotide chain
• 2 sugars of adjacent
nucleotides are joined by a
phosphodiester bond
• the phosphodiester bond can
only form between a 3’OH
“down” to a 5’PO4 group
• so that the DNA chain grows
in a 5’ to 3’ direction
-the DNA strand must “grow” in a specific direction because of the nature of the phosphodiester
bond
-this direction is called 5’ to 3’ because one end is a 5’phosphate group, the other end is a 3’ OH
group
-the complementary strand also grows this way = two anti-parallel strands of DNA (opposite
directions of growth)
Why 5’ to 3’??
• the DNA chain grows in a 5’ to 3’ direction
• why can’t you form a phosphodiester bond
from 3’ to 5’??
• the first thing that happens is the bases pair
up with each other
• THEN the phosphodiester bond forms
• the incoming nucleotide is in its
triphosphate form
Why 5’ to 3’??
• a magnesium ion is associated with the
outer two phosphate groups
• this weakens the phosphate bonds between
these groups
• and allows for a successful nucleophilic
attack by the 3’OH group in the nucleotide
that is already bound to the DNA chain
• results in the loss of the outer 2 phosphate
groups (due to a shift in electrons) and the
formation of a 3’-5’ phosphodiester bond
Nucleophilic attack
Mg2+
• single DNA chain is 2.2 to 2.6nm (22 to 26
Angstroms) wide – each nucleotide is 3.3A
long
• DNA exists as a double helix – backbone
made of alternating sugars and
phosphates from the nucleotide
• bases are inside the helix – away from the
aqueous environment of the cell
• helix makes 1 full turn every 3.4nm
– ~10bp every turn
– so one base is 0.34nm wide
• helix is a right hand twist
– clockwise twist
•
2 DNA strands are held together by Hydrogen
bonds between the bases
– not very strong, so DNA helix is easily separated
by high heat, salt, mechanical forces etc…
– this is known as denaturation
•
Watson and Crick found that the helix was
uniform in its diameter
– proposed that the base pairing had to be
consistent throughout the helix
•
•
proposed = DNA held together by
complementary base pairs
the base pairing rule is purine-pyrimidine
– purine-purine pairing would produce a bulge in
the helix
– pyrimidine-pyrimidine pairing would produce a
“dent”
– A to T – two H bonds
– C to G - three H bonds
– C-G pairings are stronger – DNA is harder to
separate in those regions (“GC rich”)
• helix also exhibits additional levels of
coiling = supercoiling
– DNA can either supercoil in the direction of
the helix (positive) or in the opposite
direction (negative)
– degree of supercoiling can determine the
strength of the interaction between the two
DNA chains
• three forms of DNA that have been
observed
– form depends on hydration levels, levels of
supercoiling
– A
– B - form found in cells
– Z
Naming the DNA strands
• DNA is a double helix of complementary sequence
• to simplify things we call one strand “sense” the
other “anti-sense”
• sense strand – has same sequence as the mRNA
strand that will be translated into a protein
• anti-sense strand – template used to create this
mRNA
• when written out – the sense strand is the top strand
– with the 5’PO4 on the left and the 3’OH on the
right
-double stranded DNA helix is
unwound into individual strands
-enzyme = DNA polymerase attaches
to each strand
-polymerase moves along the DNA
strand and attaches the complementary
base
e.g. C is read – attaches a G
Models of DNA replication
• conservative: means
that the replicated
DNA helices are
entirely parental or
daughter
• semi-conservative:
means that the
replicated DNA
helices are 50%
parental DNA and
50% daughter DNA
• dispersive: means
that the replicated
DNA helices are a
mixture of parental
and daughter DNA
Replication is Semi-conservative
•
•
•
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labelled bacterial DNA first with a heavy 15N isotope to label the parental strands
replication - added a labelled a lighter 14N isotope that would label the replicated DNA
daughter strands
continued to label bacteria with the lighter isotope for another round of replication
isolated the DNA & based on their “weight” they could tell which DNA strands had the
15N label or the 14N label



found that after the 1st
replication – hybrids of both
15/14N strands
after the second replication –
mixture of hybrid DNA and light
14N DNA helices
only way to explain this = Semiconservative replication
DNA replication – the players
• DNA polymerase III = reads the DNA strand and lays down a
complementary base to create a complementary “daughter”
strand of new DNA
• Helicase/dnaB= enzyme that “melts” or unzips the doublehelix of the parental DNA
• single stranded binding proteins/SSBs – hold the unwound
DNA helix “open” – allows for the action of the replication
machinery
• RNA polymerase (Primase/dnaG) = creates a small RNA
“oligo” primer which binds onto the DNA template and allows
the DNA polymerase III to attach to the DNA template
• DNA ligase = joins up the fragments of DNA made during
replication
DNA polymerase
• polymerase = enzyme that catalyzes the addition of
bonds between two nucleotides of either DNA or RNA
– catalyzes the bond between bases
– also catalyzes the phosphodiester bond within the
backbone
• in bacteria – three polymerases that act in replication
– replication polymerase = DNA polymerase III
– DNA polymerase I – removes RNA nucleotides found in the
primer(exonuclease activity) and fills in the gaps
• also corrects mistakes made upon replication = “proof-reading”
– DNA polymerase II – proof reading
• in eukaryotic cells – 11 polymerases !!
– same idea as bacterial replication
Problems with Replication

first problem: DNA is a helix
 DNA polymerases are unable to melt duplex DNA in order to separate
the two strands that are to be copied


second problem: unwinding the DNA produces supercoiling in the regions
ahead



solution: binding of a “helicase” enzyme unwinds the two strands
solution: action of topoisomerases to “nick” the DNA helix, unwind the
supercoils and connect the strand back together
another problem: the helix wants to re-form
 solution: single-stranded binding proteins (SSBs) also required to
prevent the DNA template from rewinding back up
still another problem: DNA polymerases cannot bind single stranded
nucleic acids



unwinding the DNA gets rid of base pairing = Problem !
DNA polymerase III requires a “primer” to elongate off of
solution: enzyme called a “primase” makes a small piece of RNA that binds to
the DNA strand and acts as a primer for the DNA polymerase – creates a
temporary double stranded structure of RNA and DNA
Problems with the DNA polymerase
• Biggest Problem: The two strands in the DNA duplex are opposite in
chemical polarity
 DNA polymerase can only form a phosphodiester bond between the
5’PO4 of an incoming new nucleotide and a 3’OH of a nucleotide already
base paired to the DNA template
• this means that new DNA strands can grow only in the 5 to 3 direction
– this is okay when replicating the anti-sense strand because the
complementary daughter strand that is being created will grow in the 5’ to 3’
direction
– but there is a problem replicating the sense strand
3’
growing daughter strand
5’
growing daughter strand
3’
DNA
poly
5’
DNA replication
• replication starts at specific sequences of
DNA = origins of replication
– called oriC in bacteria
– ~240bp sequence – containing repetitive
sequences – rich in As and Ts
– also found in viruses
– multiple origins are found in eukaryotic
chromosomes
– oriC is recognized by a protein complex
that docks onto the oriC - helps position
the helicase and DNA polymerase III near
the origin
– this complex is comprised of at least 5
types of proteins –the helicase, the
primase, DNA polymerase, clamp loading
protein, infrastructure proteins that
‘tether’ the two DNA polymerases to each
other
DNA replication
• oriC is recognized by the helicase that unwinds the DNA
– “melts” the DNA
• forms a replication “bubble”
– comprised of two replication forks
• the forks are held open by SSBs
• the helicase can actually act as a molecular “brake” that controls how fast
replication happens
the helicase/primase/DNA polymerase
moves along the parental DNA strand in
the 3’ to 5’ direction
DNA replication: The Leading strand
•
•
REMEMBER – a complex of proteins binds at the oriC
and moves along the parental DNA in the 3’ to 5’
direction
because of this one strand of parental DNA is able to
be replicated continuously without any problems
– new daughter strand grows in the 5’ to 3’ direction
•
the DNA strand that is made continuously = Leading
Strand
•
the leading strand is uses the anti-sense strand of
parental DNA as its template
the leading strand also grows in the same direction
as the replication fork
•
Replication fork
direction
DNA replication: The Lagging strand
• but since the second strand of DNA is in the opposite direction – it cannot be
replicated continuously
– this would require the daughter strand be made in the 3’ to 5’ direction
– solution – sense strand is replicated in “chunks”
• this daughter DNA strand being made is called the Lagging strand
– ‘chunks’ are called Okazaki fragments – 1000 to 2000 bps
• (Reiji Okazaki)
• as the helicase unwinds the parental duplex,
primase creates multiple primers along the
sense parent strand
• DNA polymerase “clamps” onto the 3’ end of
this DNA-RNA hybrid duplex and makes an
Okazaki fragment
•
•
DNA polymerase III – large holoenzyme of 10 different proteins!!!!!
core polymerase – three subunits:
– alpha – active site for nucleotide addition – polymerase action
– epsilon – exonuclease activity – part of the enzyme that removes incorrectly added nucleotides –
“proof-reading”
•
•
can only back-up one nucleotide and correct it
so DNA polymerase III has to “catch” its mistakes as it makes them
– beta – forms a beta-clamp – forms a donut-like clamp around the parental DNA
•
remaining 6 subunits convert the core polymerase from a distributive enzyme (which tends
to fall of the DNA frequently) into a processive enzyme that can replicate long stretches of
DNA (T4 bacteriophage - 45,000 nts per minute!!!!!)
– form a gamma complex which: 1) loads the b-clamp onto the DNA strand at the RNA primers and
2) takes the clamp back off
– now known as the Clamp-loading Protein
The big picture!!
•
•
•
•
•
leading and lagging strands are made at the same time
two core DNA polymerases III bind at each fork and replicate the DNA in the same direction!
how is this possible??
the parental/lagging strand is “looped” within the complex so that its orientation is the same as
the parental/leading strand
IMPORTANT – the DNA polymerase III complex doesn’t move – the DNA template is “fed into” the
complex and then fed back out
–
DNA polymerase III is anchored to the nuclear matrix
SSBs
DNA
polymerase
(topoisomerase)
Helicase & Primase
direction of polymerase movement (3’ to 5’)
The big picture animation
• for the “big picture”: http://www.youtube.com/watch?v=-mtLXpgjHL0
Leading
DaughterAntisense
parent
Sense parent
Beta clamp
Anti- Sense parent
alpha
helicase
primase
SSBs
RNA primer #2
alpha
RNA primer #1
Beta clamp
Okazaki
Fragment #1
Leading
DaughterAntisense
parent
Sense parent
Anti- Sense parent
alpha
RNA primer #3
alpha
SSBs
helicase
primase
RNA primer #2
Okazaki
Fragment #2
RNA primer #1
Okazaki
Fragment #1
Check out these animations!!
• http://www.ncc.gmu.edu/dna/repanim.htm
• http://www.johnkyrk.com/DNAreplication.ht
ml
• http://www.bioteach.ubc.ca/TeachingResourc
es/MolecularBiology/DNAReplication.swf
• http://bioweb.uwlax.edu/GenWeb/Molecular
/Theory/Replication/replicat.mov