1

Forms of DNA
B - DNA: Watson-Crick Structure
10 residues/turn
A - DNA: wider helix - tilted bases
no minor groove
RNA-DNA hybrids
Z - DNA: High salt form - left handed
antibodies to Z - DNA exist
in nature
 3
Procaryotic DNA
no structural proteins bound
circular DNA
supercoiling: conserves space
easier to unwind helix

L=T+W
 L = 25 + 0 L = 23 + 0 4

DNA Gyrase

Helicase
Topoisomerase
I
L = 25 - 2
 5
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

 7
Histones are highly conserved

H2A H2B

=
H3 H4

2/102 base changes in H4 between pea and cow

1% divergence every 600 million years

(compare to 6 million for Hb)
 8
Nucleosome

P
H1

~ 200 bp per nucleosome

 9
nucleosomes -
like beads on string

additional coiling &

supercoiling makes
compact chromatin.

Eucaryotic Chromosome
 23
Genetic Information
DNA Replication
Transcription
DNA
RNA
Gene
Translation Expression

Protei
n
 24
Substrates Enzyme
Replication

DNA Polymerase
dATP
dGTP DNA
dTTP
dCTP DNA
Product

Template
 25
REPLICATION
Connects dNTP’s
(3’-5’ Phosphodiester bond)
Template instructions:
parent cell’s DNA
Single copy only:
prior to cell division

Error Rate = 1 in 1 x 109

 27
Procaryotic DNA is circular

DNA Gyrase +
Helicase + replication fork
SSB Protein

Origin of Replication = Replicon Site

 Replication Fork 28

Primer = ~ 10 base oligo RNA

inserted by Primase
5

5

3 DNA Polymerase
The new strand is formed 5  3
 29
In Real System DNA Pol
“dimers” act in tandem

5’ 3’

5’ 3’
Thymine has an equilibrium between 31
keto & enol forms
O
1
H CH3
HO N
CH3
N O N
keto

O N
enol
10,000 or
10 4
 Imino form of Adenine pairs with C
NH
H N
N
H2N

N N N
N
Adenine (imino)
N N
32 Adenine (amino)
 33
Fidelity of T C
Replication G
A G T A

Chance of error
C C
1 in 104 C G
T AT
C A
G G
G C
A C
C T
U G
A A
T
 34
DNA Polymerase “Activities”
5’-3’ Polymerase: (normal)
3’-5’ exonuclease: removes last base
in growing strand if incorrect

5’-3’ exonuclease: removes RNA primer

(while 5’-3’ Polymerase fills in gap)
 Fidelity of 35
Replication T C
G
Chance of error A G T A
1 in 104
C C
Chance of correction C G
failure : 1 in 10 4
T AT
C A
G G
G C
A C
C T
U G
A A error rate = 1 in 1 x 10 8/9

T
 discontinuous (lagging) strand
requires new primer

Continuation
of Replication

continuous strand
38 no additional primer
 39
Okasaki Fragments

discontinuous ~2,000 bps

continuous
 40
E coli DNA Polymerases
DNA Pol I: primer removal & gap filling
DNA Pol II: DNA repair?
DNA Pol III: main polymerase
()2 complex
proof reading processivity
5’-3’ polymerase dimerization
 41
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.
 DNA Polymerase fills in gaps 42

S-P-S-P S-P-S-P
T A
T G T G C T A C A
S-P-S-P-S-P-S-P-S-P-S-P-S-P

Gap: missing bases

filled in by 5’-3’ Polymerase activity
 43
Primer Excision:
5’-3’ Exonuclease Activity....

3’ 5’

.... removes RNA primer

while regular polymerase
activity fills in gap
 DNA Ligase seal nicks 44

S-P-S-P-S-P-S-P S-P-S-P-S-P
T A G C A C A
T G T G C T A
S-P-S-P-S-P-S-P-S-P-S-P-S-P

Nick: no bases missing

one phosphate ester bond not formed
 46
Mutations
 in DNA Sequence
Can occur due to uncorrected
enol/imino pairing during replication:
rate = 1 in 1 x 109

Can also occur due to DNA damage

during interphase
 47
DNA Repair

mutations or DNA damage show up as

structural deviations in DNA helix
DNA Repair Enzyme complexes recognize
distortions in DNA structure & removes
~12 bp segment from damaged side!
DNA Polymerase/Ligase fills in Gap/nick.
 H2N 48
Spontaneous O
deamination
N H
N U

O N
O N
C
The lack of CH3 on U in DNA marks as error
and instigates removing and repair to restore C
Why both T & U?
 10
Restriction Endonucleases/Methylases
Recognize and bind to specific DNA sequence
Function in bacteria is to destroy foreign DNA
1) if methylated – ignore
2) if ½ methylated – methylate other strand
3) if unmethylated - cut

Feature #2 allows for marking of ‘self’ DNA

Following replication/Cell Division
 11
Restriction Endonucleases
Enz Seq Enz Seq
AluI AG’CT HpaII C’CGG
TC’GA GGC’C
BamHI G’GATCC KpaI GGTAC’C
CCTAG’G CCATG’G
BglII A’GATCT MboI ‘GATC
TCTAG’A CTAG’
ClaI AT’CGAT PstI CTGCA’G
TAGC’TA G’ACGTC
EcoRI G’AATTC PvuI CGAT’GC
CTTAA’G GC’TACG
HaeII CG’CC SalI G’TGCAC
GC’GG CACGT’G
HindII GTp’PAC SmaI CCC’GGG
CAP’pTG GGG’CCC
HindIII A’AGCTT XmaI C’CCGGG
TTCGA’A GGGCC’C
 12
RFLP’s

4
4
5 5
4 4
2 2
2’
1 1 3 3

3 3
2

1 1
5 5
 13
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
 Southern Blot add 32P 2′ to 14
nitrocellulose blot

4
5
4
2

1 3

3
2

1
5
 15
Polymerase Chain Reaction (PCR)

Heat +
0 16

Long strands add while short strands double

 17
Generation Parent Long Short
0 1 0 0
1 1 1 0
2 1 2 1
3 1 3 4
4 1 4 11
5 1 5 26
6 1 6 57
7 1 7 120
8 1 8 247
=1 l+1 2s + l
 Sequencing DNA fragment : 18
Sanger Method
DNA ‘Piece” used as template
Radioactive Primer Added
One dideoxy-nucleotide (ddNTP)
added to mixture of 4 dNTP’s
Chain terminates at ddNTP
Electrophoresis run on
synthesized fragments.
dideoxy nucleotides will terminate DNA
chain because there is no 3 end to add to.

5’ end O O O
|| || || A
O - P - O - P - O - P -O
|
-
O
| | |
O_ O_ O_

O
|
C
_
O - P -O O |
|
O_
19 OH
3’ end
 20
3’- XXXXACGTAGCTTACC - 5’
5’-32PXXXX
Add ddTTP + dTTP, dATP, dCTP, dGTP

Get : XXXXT’
XXXXTGCAT’
XXXXTGCATCGAAT’
piece length = Primer + 1,5,10
 1 2 3 4 5 6 7 8 9 10 11 12
21
3’- XXXXACGTAGCTTACC - 5’
5’ PXXXX
-32

Fragment Sizes :
ddT : 1, 5, 10
ddA : 4, 8, 9
ddC : 3, 6
ddG : 2, 7, 11, 12
 22
T A C G

12
11
10
9
8
7
6
5
4
3
2
1

5’-XXXXTGCATCGAATGG
Hasil Agarose Elektroforesis