DNA Analysis, Measurement, and Replication Flashcards
Common techniques for measuring and analyzing DNA
Absorption of UV light (Spectroscopy)
Centrifugation
Denaturation and renaturation
Radioactivity
Gel electrophoresis
DNA absorbs UV light with a wavelength of 260 nm very well Other macromolecules (proteins, carbs, lipids) do not
Absorption of UV light (Spectroscopy
More absorbance =
more quantity
Machine with an internal rotor that spins at very high speeds
The force generated from the spinning causes any particle with mass to move to the bottom of the tube
Centrifugation
two types of Centrifugation
Sedimentation equilibrium centrifugation
Sedimentation velocity centrifugation
A density gradient (usually CsCl) is created and mixture is applied
- Mixture is spun for a fixed time
- Molecules separate from one another based on their density
- They move to their buoyant density and form a band in the tube
Sedimentation equilibrium centrifugation
This spins and measures how “fast” a molecule moves down a tube during centrifugation (analyzes and separates)
- DNA/RNA shape and mass both influence how fast it moves
- Expressed in Svedberg units (S) –used to calculate mass
Sedimentation velocity centrifugation
separating the two strands and allowing them to get back together
Denaturation and renaturation
by heating DNA Hydrogen bonds between the two
strands break and the 2 strands separate
Denaturation
heating DNA
denaturation
what happens when you cool DNA
Strands go right back together
(forming proper hydrogen bonds)
Cool DNA and Strands go right back together (forming proper hydrogen bonds)
renaturation or annealing
G and C form how many hydrogen bonds
3
A and T form how many hydrogen bonds
2
DNA with more G-C pairs will require what temps to denature
hotter
Can tag nucleic acids with various radioisotopes
Radioactivity
Method of separating molecules in a mixture by adding the mixture to a semi-
solid gel and applying an electric current through it
Gel electrophoresis
Eukaryotic cells do cell division and replication in what
S phase of interphase
Bacterial cells do cell division and replication in what
binary fission
how many chromosomes do we have
46
Some possible mechanisms of DNA replication:
Semiconservative
Conservative
Dispersive
Each strand serves
as a template for a new strand
- Each new double-stranded DNA molecule
contains 1 old strand and 1 new strand
Semiconservative
Would result in 1 completely
old and 1 new double- stranded DNA molecule.
Conservative
Would produce two DNA molecules with sections of both old and new DNA
interspersed along each strand
Dispersive
Meselson-Stahl experiment
DNA replication
another name for Bacterial DNA replication
Theta replication
steps of Bacterial DNA replication
- Initiation
- Unwinding
- Primer Synthesis
- Elongation
- Primer removal and ligation
where replication starts in bacteria
origins of replication (oriC)
OriC (and other prokaryotic origins of rep.) contain binding sites for an initiator protein called
DnaA
what does the introduction of DnaA do
a slight “bend” to be introduced
The bubble serves as a binding site for
DnaB and DnaC
Type of enzyme that uses ATP
in order to denature DNA
Helicase
bind to the individual strands and keep them apart
single-strand-binding proteins
(SSB proteins)
binds to the chromosome “ahead” of the replication fork and relieves the tension
An enzyme called DNA gyrase (a type of topoisomerase)
how does gyrase relieve tension
they introduce small cuts in the strands, unwind, and reseal
how does unwinding proceed
Proceeds in both directions
(aka bidirectional)
The enzyme that makes a new DNA strand from the template called
DNA polymerase II
It only can add nucleotides to an existing
3’ OH group (It needs help getting things started)
what makes the primer
primase
a short (5-10 nucleotides) RNA
chain
primer
DNA polymerase III can then come in and create a new strand by extending
from the primer
elongation
Only adds new nucleotides onto the 3’ end of a
growing strand
DNA polymerase III
Makes a new DNA strand in the what direction
5’ 3’
it reads what is on the template strand and adds
complementary nucleotide
- Reads T, adds A; reads G, adds C
what direction does polymerase III go during exonuclease activity
3’ to 5’
Has the ability to proofread itself and correct its mistakes
DNA polymerase III
Three subunits make up polymerase III
α synthesizer
ε proofreader
θ stimulator
polymerization of nucleotides
α synthesizer
corrects mistakes made
ε proofreader
stimulates ε to do its job
θ stimulator
Sliding clamp structure that locks the whole complex onto the DNA strands
- Prevents the above core enzyme from falling off
- β clamp
Tethers two DNA pol. III together
(creates a dimer
Tau (τ) subunit -
Helps to load the whole enzyme onto the DNA template at the replication fork (lagging strand)
Gamma (γ) loader complex
Two interacting DNA strands must always run in what direction
opposite directions
unwinding at
oriC and the addition
of RNA primers (in
green) by primase
localized DNA
adds primers
primase
adds nucleotides
DNA pol III
Synthesis continues in the
5’ —–> 3’ direction
Notice one is moving
towards and the other
Notice one is moving
towards the replication
fork and the other away
One strand is being made continuously as DNA pol III moves
towards the replication fork
(this is called the LEADING STRAND)
small fragments called
Okazaki fragments
comes in and removes the RNA primers from both the leading and lagging strands and fills fills in the gaps with DNA
(DNA polymerase I)
seals the spaces
between each Okazaki
fragment on the lagging
strand
DNA ligase
leads to cell death because if the wrong gene is
crippled, the cell can’t function properly
Mutation of DNA
can abnormally lead a cell to divide uncontrollably and prevent the cell from dying properly —–> TUMOR
mutation of specific genes
what kind of chromosomes does Bacteria have
Single, circular chromosome (Avg size: 4.6 x 106 base pairs)
what kind of chromosomes does Humans have
Multiple, linear chromosomes
differences bt. eukaryotic and bacteria DNA replication
1) Eukaryotic chromosomes have multiple origins of replication
2) Eukaryotic DNA polymerases
3) Removal/assembly of histones
4) Chromosome ends/telomeres
Eukaryotic chromosomes have multiple
origins of replication
In order to function, origins must be “licensed” or approved for replication by
origin recognition complexes (ORCs)
origin recognition complexes (ORCs) recruit
helicase, SSB protein, DNA pols,
after replication what happens to (ORCs)
it comes off
Unreplicated DNA is coated with
MCM proteins (helicases)
one way that the cell has of distinguishing between replicated and unreplicated DNA
Unreplicated DNA is coated with MCM proteins (helicases)
No MCM proteins = what
no replication
what happens to MCM after replication
MCM proteins come off the DNA and are prevented
from going back on
different DNA polymerases in Eukaryotes
Pol α
Pol δ
Pol ε
Pol γ
Major polymerases involved in nuclear DNA replication
Polymerase α (Pol α) and Pol δ
Two of its 4 subunits function as the primase
Adds primer to leading and lagging strand templates and starts extending them
- This enzyme has low processivity an
Pol α
what kind of processivity does Pol α
low
Replaces pol α and finishes the job (lagging strand)
Pol δ
Pol δ Replaces pol α and finishes the job (lagging strand)
This is called polymerase switching
what does Pol δ that Pol α doesn’t
3’ —> 5’ exonuclease activity
what kind of processivity does Pol δ
high
Similar abilities as pol δ, may be major leading strand polymerase
Pol ε
Functions to replicate mitochondrial DNA
Pol γ
This DNA contains genes that are required for life
Pol γ
Pol γ is Passed from
mom to child
Eukaryotic nuclear DNA is tightly coiled around proteins
called
HISTONES
Histones are very basic proteins (+ charged) that interact with the what-charged DNA
negatively
Histones are typically altered by adding an
acetyl group (called
acetylation
what happens to histones after replication
histones are deacetylated and go back onto the DNA (and new histones are made)
In theory, our chromosomes
should shorten each time they
are replicated because
Once DNA pol I removes
the primer at the absolute end of the chromosome on the lagging
strand, there is no free 3’ end
ahead of it
The end of our
chromosomes (called what) are modified to
prevent the loss of important genes
telomeres
is activated and adds
>1,000 copies of a repeated sequence (e.g. TTAGGG)
onto the ends of each chromosome
telomerase
telomerase is only active in
gametes, embryos, and stem cells
Telomerase has a what in a pocket that
binds to the chromosome end
RNA template
Uses this to keep adding more copies of the
repeated sequence onto the telomere
RNA template
still shorten each time the DNA
is replicated
Telomeres
nstead of losing important genes, chromosomes only lose a portion of the
“garbage” repeated sequence
how repeated sequences at our telomeres also function to protect chromosomes
1) Serving as binding sites for protective proteins
2) Prevent chromosomes from fusing
The more a cell divides, the
shorter the telomeres get
what happens when the repeated sequence runs out and important genes in the
chromosomes get removed
the cell dies!
Telomere shortening acts as a
cell clock
telomere shortening could lead to
human aging
