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8/9/2019 Structure Dna Lecture
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Forms of DNA
B - DNA: Watson-Crick Structure10 residues/turn
A - DNA: wider helix - tilted basesno minor groove
RNA-DNA hybrids
Z - DNA: High salt form - left handedantibodies to Z - DNA exist
in nature
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Procaryotic DNA
no structural proteins bound
circular DNA
supercoiling: conserves spaceeasier to unwind helix
L = T + W
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Topoisomerase
I
L = 25 + 0
Helicase
L = 23 + 0
DNA Gyrase
L = 25 - 2
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Eucaryotic DNA
50:50 DNA:Protein
Histones: basic proteins
octamer core = (H2A, H2B, H3, H4)2
H1: phophorylated prior to mitosis
Histones are highly conserved
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=
H2A H2B
H3 H4
Histones are highly conserved
2/102 base changes in H4 between pea and cow
1% divergence every 600 million years
(compare to 6 million for Hb)
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H1
P
Nucleosome
~ 200 bp per nucleosome
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Eucaryotic Chromosome
nucleosomes -
like beads on string
additional coiling &
supercoiling makes
compact chromatin.
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Genetic Information
RNA
Transcription
Protei
n
Translation
DNA
DNA
Replication
Gene
Expression
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Replication
dATP
dGTP
dTTP
dCTP
DNA
DNA Polymerase
DNA
Substrates
Template
Enzyme
Product
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REPLICATION
Connects dNTPs(3-5 Phosphodiester bond)
Template instructions: parent cells DNA
Single copy only: prior to cell division
Error Rate = 1 in 1 x 109
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Procaryotic DNA is circular
Origin ofReplication = Replicon Site
DNA Gyrase +
Helicase +SSB Protein
replication fork
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DNA Polymerase
Replication Fork
Primer = ~ 10 base oligo RNAinserted by Primase
5
35
The new strand is formed 5 3
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In Real System DNA Pol
dimers act in tandem
5 3
5 3
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Thymine has an equilibrium between
keto & enol forms
N
NO
O
H CH3
keto
N
NO
HO
CH3
enol
1
10,000 or
104
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H2
N
NN
N N
Adenine (amino)
Adenine (imino)
NH
NN
N N
H
Imino form of Adenine pairs with C
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Fidelity of
Replication
AUCAG
GCT
T A
GTCCG
AATGC
T
C
A
C
GT AG
CChance of error1 in 104
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DNA Polymerase Activities
5-3 Polymerase: (normal)
3-5 exonuclease: removes last basein growing strand if incorrect
5-3 exonuclease: removes RNA primer(while 5-3 Polymerase fills in gap)
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Fidelity of
Replication
AUCAG
GCT
T A
GTCCG
AATGC
T
C
A
C
GT AG
C
Chance of error
1 in 104
Chance of correctionfailure : 1 in 104
error rate = 1 in 1 x 108/9
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continuous strand
no additional primer
Continuationof Replication
38
discontinuous (lagging) strand
requires new primer
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discontinuous ~2,000 bps
continuous
Okasaki Fragments
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E coli DNA Polymerases
DNA Pol I: primer removal & gap filling
DNA Pol II: DNA repair?
Pol III: main polymerase
) 2 2 4 ( )2 compl
5-3 polymerase
proof reading
dimerization
processivity
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E coli DNA Polymerases
DNA Pol I: primer removal & gap filling
DOMAIN 1 323 5'-3' EXONUCLEASE.DOMAIN 324 517 3'-5' EXONUCLEASE.
DOMAIN 521 928 POLYMERASE.
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Primer Excision:
5-3 Exonuclease Activity....
53
.... removes RNA primerwhile regular polymerase
activity fills in gap
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DNA Ligase seal nicks
Nick: no bases missingone phosphate ester bond not formed
S-P-S-P-S-P-S-P S-P-S-P-S-PT A G C A C A
S-P-S-P-S-P-S-P-S-P-S-P-S-P
TGTGCTA
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Mutations
in DNA SequenceCan occur due to uncorrected
enol/imino pairing during replication:rate = 1 in 1 x 109
Can also occur due to DNA damageduring interphase
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N
N
H2N
O
C
N
NO
O
HU
Spontaneous
deamination
Why both T & U?
The lack of CH3 on U in DNA marks as error
and instigates removing and repair to restore C
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Restriction Endonucleases/Methylases
Recognize and bind to specific DNA sequence
Function in bacteria is to destroy foreign DNA
1) if methylated ignore
2) if methylated methylate other strand
3) if unmethylated - cut
Feature #2 allows for marking of self DNAFollowing replication/Cell Division
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Enz Seq Enz SeqAluI AGCT
TCGA
HpaII CCGG
GGCC
BamHI GGATCC
CCTAGG
KpaI GGTACC
CCATGG
BglII AGATCT
TCTAGA
MboI GATC
CTAG
ClaI ATCGAT
TAGCTA
PstI CTGCAG
GACGTC
EcoRI GAATTC
CTTAAG
PvuI CGATGC
GCTACG
HaeII CGCCGCGG
SalI GTGCACCACGTG
HindII GTpPAC
CAPpTG
SmaI CCCGGG
GGGCCC
HindIII AAGCTT
TTCGAA
XmaI CCCGGG
GGGCCC
Restriction Endonucleases11
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RFLPs
1
2
3
4
5
5
4
3
2
1
1
2
3
4
5
5
4
2
3
1
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DNA Sequencing
Cut DNA into managable pieces
Create DNA Library of pieces
Select desired piece (Southern Blot)
Clone & amplify desired piece - PCR
Sequence cloned fragment
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Southern Blot
1
2
3
4
5
5
4
3
2
1
add 32 P 2 to
nitrocellulose blot
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Polymerase Chain Reaction (PCR)
Heat +
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0
1
2
Long strands add while short strands double
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Generation Parent Long Short
0 1 0 01 1 1 0
2 1 2 1
3 1 3 44 1 4 11
5 1 5 26
6 1 6 577 1 7 120
8 1 8 247
17
= 1 l + 1 2s + l
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|_O - P -
|
O_
O
O
O
A
|
O ||O - P -
|O_
O ||O - P -
|O_
O ||-O - P -
|O_
O
OH
O
C
|
dideoxy nucleotides will terminate DNA
chain because there is no 3 end to add to.5 end
3 end19
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3- XXXXACGTAGCTTACC - 5
5-32 PXXXX
Add ddTTP + dTTP, dATP, dCTP, dGTP
Get : XXXXT
XXXXTGCAT XXXXTGCATCGAAT
piece length = Primer + 1,5,10
20
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3- XXXXACGTAGCTTACC - 5
5-32 PXXXX
Fragment Sizes :
1 2 3 4 5 6 7 8 9 10 11 12
ddT : 1, 5, 10
ddA : 4, 8, 9
ddC : 3, 6
ddG : 2, 7, 11, 12
21
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12
11
10
98
7
6
5
4
3
2
1
T A C G
5-XXXXTGCATCGAATGG
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Hasil Agarose Elektroforesis
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