1. Lecture 7 DNA repair Chapter 10 Problems 2, 4, 6, 8, 10, 12, and 14
Molecular Biology of the Gene Lecture 7
2/6/2013
Lecture 7 DNA repair Chapter 10 Problems 2, 4, 6, 8, 10, 12, and 14
Quiz 3 due today at 4:00 PM

2. Transversion mutations
10_Figure01.jpg
Types of mutations
Transition mutations
Transversion mutations
Pyrimidine to __________
Purine to ___________
Pyrimidine to __________
Purine to ___________

3. Mutations can be permanently fixed if they are not repaired
10_Figure02.jpg
Mutations can be permanently fixed if they are not repaired
before the next round of replication
Polymerase errors cause a distortion of the DNA helix

4. Mismatch repair of mutations in E. coli
10_Figure03.jpg
Mismatch repair of mutations in E. coli
MutS protein recognizes mismatch, induces a kink in the DNA, and binds ATP
MutS recruits MutL and MutH
MutL activates MutH. MutH nicks DNA
Helicase unwinds strand. An exonuclease degrades the strand with the mutation
Polymerase III fills in the gap

5. MutS complexed with DNA
10_Figure04.jpg
MutS complexed with DNA
ATP
kink in DNA

6. which strand contains the mutated base?
10_Figure05.jpg
How does the cell know
which strand contains the
mutated base?
Dam methylase does not
methylate right after replication.
Later A in GATC sequences
are methylated.
MutH nicks the unmethylated
strand of DNA in the mismatch
repair process

7. Directionality in mismatch repair
10_Figure06.jpg
Directionality in mismatch repair
The exonuclease binds the MutH nick and degrades DNA
travelling toward MutS until MutS and the mismatch are found
Exonucleases have a distinct polarity (5’->3’ or 3’->5’) so at least
2 different exonucleases must be used in mismatch repair

8. Eukaryotic cells lack a MutH homolog
Then how do the mismatch repair enzymes recognize the newly synthesized strand of DNA?
Nicks present in unligated Okazaki fragments may play this role following lagging strand DNA synthesis.
But how nicks get introduced in the leading strand DNA is not quite clear.

9. Hydrolytic damage of DNA
10_Figure07.jpg
Hydrolytic damage of DNA
U base pairs with A
What is the result?
Adenine and guanine also spontaneously deaminate to
hypoxanthine and xanthine.
Which type of damage is worst?
Why didn’t DNA evolve to use U?
deamination
Abasic site (apurinic deoxyribose)
depurination
deamination

10. Ames Test to detect mutagens (carcinogens)
10_UnFigure01.jpg
Ames Test to detect mutagens (carcinogens)
A base substitution or frameshift
mutation is introduced in a gene
used to make the amino acid histidine.
Detects reversions
of mutation
Some mutagens need
to be activated in the liver.
So liver extract is commonly
added to the mutagen.

11. Base damage by alkylation and oxidation
10_Figure08.jpg
Base damage by alkylation and oxidation
Alkylation – introduction of methyl or ethyl groups by chemicals
(nitrosamines)
Can form O6-methylguanine
when alkylated
8-oxoguanine (Oxo-G) forms after oxidation. It can basepair with A.
Would this lead to transition or transversion mutation?

12. Radiation-induced DNA damage
10_Figure09.jpg
Radiation-induced DNA damage
UV light causes adjacent pyrimidines to covalently bond.
This blocks progression of DNA polymerase.
Gamma and X-ray radiation and some drugs (bleomycin)
induce double stranded breaks in DNA

13. Chemicals that cause mutations in DNA
10_Figure10.jpg
Chemicals that cause mutations in DNA
Base analog
of thymidine
What is result?
Intercalating agents-
insert between bases
causing insertions
& deletions in DNA
What type of
interaction with
bases?

14. 10_Table01.jpg
DNA repair systems 10

15. Direct DNA repair: Photoreactivation
10_Figure11.jpg
Direct DNA repair: Photoreactivation
Visible light is used as the energy to repair thymine dimers

16. Direct DNA repair: methyl group removal
10_Figure12.jpg
Direct DNA repair: methyl group removal
Repair of alkylation of O6-methylguanine
A cysteine on the methyltransferase binds the methyl
group on guanine.

17. Base excision repair pathway Example: uracil glycosylase
10_Figure13.jpg
Base excision repair pathway
Example: uracil glycosylase
How does uracil usually get in DNA?
AP site
What type of site is present after glycosylase action?
8 different DNA glycosylases in humans (ex. Oxo-G).
They scan minor groove looking for damaged bases
and then use base flipping to access base for repair.

18. Glycosylase protein is gray.
10_Figure14.jpg
Oxo-G glycosylase
Glycosylase protein is gray.
What is different
about the red base?
DNA is purple

19. It’s not always too late to repair DNA after replication.
10_Figure15.jpg
Oxo-G:A repair
It’s not always too late to repair DNA after replication.
A glycosylase recognizes oxo-G:A and removes the A.
Another glycosylase recognizes G:T basepairs and
removes the T which likely arose from spontaneous
deamination of 5-methylcytosine.

20. Nucleotide excision repair in E. coli
10_Figure16.jpg
Nucleotide excision repair in E. coli
Nucleotide excision repair recognizes
distortions in the double helix.
UvrA+UvrB scan DNA.
UvrA recognizes distortion and leaves.
UvrB melts DNA to form single-stranded bubble
UvrC is recruited and cuts DNA 8 nucleotides
5’ of the legion and 4-5 nucleotides 3’ of
the legion.
Helicase UvrD removes the single strand.
DNA polymerase I and DNA ligase fill the gap.

21. Nucleotide excision repair (NER) in humans
Similar but more complex than NER in E. Coli.
Mammalian NER uses around 25 proteins.
XPC recognizes distortions (like UvrA in E. coli).
XPA and XPD helicases melt DNA (like UvrB in E. coli).
Single stranded binding protein RPA binds DNA.
5’-cleavage site cut by ERCC1-XPF nuclease and 3’-cleavage site cut by XPG nuclease (similar to UvrC in E. coli)
24-32 nucleotide long DNA strand is released that is filled in
by a polymerase and sealed by DNA ligase.
Xeroderma pigmentosum disease caused by mutations in XP_ (NER) genes. Patients are susceptiple to cancer from UV light.

22. Transcription coupled DNA repair
10_Figure17.jpg
Transcription coupled DNA repair
In humans when transcription of DNA stalls due to a lesion in DNA, the RNA polymerase recruits the NER proteins.
The TFIIH complex needed for melting DNA for transcription contains XPA and
XPD. What is the significance of this?

23. Mammalian non-homologous end joining
10_Figure18.jpg
Mammalian non-homologous end joining
(NHEJ) pathway to repair double stranded
DNA breaks.
Double stranded breaks (DSB) are
the most toxic of all types of DNA damage.
Ku70/Ku80 heterodimer binds ends of DNA and recruits DNA-protein kinase cs.
Artemis, an exo/endonuclease, is phosphorylated by DNA-PKcs and
processes the DNA ends. Ligase IV
complex attaches the 2 ends together.
NHEJ also used in VDJ recombination
to produce staggering amounts of
different types of antibodies to fight infections and in Bacillus subtilis
bacterial spores to protect the DNA.

24. Translesion DNA synthesis in E. Coli
10_Figure20.jpg
Translesion DNA synthesis in E. Coli
Erroneous and used as last resort, but used
to replicate through DNA lesions.
Sliding clamp and DNA Pol III fall off DNA.
A translesion polymerase (Pol IV or Pol V)
copies across lesion (thymidine dimer).
Translesion polymerase falls off and DNA
Pol III holocomplex resumes replication.
UmuC part of Y family of DNA polymerases.
Pol V contains UmuC

25. Incoming nucleotides in red and template in blue
10_Figure21.jpg
Y family polymerase (left) & high fidelity T7 phage polymerase (right)
translesion
polymerase
normal polymerase
Incoming nucleotides in red and template in blue
What do you notice about the structure around
the active site (yellow arrow)?

26. The Y Family of translesion polymerases
10_UnFigure02.jpg
The Y Family of translesion polymerases
Translesion DNA synthesis in E. Coli is induced by SOS DNA damage response.
Adding ubiquitin peptide to sliding clamp at lesion recruits translesion polymerase.

27. 10_Table01.jpg
DNA repair systems 10

28. Double strand break repair pathway (homologous recombination)
Uses information from homologous sister chromosome and will be discussed next lecture (chapter 11)