1. The Mutability and Repair of DNA
CHAPTER 9
The Mutability and Repair of DNA
生命科学学院 04级生物学基地班 徐均阳

2. OUTLINE 1. 2. 3. Replication Errors and Their Repair DNA Damage
Repair of DNA Damage 3.

3. FOREWORD
Life and biodiversity depend on a happy balance between mutation and its repair.
Two important sources of mutation are inaccuracy in DNA replication and chemical damage to the genetic material.

4. Errors in replication and damage to DNA have two consequences.
One is, of course, permanent changes to the DNA (mutations), which can alter the coding sequence of a gene or its regulatory sequences.
The second consequence is that some chemical alterations to the DNA prevent its use as a template for replication and transcription.

5. The challenge for the cell is twofold
The challenge for the cell is twofold. First, it must scan the genome to detect errors in synthesis and damage to the DNA. Second, it must mend the lesions and do so in a way that , if possible, restores the original DNA sequence.

6. Questions to be addressed
How is the DNA mended rapidly enough to prevent errors from becoming set in the genetic material as mutation?
How does the cell distinguish the parental strand from the daughter strand in repairing replication errors?

7. How does the cell restore the proper DNA sequence when the original sequence can no longer be read?
How does the cell deal with lesions that block replication?

8. REPLICATION ERRORS AND THEIR REPAIR
CHAPTER 9
REPLICATION ERRORS AND THEIR REPAIR

9. The Nature of Mutations
Point mutations
Transitions(转换), pyrimidine -to-pyrimidine and purine -to- purine substitutions, such as T to C and A to G;
Transversions(颠换), such as T to G or A and A to C or T (Figure 9-1).

10.                                                                                                                                                                                                                                                 
a.转换 　 b.颠换
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11. Other kinds of mutations
Insertions
Deletions
Gross rearrangements of chromosome structure
Such changes might be caused by the insertion of a transposon (转座子) or by the aberrant actions of cellular recombination processes.

12. Rate of spontaneous mutation at any given site on chromosomal ranges from 10-6 to per round of DNA replication, with some sites being “hotspot” .
Mutation-prone sequence in human genome are repeats of simple di-, tri- or tetranucleotide sequences, known as DNA microsatellites (微卫星DNA). These sequences are important in human genetics and disease, hard to be copied accurately and highly polymorphic in the population.

13. Some replication errors escape proofreading
The 3’→5’ exonuclease component of the replisome, which removes wrongly incorporated nucleotides, improves the fidelity of DNA replication by a factor of about 100.
If the misincorporated nucleotide is not subsequently detected and replaced, the sequence change will become permanent in the genome. It will result in a mutation in the DNA sequence.

14. Figure 9-2 A mutation may be introduced by misincorporation of a base in the first round of replication. In the second round of replication, the mutation becomes permanently incorporated in the DNA sequence.

15. Mismatch repair removes errors that escape proofreading
Final responsibility for the fidelity of DNA replication rests with this mismatch repair system, which increases the accuracy of DNA synthesis by an additional two to three orders of magnitude.
Two challenges:
1. scan the genome for mismatches
2. correct the mismatch accurately

16. In E. coli, mismatches are detected by a dimer of the mismatch repair protein MutS (Figure 9-3).MutS scans the DNA, recognizing mismatches from the distortion they cause in the DNA backbone.
MutS embraces the mismatch-containing DNA, inducing a pronounced kink in the DNA and a conformational change in MutS itself (Figure 9-4).

17.                                                                                                                                                                           
Crystal
structure of
the
MutS-DNA
Complex.
fg09-04

18. The complex of MutS and the mismatch-containing DNA recruits MutL, MutL activates MutH, an enzyme that causes an incision or nick on one strand near the site of the mismatch. Nicking is followed by the action of a specific helicase (UvrD) and one of three exonucleases.

19.                                                                                                                                                                         
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20. Mismatch repair pathway for the repair of replication errors.
Figure 9-3

21. Question: How does the E
Question: How does the E. coli mismatch repair system know which of the two mismatched nucleotide to replace?
Answer: The E. coli tags the parental strand by transient hemimethylation. The newly synthesized strand is not methylated by Dam methylase in a few minutes after the synthesis.

22. Dam methylation at replication fork. Replication generates
                                                                                                                                                                          
Dam methylation
at replication fork.
Replication
generates
hemimethylated
DNA in E. coli.
(b) MutH makes
incision in
unmethylated
daughter strand.
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23. Detail : Different exonucleases are used to remove ssDNA between the nick created by MutH and the mismatch.
It depend on whether MutH cuts the DNA on the 5’ or the 3’ side of the misincorporated nucleotide.

24. Exonuclease removal of mismatched DNA
                                                                                                                                                                                                                                                
Exonuclease removal of mismatched DNA
Unmethylated GATC is 5’ of mutation
Unmethylated GATC is 3’ of mutation
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25. Eukaryotic cells also repair mismatches and do so using homologs to MutS (MSH) and MutL (MLH). They have multiple MutS-like proteins with different specificities.
But they lack MutH and the trick of using hemimethylation to tag the parental strand.
The underlying mechanisms are not the same and not well understood.

26. CHAPTER 9
DNA DAMAGE

27. DNA Undergoes Damage Spontaneously(自发) from Hydrolysis and Deamination1 2 3
The action of water
Radiat-ion
Mutag-ens

28. 5-methyl C generates a natural base in DNA---T
Deamination of C
creates U
Depurination of G by
Hydrolysis creates
apurinic deoxyibose
Deamination of
5-methyl C generates a natural base in DNA---T
fg09-07 Mutation due to hydrolytic damage

29. A and G are also subject to spontaneous deamination.
Under normal physiological conditions, C undergoes spontaneous deamination, thereby generating the unnatural base U in DNA. U pairs with A, instead of G that would have been directed by C.
A and G are also subject to spontaneous deamination.
All of these hydrolytic reactions result in alterations to the DNA that are unnatural. This allows changes to be recognized by the repair systems.

30. Explaining DNA has T instead of U.
Deamination of 5-methyl cytosine → T will not be recognized as an abnormal base.
So can this kind of lesion be repaired?

31. DNA is damaged by alkylation (烷基化), oxidation (氧化) and radiation (辐射)
Figure 9-8 G
modification
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32. In alkylation, methyl or ethyl groups are transferred to reactive sites on the bases and to phosphates in the DNA backbone.
Alkylating chemicals: nitrosamines and the very potent laboratory mutagen.
DNA is subject to attack from reactive oxygen species (O2-, H2O2, OH•), generated by ionizing radiation and by chemical agents that generate free radicals.

33. Another type of damage to bases is caused by ultraviolet light
Another type of damage to bases is caused by ultraviolet light. Radiation with a wavelength of about 260 nm is strongly absorbed by the bases.
One consequences is the photochemical fusion of two pyrimidines that occupy adjacent positions on the same polynucleotide. (figure 9-9)

34.                                                                                                                                                                                                                                                 
fg09-09 Thymine dimer

35. Gamma radiation and X-rays are particularly hazardous because they cause double-strand breaks in the DNA (hard to repair).

36. Mutations are also caused by base analogs and intercalating agents
Base analogs: similar enough to the proper bases to get taken up by cells and incorporated into DNA during replication. Because of the structure differences, they lead to mistakes during the replication.
One of the most mutagenic base analogs is 5-bromouracil ( 5- 溴尿嘧啶).

37. Base analogue of thymine,
5- bromouracil, can
mispair with guanine.

38. Intercalating agents are flat molecules containing several polycyclic rings that bind to the equally flat purine or pyrimidine bases of DNA, just as the bases bind or stack with each other in the double helix.
Intercalating agents, such as proflavin(原黄素), acridine(丫啶), and ethidium(溴乙锭), cause the deletion or addition of a base pair, or even a few base pairs.

39.                                                                                                                                                                                                                                                 
Intercalating agents
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40. CHAPTER 9
Repair of DNA Damage

41. Two consequence of DNA damage
1.Some damages, such as thymine dimer, nick or breaks in the DNA backbone, create impediments to replication or transcription
2.Other damages create altered bases that have no effect on replication but cause mispairing; these can result in a permanent alternation to the DNA sequence after replication.

42. Mechanisms to repair a damage
１　→　２　→　３　→　４　→　５ 3 1 2 4
Direct
Reversal
of DNA
damage
Base excision repair (切割修复)
Nucleotide
excision
repair
Translesion(移损) DNA synthesis
Recombination (DSB) repairs

43. Direct reversal of DNA damage
                                                                                                                                                                                                                                               
Photoreactivation: directly reverse the
formation of pyrimidine dimers that
result from ultraviolet irradiation.
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44. Methyl group removal
Methyl transferase catalyzes the transfer of the methyl group on O6-methyl guanine to a cysteine residue on the enzyme, thereby restoring the normal G in DNA.
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45. Base excision repair enzyme remove damaged bases by a base-flipping mechanism
Glycosylase:
recognizes and removes the damaged base by hydrolyzing the glycosidic bond → remove the resulting abasic sugar, apurinic and apyrmidinic sugars → a repair DNA polymerase and DNA ligase restore an intact strand

46. Base excision pathway: the uracil glycosylase reaction
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47. Structure of a DNA-glycosylase complex
                                                                                                                                                                          
Structure of a DNA-glycosylase complex
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48. A glycosylase recognizes the mispair and removes A.
oxoG: A repair
                                                                                                                                                                                                                                               
A glycosylase recognizes the mispair
and removes A.
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49. Another example of a fail-safe system is a glycosylase that removes T opposite a G.
The glycosylase system assumes that the T in a T:G mismatch arose from deamination of 5-methyl-cytosine and selectively removes the T so that it can be replaced with a C.

50. Don’t recognize any particular lesion.
Nucleotide excision repair enzymes cleave damaged DNA on either side of the lesion
Don’t recognize any particular lesion.
Recognize distortions to the shape of the double helix
Remove a short single-stranded segment that includes the lesion.
Use DNA polymerase to fill in the gap.

51. Figure 9-16 Nucleotide excision repair pathway UvrB melts DNA
UvrABscan DNA
to identify a distortion
UvrB melts DNA
locally around
the distortion.
UvrC forms
a complex
with UvrB
and creates
nicks.
UvrA leaves the complex
Figure 9-16
Nucleotide excision
repair pathway
Release the single stranded fragment and repair and seal the gap

52. fg09-17 Transcription coupled DNA repair
                                                                                                                                                                         
Transcribe
DNA
normally
Upon
encountering
the lesion,
transcription
stops.
Recruit proteins
to the lesion,
then back up or
dissociates to
allow the proteins
access to the
lesion.
fg09-17 Transcription coupled DNA repair

53. Recombination repairs DNA breaks by retrieving sequence information from undamaged DNA
Double-strand break (DSB) repair pathway: retrieves sequence information from the sister chromosome. (Details are in chapter 10.)
DNA recombination also helps to repair errors in DNA replication.
Nonhomologous end joining (NHEJ): don’t involve homologous recombination.

54. Translesion DNA synthesis enables replication to proceed across DNA damage
Highly error-prone and likely to introduce mutations
Be catalyzed by a specialized class of DNA polymerases that synthesize DNA directly across the damage site
Template dependent, independent of base pairing
Not be random
A system of last resort
Be introduced only in response to DNA damage
The genes are expressed as part of a pathway known as the SOS response.

55. fg09-18
Translesion
DNA
synthesis

56.                                                                                                                                                                            
Figure 9-19
Crystal
structure of
a translesion
polymerase

57. CHAPTER 9
Summery

58. Types of mutations: base substitutions , small and large additions and deletions, and gross rearrangements of DNA sequences.
Cells have a large repertoire of enzymes devoted to repairing DNA damage. Some enzymes directly reverse DNA damage, such as photolyses, which reverse pyrimidine dimer formation.
A more versatile strategy is excision repair, in which a damaged segment is removed and replaced.

59. In base excision repair, DNA glycosylases and endonucleases remove only the damaged nucleotide. In nucleotide excision repair, a short patch of single-strand DNA containing the lesion is removed.
Recombination or double-strand break repair, in which an intact DNA stand is copied from a different but homologous duplex, is available when no template for repair synthesis.

60. Finally, translesion synthesis enables replication to continue across damage. It works in an error-prone manner that doesn’t depend on base pairing.

61. Thank You !