1. Chapter 9 The mutability and repair of DNA
04生物学基地班 张欣

2. *replication errors and their repair *DNA damage *repair DNA damage
Outline
*replication errors and their repair
*DNA damage
*repair DNA damage

3. Introduction
Organisms can survive only if their DNA is replicated faithfully and is protected from chemical and physical damage that would change its coding properties.The limits of accurate replication and repair of damage are revealed by the nature mutation rate.
Thus,an average nucleotide is likely to be changed by mistake only about once every 109 times it is replicated.

4. Two important sources of mutation
*inaccuracy in DNA replication
*chemical damage to the genetic material

5. Errors in replication and damage have two consequences
1.Permanent changes to the DNA (mutations),
can alter the coding sequence of a gene or its regulatory sequences.
2.Some chemical alterations to the DNA prevent its use as a template for replication and transcription.

6. Since the mutation is so important for all the living things,now we will consider errors that occur during replication and how they are repaired. We will see that multiple overlapping systems enable the cell to cope with a wide range of insults to DNA,underscoring the investment that living organism make in the preservation of the genetic material.

7. Part ⅰ Replication errors and their repair

8. The nature Mutations
Mutations include almost every conceivable change in DNA sequence. The simplest mutations are switches of one base for anther. There are two kinds:Thansitions and
Transversions.

9. Other kinds of mutation cause more drastic changes in DNA,such as extensive insertions and deletions and gross rearrangements of chromosome structure. Such change may be caused by transposon.
One kind of sequence that is particularly prone to mutation merits special comment because of its importance in human genetics and disease. Like DNA microsatellites.

10. Some replication errors escape proofreading
As we have seen,the 3’-5’ exonuclease component of the replisome,which removes wrongly incorporated nucletides. The proofreading exonuclease is not however,foolproof. Some misincorporated nucleotides escape detection and become a mismatch between newly synthesized srand and the template srand.

11. A mutation can be permanently incorporated by replication
A mutation can be permanently incorporated by replication. In the second round the mutation becomes permanently incorporated in the DNA sequence

12. Mismatch repair removes errors that escape proofreading
Mismatch repair system can detect mismatches and repair them. There are two challenges:1,it must scan the genome for mismatches,it will rapidly find and repair .
2.the system must correct the mismatches accurately,it must replace the misincorporated nucleotide in the newly synthesized stand not the parental strand

13. In E.coli,mismatches are detected by a dimer of the mismatch repair protein MutS. MutS scans the DNA,recognizing mismatches from the distortion they cause in the DNA backbone. MutS embraces the mismatch-containing DNA, inclosing a pronounced kink in the DNA and a conformational change in itself.A key to the specificity of MutS is that DNA containing a mismatch is much more readily distorted than properly base-paired DNA.

15. E.Coli tags the parental strand by transient hemimethylation
E.Coli enzyme Dam methylase methylates a residues on both strands of the sequences 5’-GATC-3’.when a replication fork passes through DNA that is methylated at GATC sites on both strands,the resulting daughter DNA duplexes will be hemimethylation .

16. Dam methylation at replication fork (a)replication generates hemimethylated DNA in E.coli(b)MutH makes incision in unmethylated daughter strand

18. Eukaryotic cells also repair mismatches and do so using homologs to MutS and MutL. Indeed, eukaryotic have multiple MutS-like proteins with different specificities.

19. Part ⅱ DNA damage

20. DNA undergoes damage spontaneously from hydrolysis and deamination
Some damage is caused by environmental factors such as radiation and mutagens.
The most frequent and important kind of hydrolytic damage is deamination of base cytosine.
The hazard of having deamination generate a naturally occurring base is illustrated by the problem caused by presence of 5-methylcytosine.

22. DNA is damaged by alkylation oxidation and radiation
DNA is vulnerable to damage from alkylation oxidation and radiation .
In alkylation,methyl or ethyl groups are transferred to reactive sites on the bases and to phosphates in the DNA backbone.
DNA is also subject to attack from reactive oxygen species

23. Thymine dimer.uv includes the formation of a cyclobutane ring between adjacent thymines.

24. Another type of damage to bases is caused by ultraviolet light
Another type of damage to bases is caused by ultraviolet light.Reaction with a wavelength of about 260 nm is strongly absorbed by the bases
Gamma radiation and X-rays are particularly hazardous because they cause double-strand breaks in the DNA.

25. Mutations are Also Caused by Base Analogs and Intercalating Agents
Mutations are also caused by components that substitute for normal bases or slip between the bases to cause errors in replication.
Base analogs are structurally similar to proper bases but differ in ways that make them treacherous to the cell .

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

27. TOPIC ⅲ Repair of DNA damage

28. In this section, we consider the systems that repair damage to DNA
In this section, we consider the systems that repair damage to DNA. In the most direct of these systems ,a repair enzyme simply reverses the damage. One more elaborate step involves excision repair systems, in which the damaged nucleotide is not repaired but removed from the DNA.
In excision repair systems, the other, undamaged, strand serves as a template

29. for reincorporation of the correct nucleotide by DNA polymerase.
More elaborate is recombinational repair,which is employed when both strands are damaged as when the DNA is broken. In such situations, one strand cannot serve as a template for the repair of the other. Hence in recombinational repair ,sequence information is retrieved from a second undamaged copy of the chromosome

30. Direct Reversal of DNA Damage
An example of repair by simple reversal of damage is photoreactivation.
Photareactivation directly reverses the formation of pyrimidine dimers that result from ultraviolet irradiation.

31. Photoreactivation

32. Another example of direct reversal is the removal of the methyl group from the methylated O6-methylguanine .

33. Base Excision repair enzymes remove damaged bases by a base-flipping mechanism
The most prevalent way in which DNA is cleansed of damaged bases is by repair systems that remove and replace the altered bases.
The base excision repair, an enzyme called a glycosylase recognizes and removes the damaged base by hydrolyzing the glycodic bond.the resulting abasic sugar is removed from the DNA backbone in a further endonucleolytic step.

34. Base excision pathway:the uracil glycosylase reaction

35. endonucleolytid cleavage also removes apurinic and apyrimidinic sugars that arise by spontaneous hydrolysis. After the damaged nucleotide has been entirely removed from the backbone, a repair DNA polymerase and DNA ligase restore an intact strand using the undamaged strand as a template.

36. Dleansing the genome of damaged bases is a formidable problem because each base is buried in the DNA helix. Evidence indicates that these enzymes diffuse laterally along the minor groove of the DNA until a specific kind of lesion is detected.
X-ray crystallographic studies reveal that the damaged base is flipper out so that it projects away from the double helix,where it sits in the specificity pocket of the glycosylase.

37. Nucleotide excision repair enzymes cleave damaged DNA on either side of the lesion
Unlike base excision repair ,the nucleotide excision repair enzymes don't recognize any particular lesion. rather ,this system works by recognizing distortions to the shape of the double helix

38. Nucleotide excision repair pathway

39. Not only the nucleotide excision repair capable of mending damage throughout the genome, but it is also capable of rescuing RNA polymerase, the progression of which has been arrested by the presence of alesion in the transcribed strand of a gene. this phenomenon ,known as transcription-coupled repair, involves recruitment to the stalled RNA polymerase of nucleotide excision repair proteins .

40. Recombination repairs DNA breaks by retrieving sequence information from undamaged DNA
Excision repair uses the undamaged DNA strand as a template to a replace a damaged segment of DNA on the other strand.
This is accomplished by the double-strand break(DSB) repair pathway, which retrieves sequence information from the sister chromosome.
DNA recombination also helps to repair errors in DNA replication.

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