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DNA Repair Types
• Direct repair
– Alkylguanine transferase
– Photolyase
• Excision repair
– Base excision repair
– Nucleotide excision repair
– Mismatch repair
• Recombination repair
Direct repair: O6-alkylguanine DNA alkyltransferase (AGT)

Directly repaires O6-alkylguanines (e.g. O6-Me-dG, O6-Bz-dG)
In a stoichiometric reaction, the O6 alkyl group is transferred to a
Cys residue in the active site. The protein is inactivated and
degraded.

O
N
N
CH3
O
N
N
N
NH
AGT-CH2-SH
NH2
O6-methylguanine
N
N
AGT-CH2-S
NH2
CH3
AGT inhibitor O6-benzylguanine is in clinical trials to be
used in conjunction with antitumor alkylnitrosoureas
NO
N
Cl
NHR'
O
2-chloroethylnitrosoureas
Cl
O
N
Cl
dG in DNA
N
O
H
H
O
O
N
NH2
DNA-DNA cross-links
H
H
N
H
tumor cell death
AGT overexpression in tumors makes them resistant to
alkylnitrosoureas
NO
N
Cl
NHR'
O
2-chloroethylnitrosoureas
Cl
O
N
Cl
dG in DNA
N
O
H
H
O
O
N
N
NH2
DNA-DNA cross-links
H
H
H
AGT
normal dG
tumor cells survive
Combination therapy with O6-benzylguanine overcomes tumor
resistance to alkylnitrosoureas
NO
N
Cl
NHR'
O
2-chloroethylnitrosoureas
Cl
O
N
dG in DNA
Cl
N
O
H
H
O
O
N
N
DNA-DNA cross-links
H
H
NH2
H
O
N
N
H
AGT
N
N
NH2
normal dG
tumor cell death
Excision Repair
Takes advantage of the double-stranded (double information)
nature of the DNA molecule.
Four major steps:
1. Recognize damage.
2. Remove damage by excising part of one DNA strand.
3. The resulting gap is filled using the intact strand as the template.
4. Ligate the nick.
Antiparallel DNA Strands contain the same
genetic information
5'
3'
5'
3'
5'
3'
3'
A :: T
A :: T
A :: T
G ::: C
G
G ::: C
T :: A
T :: A
T :: A
5'
Original DNA duplex
3'
5'
DNA duplex with
one of the nucleotides
removed
3'
5'
Repaired DNA duplex
Base excision repair (BER)
• Used for repair of small damaged bases in DNA (AP
sites, methylated bases, deaminated bases, oxidized
bases…)
H
N
O
N
OH
N
NH
NH
O
N
8-oxo-G
NH2
O
O
O
O
NH2
Abasic site (AP
site)
N
N
H
Xanthine
O
N
N
N
N
Me
N3-Me-Ade
• Human BER gene hogg1 is frequently deleted in lung
cancer
Uracil DNA glycosylase removes deaminated C
No Me group
NH2
O
N
N
BER
NH
O
N
Cytosine
C
O
Not normally present in DNA
U rac il
O
H3C
NH
N
Thymine
O
Normal DNA base
Not recognized by BER
Nucleotide Excision Repair
•
Corrects any damage that both distorts the DNA molecule and
alters the chemistry of the DNA molecule (pyrimidine dimers,
benzo[a]pyrene-dG adducts, cisplatin-DNA cross-links).
O
H3C
O
NH
N
5'
O
O
O P OO
N
O
HO32N
CH
Pt
NH
H2N
N
Cl
Cl
O
O
3'
•
HO
O
NH
H2N N OHNH
O N
2
HOPt
OH H2N OH2
HO
OH
H
N
NH2
N
-GGH2N
H2N
N
Pt
N
O
N
N
NH
N
NH2
Xeroderma pigmentosum is a genetic disorder resulting
in defective NER
Mismatch Repair Enzymes
Nucleotide mismatches can be corrected after DNA synthesis!
Repair of nucleotide mismatches:
G
T
1. Recognize parental DNA strand (correct base) and daughter
strand (incorrect base)
Me
Parental strand is methylated:
H3C
NH2
HN
N
N
N
O
N
N
N
2. Replace a portion of the strand containing erroneous nucleotide
(between the mismatch and a nearby methylated site –up to 1000 nt)
Genetic diseases associated with defective DNA repair
Xeroderma Pigmentosum
NER
Hereditary nonpolyposis
colorectal cancer
MMR
Cockrayne’s syndrome
NER
Falconi’s anemia
DNA ligase
Bloom’s syndrome
BER, ligase
Lung cancer (?)
BER
Chapter 3. RNA synthesis (Transcription)
Required reading: Stryer 5th edition p. 129-132, 781-792
(or Stryer 4th edition p. 95-102, 841-849, 851-859)
Flow of genetic information
replication
DNA
RNA
Proteins
transcription translation
DNA
Cellular Action
RNA is a biopolymer consisting of
ribonucleotide units
NH2
5’
O
O
O
-O P O P O
P
OO- -O
O
N
N
N
N
NH
RNA is usually single stranded
but can form hairpin structures
by folding over the same strand:
N
NH2
N
NH2
U
N
O
O
O
OH
O P OO
O
H
O
OH
O P OO
O
Loop
N
N
U
O
N
O
O
N
3’
O
OH
O P OO
O
N
OH OH
NH
N
NH2
5'-U•C•C•C•A•C
G
G
G•C
A•U
C•G
C•G
G•C
C•G
C•G
G•C
O
NH
O
OH
O P OO
O
C
Doublestranded
region
(stem)
A•U•U•U-3'
Structural differences between DNA and RNA
DNA
RNA
O
O
H3C
NH
NH
N
O
H
Uracil (U)
N
O
H
Thymine (T)
HO
CH2
H
HO
O
Base
CH2
H
O
H
H
Base
H
H
H
H
O
H
O
2'-deoxyribose
O
BASE
2'
5'
O
3'
O
1'
H
OH
ribose
O
O
5'
3'
BASE
1'
O
2'
OH
RNA Types
mRNA = Messenger RNA; an RNA copy of the DNA sequence
(gene) used as a template for protein synthesis
tRNA = Transfer RNA; a small RNA that is attached to an
amino acid which can be added to a growing peptide chain
rRNA = Ribosomal RNA; component of
ribosomes with catalytic and structural
function
snRNA = Small nuclear RNA, involved in RNA splicing in
eukaryotes
RNA in E. coli
Type
Relative amount
Mass
# of Nucleotides
____________________%_________________kDa_____________________
1.2X103
0.6X103
3.6X101
3700
1700
120
15
 2.5X101
75
mRNA
5
heterogeneous
sn RNA
<1
rRNA
80
tRNA
23S
16S
5S
RNA synthesis
• RNA synthesis involves transcribing a specific portion of DNA strand
into RNA sequence
•RNA polymerases sequentially add ribonucleotides to the 3’ end
of an RNA polymer using DNA strand as a template (5’  3’ direction)
2Pi
(RNA)n bases + NTP
(RNA)n+1 bases +PPi
•RNA Pol selects ribonucleotides complementary to the DNA
template and catalyzes the formation of new phosphodiester bonds.
This process is repeated as the enzyme moves along the DNA.
RNA Synthesis: addition of new NTPs
follows Watson-Crick rules
N
O
N
H 2N
NH
N
N
N
NH 2
O
N
N
N
NH 2
N
N
O
O
A•U
G•C
Template base
G
C
U
A
HN
Incoming base
C
G
A
U
The sequence of the RNA transcript is
complementary to the transcribed DNA strand
and is the same as the coding strand
Coding strand
5’…A T G G C C T G G A C T T C A…3’
3’…T A C C G G A C C T G AA G T…5’
Non-coding (template) strand
Transcription
5’- A U G G C C U G G A C U U C A…3’
Translation
…Met-Ala-Trp-Thr-Ser…
Peptide