Lecture 4- Nucleic Acid Structure
DNA modifications
-Recall from last lecture alkylation by mustards can lead to
tautomerization and mispairing
-Alkylation by mustards can also lead to interstrand crosslinking which
blocks transcription and replication
-Alkylation at N7 can also lead to backbone cleavage
OH
CH3
N
O
O
NH
N
O
O
CH3
N
HO
O
NH
N
O
N
N
NH2
O
O P O
OR
NH2
O
O P O
OR
DNA crosslinking molecules
Mitomycin A
NH2
O
Bind DNA
(intercalation)
O
O
MeO
OMe
N
Me
O
H
CH3
N
H
OH N
CH3
N
O
O
NH
H
O
H
N
O H
O P O
OR
B
NH2
O
H
O
O P O
OR
+
H
H2N
NH
N
NH2
Alkylation leads to the
cleavage of backbone
MeO
NH
N
Me
N
Me
NH2
OH
NH2
OH
NH2
NH2
O
CH3
N
O
NH2
O
OH
MeO
O
O P O
OR
CH3
N
O
NH2
H2N dG
O
MeO
O
NH
1. - MeOH
2. 1,3-H shift
O
OH
OH H
N
Me
O
Base
O
NH
OMe
Mitomycin A
- DNA alkylator
- reductive activation
N
N
MeO
OH
O
O
2H+, 2e-
NH2
O
OH
O
O
O
O
H
N
O
MeO
OH
NH2
N
NH2
OH
H
N dG
H
N
OH
MeO
dG
N
Me
OH
NH2
dG
N
Me
NH
N
H
N
dG
N
Me
H2N dG
OH
NH2
inter- or intrastrand cross-linking
Lecture 4- Nucleic Acid Structure
Antibiotics (function by modify DNA)
DNA crosslinking molecules
O
O P O
O
Benzopyrene
- activated by p450 (Cyp1A1)
- from fuel, cigarette smoke, burnt toast
- intercalates into DNA
-
-
-
B
O
epoxide
hydrolase
O
O P O
O
B
O
O
O
O P O
O
O OH
B
O
O
O
H2N dG
dG
HN
O
OH
O
O P O
OH
HO
HO
MeO
O
O
OH
Sugar O
B
OH
O
O
O
O P O
O
O
SR
O
O
Enediyne (Neocarzinostatin)
- extremely toxic
- intercalates into DNA
- bind carrier protein
O
O P O
O
-
Or
O
-
O
B
-
O
HO O
O
O
O
O
O
O
RS
O
SR
O
Bergman
Cyclization
OH
RO
OH
O
Sugar
.
.
O
Sugar
phenyl radical
110 kcal/mol (C-H bond)
O
O P O
O
B
O
O
RO
B
cannot be repaired
OH
O
H
O
Bind DNA
(intercalation)
Me
-
OH
OH
Enediyne (Neocarzinostatin)
O
O P O
O
-
O
O P O
O
-
3'-phosphodiester cleavage;
same as
EDTA.Fe(II)
mechanism
O
HO
O
B
B
Lecture 4- Nucleic Acid Structure
Modification of DNA (Enzyme mediated)
-Epigenetic modifications passed on through multiple rounds of
cell divisions; most common is 5-methyl-C
NH2
NH2
CpG
N
N
Me
O
N
N
5.MeC
O
-modifcation of DNA causes silencing/activation of gene expression
-DNA methyl transferases (DNMT) modify hemimethylated DNA
-also 5-hydroxymethylcytosine found in brain
O
Tools for DNA
-Synthesize
-Sequence
-Amplify (PCR)
Polymerase Chain Reaction (PCR)
5'
3'
3'
5'
Denaturation
5'
3'
HO
5'
N
NH
HO
O
N
5.HOMeC
O
-N-Ac-Lys
-neutralize charged lysine (structural effects on chromatin)
-bromodomain of transcription factors binds to -N-Ac-Lys (popular
drug target) and recruits protein TFs and cofactors
5'
5'
3'
-RNA is a subject to many base modifications
-alkylate exocyclic NH2 of A/G/C
-alkylate ring N of N1A, N7G, N3C
O
S
R1
5'
3'
3'
5'
3'
O
O
O
O P O P O P O
O
O
O
O
B
OH
Extension
5'
Sequencing of DNA
Chemical sequencing
-A/G/C/T specific reactions enable specify DNA modification to
identify A/G/T/C
Sanger sequencing
5'
3'
RNA modifcations
R2
Annealing
10 cycle 210 ~103
HAT histone acetyl transferase
K14/K9 H3
3'
DNA Polymerase
Modification of histone proteins
R1
3'
O
NH
5'
ds DNA
R2
-saturate C(5)-C(4)
-glycosylate (allow phage to identify E. coli genome from phage
genome.
ss DNA
3'
5'
+
Insert into D5
Phage genome
isolate
ssDNA
+
3'
RF restriction
site
DNA
5'
3'
(+DNA Poly)
-M13 phage has ssDNA form
-RF (replicated form) ds DNA
-packaged form ssDNA
5'
Lecture 4- Nucleic Acid Structure
Sequencing of DNA
Sanger sequencing uses dideoxy chain terminators
PPPO
Find G:
add dNTPs (A/G/C/T) +
O
5'
3'
5'
3'
DNA synthesis (continue)
Base labile
R (ddGTP)
O
O
A:
C:
N
H
O
N
H
G:
N
H
OMe
CddG
GCATGC
5'
Acid Label/Base Label-orthogonal protecting groups
CGTACddG
GCATGC
5'
HO
DMTO
......
ddT
ddA
ddC
ddG
O
+
H
N
O
B'PG
O
BPG
O
NC
O
O
P
N
O
Drug for Ebola
HO
H
N
O
H2
N
DMTO
Ebola RNA polymerase will take it as a
substrate
Human RNA polymerase will not take it
NH
NH2
OH OH
B'PG
O
CN
O
P O
O
O2, I2 oxidation
O
DNA synthesis
-phosphotriester
-phosphoramidite
Solid-phase synthesis-rapid way to purify reactants from products
OMe
more methoxy group
less acid for cleavage
O
O
Acid labile
DMT dimethoxytrityl
R1
H
R2
O R4
R3
BPG
O
H
N
OMe O
O
R1
R2
HO R4
R3
stable cation
B'PG
O
BPG
O
BPG
O
O
H
N
O
CN
O
O P O
O
H
N
O
O
O
CN
RO
H
B
beta elimination
to avoid phosphodiester bond
cleavage
1. H+ deprotection
2. coupling
3. solid phase purification
NH4OH
H+