1. Definitions: ★ replication errors ★ spontaneous DNA damage
★ DNA mutations
★ double-strand break (DSB) repair pathway

2. Questions to be addressed
How does the mismatch repair system accurately detect, remove and repair the mismatch resulting from inaccurate replication?
What are the environmental factors that cause DNA damage?

3. How could a DNA damage be converted to DNA mutation?
What are the mechanisms to repair a DNA damage? Describes how base excision repair and nucleotide excision repair work?
What is translesion DNA synthesis? Why it is important?

4. The mutability and repair of DNA
Replication errors and their repair
DNA damage
Repair of DNA Damage

5. Replication errors and their repair
Rapair:
Mismatch repair
Proofreading

6. The nature of mutations
Simple mutations:
Transitions(pyrimidine-to-pyrimidine and purine-to-purine)
Transversions(pyrimidine-purine and purine-to-pyrimidine)
Insertions and deletions (a nucleotide or a small number of nucleotides)
★point mutations: mutations that alter a single nucleotide

8. Other kinds of mutation:
----cause more drastic changes in DNA
Extensive insertions and deletions
Gross rearrangements
Such changes might be caused by the insertion of a transposon or by the aberrant actions of cellular recombination processes

9. hotspots—some sites on the chromosome where mutations arise at high frequency while other sites undergoing alterations at a comparatively low frequency about 10-6 to per round of DNA.
DNA microsatellites —one kind of sequence that is particularly prone to mutation merits special comment , because of its importance in human genetics and disease . They are repeats of simple di-,tri- or tetranucleotide sequences, which are known as DNA microsatellites. (eg.dinucleotide sequence CA)

10. The replication errors escape proofreading
Proofreading improves the fidelity of DNA replication by a factor of about 100.The proofreading exonuclease is not poolproof.
If the misincorporated nucleotide is not subsequently detected and replaced, the sequence change will become permanent in the genome.

12. Mismatch repair removes errors that escape proofreading
★Increase the accuracy of DNA by an additional 2-3 orders of magnitude
★Two challenges:
●scan the genome rapidly
●correct the mismatch accurately ( that is it must recognize the newly synthesized strand)
E..coli
we take for example

13. MutS scans the DNA, recognizing the mismatch from the distortion they cause in the DNA backbone

15. MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself.
The complex of MutS and the mismatch-containing DNA recruits MurL.
MutL,in turn, activates MutH, an enzyme that causes an incision or nick on one strand near the site of the mismatch.
Necking is followed by the action of a specific helicase(UvrD) and one of three exonucleases.

16. The helicase unwinds the DNA ,starting from the incision and moving in the direction of the site of the mismatch ,and the exonuclease progressively digests the displaced nucleotide.
This action produces a single-strand gap, which is then filled in by DNA polymeraseⅢ and sealed with DNA ligase.

17. ATP The MutS protein of Escherichia coli
MutS is responsible for recognizing and binding to base pair mismatches, and recruits other key proteins (MutH and MutL) required for repair to the mismatch site.

18. ?
How does the E..coli mismatch repair system know which of the two mismatched nucleotides to replace？
Dam methylases tags the parental strand by transient hemimethylation and methylates A residues on both strands of the sequence 5’-GATC-3’.
MutH protein become activated only when it is contacted by MutL and MutS located at a nearby mismatch.
NEXT PAGE

20. Different exonucleases are used to remove single-stranded DNA between the nick created by MutH and the mismatch.
It’s all depending on whether MutH cuts the DNA on the 5’ or the 3’ side of the misincorporated nucleotide.
NEXT PAGE

21. Unmethylated GATC is
5’ of mutation
Unmethylated GATC is
3’ of mutation

22. When it comes to eukaryotic cells
MSH == MutS homologs
MLH or PMS == MutL homologs
Eukaryotes have multiple MutS-like proteins with different specificities.

23. DNA damage Three reasons for DNA damage
hydrolysis(水解) and deamination(去氨基)
Alkylation, Oxidation, and Radiation
base analogs and intercalating agents

24. DNA undergoes damage spontaneously from hydrolysis(水解) and deamination(去氨基)
This is ironic since the proper structure of the double helix depends on an aqueous environment.

25. Deamination
C-U
Depurination
---->
an abasic site
Deamination of
5-mC---->T

26. DNA is damaged by Alkylation, Oxidation, and Radiation
Often mispqir with thymine
G:C –A:T
Reactive oxygen species
O2-, H2O2, OH•
G modification (alkylation & oxidation)

27. Thymine dimer by ultraviolet light
Incapable of base-pairing and cause the DNA
polymerse to stop during replication
Thymine dimer by ultraviolet light

28. Clastogenic – ionizing radiation and agents like bleomycin that cause DNA to break are said to be clastogenic.

29. Mutations are also caused by base analogs and intercalating agents
Base analogues

30. Intercalating agents which cause the deletion or addition of a base pair or a few base pairs

31. Repair of DNA damage The consequences of damage to DNA
Impediments (not permanent)
Mispairing(can cause permanent alteration)

32. Direct reversal of DNA damage
Photoreactivation (the enzyme DNA potolyase captures energy from light )

33. Methyl group removal
(a methyltransferase removes the methyl group by transferring it to one of its own cysteine residues)

34. Base exicision repair enzymes remove damaged bases by a base-flipping mechanism
Base excision repair
&
Nucleotide excision repair

35. Base excision pathway
A glycosylase acts by hydrolyzing the glycosidic bond
& then DNA polymerase and DNA ligase restore an intact strand

36. Structure of a DNA-glycosylase complex
DNA glycosylase leision-specific and cells have multiple DNA glycosylases with different specificities

37. ? & The DNA-glycosylase complexs diffuse
How do DNA glycosylases detect damaged bases while scanning the genome?
Due to the flexibility of DNA ,the damaged
base is flipped out so that it projects away
from the double helix
&
The DNA-glycosylase complexs diffuse
laterally along the minor groove of the DNA
until a specific kind of lesion is detected

38. If a damaged base is not removed by base excision before DNA replication
A fail-safe system

39. Use the undamaged DNA as a template
Nucleotide excision repair enzymes cleave damaged DNA on either side of the lesion
★recognize distortions
&
★ a single-stranded gap in the DNA
★ DNA polymerase or ligase fill in the gap.
Use the undamaged DNA as a template

40. Look at the next picture for detail
E..coli
When it comes to
There are four proteins about nucleotide exicision: UvrA, UvrB, UvrC, UvrD
Look at the next picture for detail

41. 2 3 1 4

42. 1.UvrA and UvrB scan DNA to identify a distortion
2. UvrA leaves the complex,and UvrB melts DNA locally round the distortion
3. UvrC forms a complex with UvrB and creates nicks to the 5’ side of the lesion
4. DNA helicase UvrD releases the single stranded fragment from the duplex, and DNA Pol I and ligase repair and seal the gap

43. Transcription coupled DNA repair:
nucleotide excision repair system is capable of rescuing RNA polymerase that has been arrested by the presence of lesions in the DNA template

44. Recombination repairs DNA breaks by retrieving sequence information from undamaged DNA
This is accomplished by the double-strand break (DSB) repair pathway

45. Damage in the DNA
template can lead to
DSB formation during
replication

46. DSB repair model for homologous
recombination

47. Translesion DNA synthesis enables replication to proceed across DNA damage
Occurs when the above repairs are not efficient enough
a fail-safe or last resort mechanism, which spares the cell the worse fate of an incompletely replicated chromosome

48. Translesion DNA synthesis
Catlyzed by a specialized class of DMA polymerases that synthesize DNA directily across the site of the damage

49. Crustal structure of a translesion polymerase

50. The enzyme is not ‘reading’ sequence information from the template
SOS response
Translesion synthesis is often
highly error-prone

51. THAT'S ALL