4. DNA damage and repair

Question 1

Name 4 environmental DNA damage agents

Answer 1

1. Cellular metabolism
2. Radiation
3. Chemicals
4. Replication errors

Question 2

Name 4 consequences of DNA damage

Answer 2

1. Cell cycle checkpoint activation
2. Transcriptional program activation
3. Activation of DNA repair machinery
4. Apoptosis

Question 3

Name 2 examples of radiation that damages DNA

Answer 3

Ionizing, UV

Question 4

Name 4 types of chemicals that damage DNA

Answer 4

1. Alkylating agents (e.g. MMS used in mutagenesis)
2. DNA crosslinking agents (e.g. cisplatin, nitrogen mustard)
3. Aldehydes that crosslink DNA and proteins (e.g. formaldehyde used in ChIP)
4. Chemicals that cause dsDNA breaks (e.g. bleomycin, topoisomerase poisons)

Question 5

Mechanistically, what 3 types of damage can happen to DNA?

Answer 5

1. Oxidative damage
2. Hydrolysis
3. Depurination

Question 6

Name 5 endogenous enzymes that can cause DNA damage when misfunctioning

Answer 6

DNA polymerase; topoisomerases; DNA repair enzymes; cytosine deaminase; covalently trapped enzymes

Question 7

What is the result of defective DNA repair?

Answer 7

Genome instability and eventually cancer

Question 8

Name a neurodegenerative disease that results from defective DNA damage repair

Answer 8

Spinocerebellar ataxia with axonal neuropathy (SCAN1)

Question 9

What are the two main differences between type I and type II topoisomerases?

Answer 9

Type I topoisomerases are monomeric and cleave one strand of DNA. Type II topoisomerases are dimeric and cleave both strands of DNA.

Question 10

What is the similarity between all topoisomerases except topoisomerase IB?

Answer 10

All go through a covalent reaction intermediate with the DNA, using a 5’phospho-tyrosyl bond

Question 11

Which type of topoisomerase is a target for anticancer drugs?

Answer 11

Both

Question 12

Which type of topoisomerase is a target for antibiotic drugs?

Answer 12

Topoisomerase II

Question 13

Describe the steps of topoisomerase IB catalytic cycle

Answer 13

1) Cleavage by covalently attaching to 3’ end
2) DNA relaxation through free rotation of the strands
3) Religation of the ssDNA nick

Question 14

Name 6 conditions (4 DNA damage, 2 exogenous) that trap topoisomerase IB on the DNA

Answer 14

1) Nicks or gaps
2) Base mismatches
3) Abasic sites
4) Alkylated bases
5) Chemotherapy drugs
6) K532A mutation in the topoisomerase

Question 15

What is the mechanism of action of camptothecin

Answer 15

Intercalates into cleaved DNA via base stacking

Question 16

What is the consequence of stalled topoisomerase IB-DNA complexes in DNA replication?

Answer 16

Replication fork runs into a barrier and results in multiple, complex dsDNA breaks

Question 17

What is the consequence of stalled topoisomerase IB-DNA complexes in transcription?

Answer 17

RNA polymerase becomes stalled, causes a persistent ssDNA break that eventually is converted into a dsDNA break

Question 18

Name two repair pathways for stalled DNA-topoisomerase IB complexes

Answer 18

1) Endonuclease 2) Tyrosyl-DNA phosphodiesterase

Question 19

What does endonuclease do for stalled DNA-topoisomerase IB complexes?

Answer 19

Removes a snippet of DNA with the stuck protein

Question 20

What does tyrosyl-DNA phosphodiesterase do for stalled DNA-topoisomerase IB complexes?

Answer 20

Specifically cleaves the tyrosyl-3’DNA bond

Question 21

What does Tdp1 stand for?

Answer 21

Human tyrosyl-DNA phosphodiesterase 1

Question 22

Is Tdp1 ATP-dependent?

Answer 22

No

Question 23

Name the main steps of Tdp1 complex catalytic cycle for removing stalled Topo1B

Answer 23

1) Proteolysis of TopoIB to remove bulk of protein
2) Cleavage of the tyrosyl-3’DNA bond
3) Kinase and phosphatase generate ligatable ends post-cleavage
4) Ligase seals the ssDNA break

Question 24

What residues are contributed by each lobe of Tdp1 into the active site?

Answer 24

Each contributes a Lys and a His residue

Question 25

What is the nucleophile in the Tdp1 cleavage reaction?

Answer 25

His263 residue

Question 26

What is the general acid in Tdp1 cleavage reaction?

Answer 26

His493 residue

Question 27

How are DNA and topoisomerase binding sites distinguished from strucutral studies of Tdp1?

Answer 27

By mapping the net charge on its surface

Question 28

What 3 reagents are required to assemble a Tdp1 transition state analog?

Answer 28

Vanadate, DNA and peptide

Question 29

What transition state does vanadate represent?

Answer 29

Trigonal bipyramidal

Question 30

What other functionality does Tdp1 have that is important for setting up a crystallisation experiment?

Answer 30

It is able to remove a 3'OH base therefore needs to be accounted for when designing DNA substrates to be crystallised

Question 31

What structural feature of Tdp1 separates the 2 DNA strands?

Answer 31

A hydrophobic loop that wedges between the strands

Question 32

Name the clinical manifestations of SCAN1

Answer 32

Peripheral axonal motor and sensory neuropathy Muscular and cerebellar atrophy Onset during late teenage year Affects large, differentiated, non-dividing neuronal cells

Question 33

What is the genetic cause of SCAN1?

Answer 33

Tdp1 H493R mutations

Question 34

When Tdp1 repair is inactive, would the cells be more or less sensitive to camptothecin? Why?

Answer 34

More sensitive. Camptothecin intercalates into broken DNA and stabilises the lesion, so if Tdp1 repair is inactive, there will be more ssDNA breaks due to stalled enzymes for camptothecin to intercalate into.

Question 35

What technique was used to assay Tdp1 activity in WT and SCAN1 cells?

Answer 35

Denaturing urea PAGE of labelled DNA oligomer with an attached 3'-tyrosyl over time

Question 36

What technique was used to prove that Tdp1 H493R mutants accumulate a covalent Tdp1-DNA intermediate?

Answer 36

SDS-PAGE of labelled DNA oligomer with an attached topoisomerase peptide with and without trypsin digestion. If the intermediate is stalled and accumulated, a band is seen; but if it is digested with trypsin, no band is seen suggesting that the retention is caused by protein binding

Question 37

What technique was used to prove that WT Tdp1 can remove H493R Tdp1 from DNA?

Answer 37

SDS-PAGE of two molecules: 1) protein/labelled DNA complex with attached H493R Tdp1 2) Tdp1, either WT or mutant As band intensity decreases over time, that suggests that Tdp1 can remove its own stalled complex. This happens in both WT and H493R states

Question 38

What technique was used to show the hand-off mechanism when only mutant Tdp1 are involved?

Answer 38

SDS-PAGE of two molecules: 1) Protein/labelled DNA complex with attached H493R; 2) truncated H493R that can be distinguished by size DNA label shifts downwards and eventually disappears, showing that mutant Tdp1 can remove other mutant Tdp1s (although probably at a slowed rate than WT)