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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

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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....

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.... 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

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= 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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