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reason #1 that DNA structure is stable
hydrogen bonds between complimentary bases and between sugar phosphate backbone and H2O
reason #2 that DNA structure is stable = electrostatic interactions
negatively charged phosphate groups repel one another and interact with Mg2+
reason #3 that DNA structure is stable = van der waals and hydrophobic interactions
stacking of base pairs
how many bonds do A and T share
2 H bonds
how many bonds do G and C share
3 H bonds
which regions of DNA are more stable
G and C
helix is anti-parallel meaning
sugar-phosphates outside and bases stack inside
helix dimensions
10 bp per turn
0.34nm bp spacing
2.37nm diameter
3.4nm pitch
tops of bases line
floor of the major groove
bp edges nearest to the glycosidic bond form
interior surface of minor groove
major groove can accomodate
a protein
regulatory proteins can recognize
pattern of bases and H-bonding possibilities in major groove
what happens when heating DNA >80C
bp interactions disrupted
why does denaturation increase UV absorbance
pi-electrons of unstacked bases
midpoint of absorbance increase
the melting temperature
DNA can also be denatured using
an alkali and/or pH
when temperature is lowered DNA
absorbance drops = re-establishment of stacking
why do DNA's differ in TM values
due to relative G:C content
higher g:c content of DNA results in
higher Tm because G:C pairs have more bonds
what does DNA assume
circular higher order structure
plasmid DNA
bacterial extrachromosomal DNA - closed DNA duplexes
supercoiled state
circular DNA sometimes has more or less than 10 bp per turn
What do topoisomerases/gyrases do?
introduce or remove supercoils
E.coli chromosomal DNA bp
4.64*10^6
length of e.coli chromosome
1.6mm
length of e.coli
0.002mm
how does the chromosome fit in the bacterial cell
supercoiling extensively packages circular DNA to fit
diameter of a typical human cell
20um
genetic material consists of
23 pairs of dsDNA in the form of chromosomes
total length of human DNA
2 meters
How much is DNA condensed?
x100000
how DNA condensation made possible
by wrapping DNA around nucleosomes and then packing them with DNA into helical filaments
chromatin
nucleoprotein complex of DNA
chromatin protein constituents
histones and non-histone chromosomal proteins
what are the 4 pairs in the histone octamer structure
H2A, H2B, H3 and H4
what are the regulators of gene expression
the lesser known histone proteins
what gives rise to chromosomes
higher-order structural organization of chromatin
why does replication of DNA give identical progeny molecules
because base-pairing is the mechanism determining the nucleotide sequence synthesized in each of the new strands during replication
each original strand acts as what for the new strand?
template
what happens with the template strand
it is used to from a new complementary strand by enzyme DNA polymerase
How is DNA replication semi-conservative?
It creates two strands DNA each with 1 new strand and 1 original strand
Where does DNA replication occur?
origins of replication
which way is replication directed
bi-directional = 2 replication forks
What end are nucleotides added to?
3' end
which way does replication occur
5' to 3'
why must double helix be unwound by helicases
to expose single strands
leading strand copies
continuously
lagging strand copies
in segments which must be subsequently join
what happens at the origin of replication
Helicase separates the strands of DNA.
what can unwinding result in
introduction of positive supercoils
gyrase introduces
negative supercoils using atp hydrolysis, this relaxes positive supercoils and seals breakage
single strand DNA binding proteins
bind to ssDNA and stops strands from binding again
DNA primase
primer required to bind at ori
primers
short sequences of RNA
primase
synthesizes primers
DNA polymerase 3
makes new strand by reading template strand and adding 1 nucleotide after the other
leading strand replication
fork moves 5' to 3'
parent antisense strand acting as template for continuous synthesis
lagging strand
replication fork moving right to left
parent sense strand acting as template for discontinuous lagging strand synthesis
DNA polymerase
enzymes that replicate DNA
pol 1 needs
all 4 deoxynucleotides
a template
a primer
pol 1 has 3 active sites
has polymerase activity
has proofreading and editing function
pol 3 is the chief DNA-replicating enzyme of E.coli
sits at each replication fork
why does DNA pol1 recall RNA primer
to initiate strand synthesis
what does DNA pol1 do
replaces RNA primers with DNA during replication using 5' to 3' polymerase activity
what does pol 1 polymerise
about 200 bases before it dissociates from template
repair functions of pol 1
has 3'-5' exonuclease function
has 5'-3' exonuclease activities
why the 3'-5' exonuclease activity
enhances accuracy of DNA replication
3' exonuclease activity
removes nucleotides from 3' end of growing chain
proofreading function of 3' to 5' exonuclease activity
removes incorrectly matched bases. enhances fidelity of replication process
alpha subunit
polymerase
epsilon subunit
3' to 5' exonuclease
theta subunit
stabilization of epsilon subunit
dimeric polymerase
1 unit synthesis leading strand and lagging strand each
beta subunit
forms a sliding clamp around DNA so that pol can move along
clamp loader
responsible for adding and anchoring beta subunit's core structure
replisome consists of
dna unwinding proteins
priming complex
DNA polymerase 3 holoenzyme comprising 2 replicative polymerases
initiation
DNA protein binds to repeats in ori, initiating strand separation
primase binds and constructs RNA primer
elongation
DNA gyrase relieves supercoiling
DnaB unwinds DNA
SSB binds to keep strands separated
DNA pol 3 replicates each strand
termination
ter locus opposite of ori, rich in G and T consensus sequence signals end of replication
tus protein
a contrahelicase that prevents further unwinding
how does tus protein work
blocks helicase and replication fork
lagging strand replication
DNA pol 2 is 1 enzyme with 2 units for leading and lagging
lagging strand is looped around and replication occurs 5 to 3
DNA pol 3 unclamps and reclamps periodically on lagging strand when it encounter primer of okazaki fragments
DNA pol1 eexcises RNA primer and replaces it with DNA
ligase seals remaining nick
DNA polymerase alpha
initiation of nuclear DNA replication, processivity = 200 nucleotides
DNA polymerase epsilon
leading and lagging strand synthesis, checkpoint control
DNA polymerase sigma
principal polymerase in leading and lagging strand synthesis; highly processive
multiple origins of replication
2 replication forks at each origin
mutations that arise due to environmental factors or endogenous errors during synthesis
deletions
insertions
substitutions
replication errors
base mismatches
UV-light
chemical mutagens
integrity of DNA is vital to cell survival and reproduction
repair systems to correct DNA damage
types of substitution mutations
transition and transversion
transition
purine to purine or pyrimidine to pyrimidine
transversion
purine to pyrimidine or pyrimidine to purine
insertions and deletions
insertion or deletion of one or more bases
deamination of cytosine
forms uracil which base pairs with adenine. incorrect base-pairing: C-G becomes U-A
deamination of adenine
forms hypoxanthine which base pairs with cytosine. incorrect base-pairing: A-T becomes C-G
depurination
loss of purines from DNA resulting from hydrolysis of the glycosidic bond between deoxyribose and the base, leaving an apurinic site in DNA
in amino-imino tautomers
an amino group, usually protonated, can tautomerise to an imino group and become deprotonated
when an imino tautomer of adenine base-pairs with cytosine
A-T pair changes to mismatched A-C pair = point mutation, non-coding
what does ultraviolet light promote
formation of covalent bonds between adjacent thymine residues in a DNA strand
what does it mean if a thymine dimer cannot fit into a double helix
replication and gene expression are impaired
chemical mutagens
chemical compounds and alkylating agents
