Lecture 2: DNA Repair and Transcription Regulation Flashcards
1
Q
What is DNA damage?
A
Any change from the normal nucleotide sequence n supercoiled double helical state
2
Q
Causes of DNA damage
A
- Physical n chemical agents in the environment
- UV light, free radicals produced during metabolism
- Errors in DNA replication
3
Q
What 2 general classes does DNA damage fall into?
A
-
Single base changes – produces mutations but have no effect on physical process of transcription or replication
- Replication errors due to keto-enol type tautomerization
- Deamination of cytosine to uracil
- Incorporation of U rather than T during replication
- Chemical modification of bases
-
Structural distortions may impede transcription and/or replication
- Single strand breaks
- Covalent modification of bases e.g. alkylation
- Removal of a base
- Interstrand and intrastrand covalent bonds
4
Q
Cause of single base changes
A
- Replication errors due to keto-enol type tautomerization
- Deamination of cytosine to uracil → changes how they bind
- Incorporation of U rather than T during replication
- Chemical modification of bases
5
Q
Give an example of structural distorNon in the context of DNA damage
A
- Thymine dimer formation caused by UV light
- 2 T on the same strand become covalently linked
- Forms either
- Cyclobutene structure
- (6-4) photoproduct
6
Q
Cause of structural distortions
A
- Single strand breaks
- Covalent modifications of bases (e.g. alkylation)
- Removal of a base
- Interstrand n intrastrand covalent bands
7
Q
How are mismatches and structural distortions in DNA dealt with?
A
- Direct repair: doesn’t require nucleotide template, cleavage or synthesis
- Reversal or simple removal of the damage
- Mismatch repair: bidirectional excision-resynthesis, detects + removes mismatch
- Detection n repair of mismatched bases
- Excision repair: large parts of the DNA are removed n replaced (removal +synthesis)
- Recognition of the damage followed by excision of a patch of DNA n its replacement by undamaged DNA
- Tolerance systems: allows DNA replication to proceed thru damaged regions of DNA
- Retrieval systems: recombinational processes to repair damaged DNA
8
Q
Give an example of direct repair
A
Photoreactivation
- Repairs UV induced T-T dimers
- Photolyase binds to T-T dimers in the dark
- Contains 2 chromophores that absorb light energy
- Uses this energy to split cyclobutene structures
9
Q
Give an example of mismatch repair
A
Uracil DNA glycosylase
- Context: U is sometimes incorporated into DNA instead of T
- Uracil DNA glycosylase removes U
- RESULT: AP site (gap in DNA where there’s no base)
- AP endonuclease nicks the AP site
- Makes a break in the phosphodiester backhone
- DNA Pol I binds to the break
- Adds new nucleotide
- DNA ligase seals the gap
10
Q
What is the mut system?
A
- MutS: recognizes mismatches and short insertion/deletions (indels) on hemi-methylated DNA and binds to them
- MutL binds and stabilizes the complex
- MutS-MutL complex activates MutH
- MutH locates a nearby methyl group and nicks the newly synthesized strand opposite the methyl group
- MutU (Helicase II) unwinds the DNA from the nick in the direction of the mismatch
- DNA PolI degrades and replaces the unwound DNA and DNA ligase seals the single strand break
11
Q
What are the 3 excision repair modes found in E.coli?
A
- Very short patch (deals with mismatches between bases)
- Short patch: ~20 nucleotides
- Long patch: 1500 - 10,000 bps
12
Q
What does short and long patch repair utilize and what are they encoded by?
A
- Both short and long patch repair utilize the repair endonuclease
- Encoded by the uvrA, uvrB and uvrC genes
13
Q
Give an example of excision repair
A
- The enzyme (uvrABC) binds to damaged regions
- Makes an incision on both sides of the damage
- UvrD (i.e. MutU, DNA helicase II) separates strands n removes damaged DNA
- DNA polI replaces the DNA and DNA ligase fills the gap
- Short patch repair accounts for 99% of bulky lesions repair events
14
Q
Give an example of tolerance systems
A
Inducible error prone repair
- Low-fidelity DNA polymerases (translesion synthesis polymerases (TSPs)) can synthesise DNA past damaged bases
- Not efficient at replicating undamaged DNA accurately
- Most lack proof-reading ability
- Two in E. coli, polymerases IV and V, and five in human cells
- Almost all are members of a new DNA pol family, the Y-family
- In some circumstances make many errors, so can generate mutations
15
Q
How does human polymerase η (eta) contribute to preventing UV-induced mutations and cancer?
A
- Human polymerase η (eta) efficiently bypasses the major UV photoproduct, typically inserting the correct nucleotides.
- It is less effective with other types of damage.
- In individuals with xeroderma pigmentosum, a highly skin-cancer-prone genetic disorder, polymerase η is defective.
- Its absence leads to an increased risk of UV-induced mutations and cancer.
- Although one of its counterparts may substitute for it in its absence, it is less efficient, resulting in increased mutations and cancer susceptibility.
