1. DNA Repair Types Direct repair Excision repair Recombination repair
Alkylguanine transferase
Photolyase
Excision repair
Base excision repair
Nucleotide excision repair
Mismatch repair
Recombination repair

2. 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.

3. AGT inhibitor O6-benzylguanine is in clinical trials to be
used in conjunction with antitumor alkylnitrosoureas

4. AGT overexpression in tumors makes them resistant to
alkylnitrosoureas

5. Combination therapy with O6-benzylguanine overcomes tumor
resistance to alkylnitrosoureas

6. Excision Repair
Takes advantage of the double-stranded (double information) nature of the DNA molecule.
Four major steps:
Recognize damage.
Remove damage by excising part of one DNA strand.
The resulting gap is filled using the intact strand as the template.
Ligate the nick.

7. Antiparallel DNA Strands contain the same genetic information5' 3' 5' 3' 5' 3'
A :: T
A :: T
A :: T
G ::: C G
G ::: C
Two polynucleotide chains running in the opposite directions
Each base is H-bonded to a complementary base in the opposite strand
2. The two chains are held together by stacking (hydrophobic) interactions, hydrogen bonds, and electrostatic forces.
3. Base pairing is in the order A:T, which contains two hydrogen bonds, and G:C, which contains three hydrogen bonds.
T :: A
T :: A
T :: A 3' 5' 3' 5' 3' 5'
Original DNA duplex
DNA duplex with
one of the nucleotides
removed
Repaired DNA duplex

8. Base excision repair (BER)
Used for repair of small damaged bases in DNA (AP sites, methylated bases, deaminated bases, oxidized bases…)
Human BER gene hogg1 is frequently deleted in lung cancer O N N H N N O H X a n t h i e

9. Uracil DNA glycosylase removes deaminated C
No Me group N H 2 O
BER C N N H N O N O
Cytosine
Not normally present in DNA U r a c i l
Normal DNA base
Not recognized by BER

10. 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).
Xeroderma pigmentosum is a genetic disorder resulting
in defective NER

11. Mismatch Repair Enzymes
Nucleotide mismatches can be corrected after DNA synthesis!
Repair of nucleotide mismatches:
Recognize parental DNA strand (correct base) and daughter
strand (incorrect base)
Parental strand is methylated:
2. Replace a portion of the strand containing erroneous nucleotide
(between the mismatch and a nearby methylated site –up to 1000 nt)
G T

12. Genetic diseases associated with defective DNA repair
Xeroderma Pigmentosum NER
Hereditary nonpolyposis MMR
colorectal cancer
Cockrayne’s syndrome NER
Falconi’s anemia DNA ligase
Bloom’s syndrome BER, ligase
Lung cancer (?) BER

13. Chapter 3. RNA synthesis (Transcription)
Required reading: Stryer 5th edition p ,
(or Stryer 4th edition p , , )

14. Flow of genetic information
DNA
RNA
Proteins
Cellular Action
replication
transcription
translation
Recall that the genetic information stored in DNA is retrieved in a two step process. Transcription generates a copy of RNA identical in sequence to one of the DNA strands, and translation converts the nucleotide sequence of RNA into aa sequence of the corresponding protein.
DNA

15. RNA is a biopolymer consisting of ribonucleotide units5’
RNA is usually single stranded
but can form hairpin structures
by folding over the same strand: G C U
5'-U•C•C•C•A•C
A•U•U•U-3'
G•C
A•U
C•G
Loop
Double-
stranded
region
(stem) 3’

16. Structural differences between DNA and RNA

17. 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

18. RNA in E. coli

19. 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)
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.
2Pi

20. RNA Synthesis: addition of new NTPs follows Watson-Crick rulesNH 2 N H 2 N O N N NH N N HN N N N N NH 2 O O
A•U
G•C
Template base Incoming base
G C
C G
U A
A U
The addition of nucleotides follows Watson-Crick base pairing rules (G:C, A:T) by forming specific hydrogen bonds between nucleobases in the template strand and the incoming dNTPs. The likelyhood of phosphodiester bond formation is very low unless WC bonds are formed

21. Non-coding (template) strand
The sequence of the RNA transcript is complementary to the transcribed DNA strand and is the same as the coding strand
Coding strand
Non-coding (template) 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’
5’- A U G G C C U G G A C U U C A…3’
Transcription
Translation
…Met-Ala-Trp-Thr-Ser…
Peptide