Repair Mechanisms

Question 1

What are intercalating agents?

Answer 1

Mutagens that modify DNA causing indels by mimicking base pairs and slipping between stacked bases causing single nucleotide pair insertions or deletions.

Question 2

What are some examples of intercalating agents?

Answer 2

Proflavin

Acridine orange

ICR

Question 3

What light causes DNA damage response (DDR)?

Answer 3

UV light

Question 4

Which UV radiation can penetrate earth’s atmosphere?

Answer 4

UV-A and UV-B

Question 5

What types of DNA lesions does UV light trigger?

Answer 5

Cyclobutane pyrimidine dimers (CPDs)

6-4 photoproducts (6 - 4 PPs)

Question 6

What do the 2 lesions created by UV light do to DNA?

Answer 6

Both distort DNA’s structure and impede transcription and replication.

Question 7

Which part of the DNA is most susceptible to damage?

Answer 7

Relatively flexible areas of DNA double helix. One hotspot is within the commonly mutated p53 gene.

Question 8

What happens if p53 is overexpressed?

Answer 8

The cell cycle is halted to allow for repair of mutation in p53.

Question 9

How do pyrimidine dimers cause issues?

Answer 9

Dimers distort DNA helix and can block replication.

Question 10

How are pyrimidine dimers repaired?

Answer 10

Sensor activates repair mechanisms by recruiting multiple proteins.

Effector proteins return DNA to native sequence or introduce variation which may cause disease or it can be tolerated.

Question 11

What happens when DNA replication is halted?

Answer 11

DNA polymerase stops and some error prone polymerases (ER polymerases) take over replication and these finish of replication and are more likely to form mutated DNA strands.

Question 12

What test is used to test mutagenicity?

Answer 12

The Ames test for mutagenicity.

This test uses auxotrophic histidine mutants of salmonella to test for reversion to prototrophy in presence of chemical being tested.

Without histidine these salmonella can’t grow but if the mutagen caused a mutation then they can survive.

Question 13

Many mutagens or their metabolites are also:

Answer 13

Carcinogens

Question 14

How can the ames test be modified to test for metabolites that are potential mutagens?

Answer 14

Using ground liver from another mammal (eg rats)

Question 15

What types of mutations are required for ames test?

Answer 15

In TA1538 and TA1535: Indel mutations for reversion to prototroph

TA100: Base substitution GC -> TA transversions

Question 16

Which DNA polymerases are involved in DNA repair?

Answer 16

Some DNA pols have 5’ - 3’ polymerase activity and have some proofreading 3’ - 5’ exonuclease activity. These are γ, δ, and ε.

Other DNA pols do not have proofreading such as: DNA pol α (primase activity and used in DNA repair) and DNA pol β (has a role in excision repair)

Lots of DNA pols which are used in translesion synthesis allowing replication when DNA is damaged and other repair processes include zeta, eta, theta, iota, kappa, lambda, mu, sigma, phi, Rev1

Question 17

When are most important repairs made?

Answer 17

During DNA replication, proofreading repairs DNA mismatches and decreases number of mutations.

Question 18

What are the general pathways for DNA repair?

Answer 18

Most repair requires 2 DNA strands so that one can act as a template for synthesis of the other.

Redundancy, most DNA damage can be repaired in multiple ways.

Question 19

What is homology dependent repair?

Answer 19

Some repair systems utilise the antiparallel complementarity of the DNA double helix to restore damaged segments to original sequence. Section of DNA is removed and replaced with newly synthesized complementary DNA then DNA ligase seals the gap in the sugar phosphate backbone

Question 20

What is mismatch repair?

Answer 20

Mismatch repair systems recognise mismatched bases by detecting distortions in DNA usually during replication.

Parental strand sequence is used to correct newly synthesized strand error.

Incorrect nt is detected and excised as well as nearby nt. Then DNA polymerase is used to repair gap and then this is joined by DNA ligase.

Mismatch repair can also recognize small unpaired DNA loops caused by slippage during DNA replication.

Question 21

What is base-excision repair?

Answer 21

AP endonuclease excises damaged base and then entire nt is replaced.

Eukaryotes replace excised DNA with DNA pol beta.

DNA ligases that join sugar phosphate backbone at the end require the correct orientation of nucleotides for it to finish the process.

Question 22

What is the problem with using base-excision repair to fix errors in DNA replication? How is this problem countered?

Answer 22

Eukaryotes use DNA pol beta with no proof reading activity so errors can be introduced. This can be significant given the number of base-excision repairs occurring in a human cell.

DNA ligases cannot join the sugar phosphate backbone if there is a mismatch because orientation is important.

Question 23

How does base-excision repair take place?

Answer 23

Each DNA glycosylase recognizes and removes specific damaged bases producing apurinic/apyrimidinic site

AP endonuclease cleaves phosphodiester bond on 5’ side and removes deoxyribose sugar

DNA polymerase adds new nucleotides to the exposed 3’-OH group

Nick in the sugar-phosphate backbone is sealed with DNA ligase.