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Genetics Gary > Lecture 6- DNA repair > Flashcards

Lecture 6- DNA repair Flashcards

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1
Q

What determines the mechanism used to fix DNA damage?

A

The nature of the damage

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2
Q

What do the pathways for repair include?

A

Direct repair: methylated bases and pyrimidine dimers
Base excision repair: modified bases and abasic site
Nucleotide excision repair: bulky lesions
Mismatch repair: mis-paired bases, short insertions and delection
Recombination repair: homologous recombination

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3
Q

What repair pathway is used if there are multiple repair pathways that could be used?

A

It depends on which enzyme arrives to the site first.

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4
Q

What is direct repair?

A

The elimination of DNA damage using chemical reversion that does not require:
nucleotide template
breakage of phosphodiester bond
DNA synthesis

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5
Q

What is an advantage of direct repair?

A

Error-free and preserves genetic information

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6
Q

How could you describe DNA damage?

A

Simple (energetic advantage)
Safe
Minimises the change of mistakes induced by the repair

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7
Q

What are the 3 examples of direct repair?

A

Repair of single strand breaks
Repair of pyrimidine dimers
Repair of methyl groups

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8
Q

What do single strand breaks result from?

Repair of single strand break

A

DNA replication, recombination and DNA repair, exposure to radiation

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9
Q

When are single stranded breaks are non-problematic?

Repair of single strand break

A

When the cell is not dividing

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10
Q

What generates problems with a single stranded break?

Repair of single strand break

A

Breaks that occur close to each other in opposing strands or replication of a single strand break can generate problems - in replication part of the arm is lost in the replication fork.

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11
Q

What repairs single stranded breaks?

Repair of single strand break

A

DNA ligase,
(Bacterial DNA ligase uses NAD, Eukaryotic DNA ligases use ATP).

Recognises nicks and adds a phosphate group/restores the phosphodiester bond

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12
Q

How do pyrimidine dimers form?

Repair of pyrimidine dimers

A

With exposure to UV light

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13
Q

How is DNA photolyase adapted to repair pyrimidine dimers?

Repair of pyrimidine dimers

A

It have light harvesting molecules that absorb light to activate the enzyme and can donate electrons to break the dimer

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14
Q

Outline the events involved in repairing a pyrimidine dimer

Repair of pyrimidine dimers

A

Pyrimidine dimer in DNA induced by UV e.g. thymine dimers
Complex of DNA with DNA photolyase enzyme.
Folate harvests light energy and transfers it to flavin.
Flavin breaks apart the dimer by donating an electron
Release of enzyme to restore native DNA

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15
Q

What is a cyclobutane pyrimidine dimer?

Repair of pyrimidine dimers

A

TT
TC
CC

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16
Q

Describe DNA photolyase

Repair of pyrimidine dimers

A

2 light harvesting cofactors to absorb light energy:
5-10 methenyl-tetrahydrofolate and 1,5-dihydroflavin adenine dinucleotide (FAD)

Light dependent enzyme that eliminates thymine dimers by binding to them and transferring an electron to the dimer

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17
Q

What is used to repair damage caused by UV?

Repair of pyrimidine dimers

A

Light

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18
Q

How is FAD involved?

Repair of pyrimidine dimers

A

The catalytic FAD factor is in a reduced state so when the absorbed blue light energy is transferred the FADH donates an electron to the dimer. Addition of this electron to the pyrimidine dimer breaks the covalent bonds and the pyrimidine molecules are returned to their undamdaged states. Once the damage is repaired the electron is transferred back to the FADH cofactor so that it returns to its reduced state and regains catalytic activity

19
Q

What is the problem with pyrimidine dimers?

Repair of pyrimidine dimers

A

They weaken hydrogen bonding and lead to deformation of the double helix creating problems in replication

20
Q

Why are mis-match repair bases difficult to identify?

A

They appear normal

21
Q

Proof-reading

A

Bacterial DNA polymerase III back tracks and incorrectly inserted nucleotides are removed by the 3’-5’ exonuclease activity of epsilon.

22
Q

What does a mutation of dnaQ (epsilon proof reading subunit) result in

A

Elevated mutation rate

23
Q

What is the main repair system in bacteria?

A

Using MutHLS proteins and DNA methylation to distingush between template and newly synthesised strands

24
Q

What does Dam methylase do in E.coli?

A

Adds a methyl group to adenine in the sequence 5’-GATC-3’
The short delay of methylation of the newly synthesised strand allows enzymes to identify new DNA and target repair to this strand.

25
Q

What are the key enzymes involved in methyl-directed MMR

A 
MutS: binds DNA mismatch 
MutH: Cuts unmethylated strand at GATC sites
MutL: stimulates MutS & MutH
MutU: unwinds nicked DNA strands
Dam: methylates GATC sites
26
Q

MMR

A

Mismatch repair

27
Q

Explain how methyl directed mismatch repair works

A

Incorporation of base pair mismatch during DNA replication. Mismatch is detected shortly after replication.
Methylation does not occur until several minutes after a strand has been made.
The MutS protein clamps onto DNA & scans it until it finds a distortion in the DNA backbone caused by a mis-paired base. MutS binds to the site
Complex between MutS and MutL formed. The formation of this complex attracts MutH which binds to a hemi-methylated site.
DNA looping allows the MutS and MutL complex to interact with MutH. MutH makes a cut in the non-methylated strand.
An exonuclease digests the non-methylated strand from the cleavage site to just beyond the mis-match site. This leaves a gap in the daugher strand.
DNA polymerase III seals the gap in their daughter strand.
DNA ligase seals the nick.

28
Q

What happens when methylating agents react with DNA

Repair of methyl groups

A

Methylating agents (methy-methane-sulphonate MMS) react with DNA to produce O-alkylated and N alkylated products including O6 methylguanine, which can pair with thymine.

29
Q

Role of methyltransferase in repair of methyl groups

Repair of methyl groups

A

methyltransferase recognises the distortion in the backbone. The methyltransferase accepts the methyl group onto a cysteine in the protein which inactivates it. Suicide methyltransferase.

30
Q

E.coli Ada protein

Repair of methyl groups

A

Regulates a set of genes involved in repairing alkylating damage.
The N-terminal half of Ada switches on adaptive response once it has been methylated.
Removal of methyl groups is achieved by a single step methyltransferase reaction- Ada accepts the adducts from the modified oxygen molecule onto internal cysteine residues restoring the DNA damage and inactivating the protein.
Once modified the inactivated protein is targeted for degradation.

31
Q

What is Ada made up of?

Repair of methyl groups

A

Two active cysteines, one for the repair of O6-methylguanine and the other for restoration of methylphosphotriesters.

32
Q

Explain methyltransferases

Repair of methyl groups

A

Chemical agents induce the methylation and alkylation of guanine residues at the oxygen attached to carbon 6 of the purine ring.
Mutation
Methylguanine-DNA methyltransferase eliminates these methylations
Enzyme restores the correct structure of guanine to prevent mutations from occurring

33
Q

What is base excision repair?

A

Removal of a damaged base - protects DNA in cells from oxidation, alkylation and deamination.
Fixes single base leasions that contain small chemical modifications.

34
Q

How is the repair pathway initiated in base excision repair?

A

The removal of damaged bases followed by incision at the site of the missing base.

35
Q

Summarise the steps in base excision repair

A

Damaged base
DNA glycosylase or N glycosylase
A basic site
AP endonuclease which cleaves on the 5’ or 3’ site
Deoxyribose phosphate removes the attached phosphate
DNA polymerase inserts the correct nucleotide
DNA ligase seals the nick

36
Q

What recognises the type of damage?

base excision repair

A

DNA glycosylases.

These cleave the bond between the base and sugar backbone

37
Q

AP endonucleases

base excision repair

A

recognises teh abasic site (AP iste) and cleave the phosphodiester backbone on the 3’ and 5’ site.

38
Q

Sealing the gap

base excision repair

A

The single nucleotide gap filled in by a DNA polymerase and the nick sealed by a DNA ligase.

39
Q

What do the types of damage include in base excision repair?

A

Deamination bases e.g. cytosine to uracil, adenine to hypoxanthine
Spontaneous loss of bases e.g. hydrolytic loss of purines
Oxygen free radical damage e.g. thymine glycol
Methylated bases e.g. O6 methylguanine

40
Q

DNA glycosylases

base excision repair

A

Small proteins that target dsDNA
E.coli has 6 different DNA glycosylases that target specific types of damaged bases.
Can be monofunctional or bifunctional

41
Q

Uracil DNA glycosylase

base excision repair

A

Uracil gets into DNA in two main ways:

  1. Deamination of cytosine to uracil
  2. Misincorporation of dUTP during DNA synthesis. Bacteria have an enzyme called Dut which has dUTPASE activity degrading dUTP. UDG flips uracil out of the double helix before cleavage. The bacterial and human enzymes are closely related. Base flipping is a common feature of DNA glycosylases.
42
Q

AP endonuclease:

Exonuclease III

A

Exonuclease III (Xth): Monofunctional and cleaves by hydrolysis on the 5’ side AP sites. Xth has the capacity to repair various blocking groups on the 3’ terminus of DNA which includes the products of AP lyases. The cleavage product can be eliminated by 5’-phosphodiesterase

43
Q

AP endonuclease:

Exonuclease IV

A

Bifunctional, has DNA glycosylase activity on urea and thymine glycol and cleaves in the 3’ side of AP sites. Bifunctional AP endonucleases cleave by a beta-elimination reaction. The resulting alpha-beta-unsaturated aldehyde can be removed by Xth. The single nucleotide gap created can be filled in by DNA polymerase and the gap sealed by DNA ligase

Genetics Gary (9 decks)

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