(Dr. Heinemann) (Unit C) Topic 16

Question 1

How is “DNA damage” defined?

Answer 1

Unintended changes in the sequence or structure of DNA

Question 2

Main types of DNA damage

Answer 2

1. Copying mistakes
2. Depurination
3. Deamination
4. Pyrimidine dimers
5. Other base modifications
6. Strand breaks

Question 3

Copying mistakes

Answer 3

Mistakes may still happen even with DNA polymerase proofreading
* Results in a change of sequence or a mutation

Question 4

Depurination

Answer 4

Spontaneous hydrolyzation of N-glycosidic bond
* Results in the loss of A or G base

Question 5

In depurination, is the sugar-phosphate backbone affected?

Answer 5

No, it remains intact

Question 6

What happens as a result of depurination?

Answer 6

Abasic sites
* Block replication by the normal replicative DNA polymerase

Question 7

Translesion DNA polymerase

Answer 7

Allows synthesis of DNA past abasic sites
* Either mutates that position or completely skips that position

Question 8

Does translesion DNA polymerase repair damage?

Answer 8

No, it just allows DNA synthesis by jumping past damage and letting normal DNA polymerase do its job

Question 9

Deamination

Answer 9

Amine group of a nitrogenous base (most commonly C) is changed to a carbonyl
* C is converted to U in this process

Question 10

Is deamination:
* Spontaneous?

Answer 10

Yes, and it may cause a mutation

Question 11

Pyrimidine dimers

Answer 11

Double bonds in adjacent pyrimidines (most commonly Thymines) react to form a four-membered ring structure

Question 12

What are pyrimidine dimers usually caused by?

Answer 12

UV radiation

Question 13

What allows replication past pyrimidine dimers?

Answer 13

Translesion DNA polymerases
* More error prone than normal replicative DNA polymerases
* Increases the risk of mutation

Question 14

Other base modifications

Answer 14

• Ionizing radiation causes modifications to bases
• Mutagens react with DNA bases and leads to changes (ROS, polycyclic aromatic hydrocarbons etc.)

Question 15

Strand breaks

Answer 15

Single or double stranded breaks caused by a wide variety of factors

Question 16

What can cause strand breaks?

Answer 16

• Ionizing radiation
• Reactive oxygen species
• Mechanical stress

Question 17

True or False:

Translesion polymerases can synthesize DNA past a break in the template

Answer 17

False, neither DNA polymerase can synthesize past breaks

Question 18

What do strand breaks cause?

Answer 18

Chromosomal abnormalities, or result in cell death

Question 19

What forms of DNA repair systems are there?

Answer 19

1. Proofreading during DNA replication
2. Mismatch repair
3. Direct repair
4. Base excision repair
5. Nucleotide excision repair
6. Non-homologous end-joining (NHEJ)
7. Homologous recombination

Question 20

Mismatch repair

Answer 20

Fix mistakes made by DNA polymerase that escaped correction by proofreading
* Success rate of 99%

Question 21

In mismatch repair:

What protein detects a site of mismatch?

Answer 21

MutS

Question 22

In mismatch repair:

In bacteria, how is it determined which strand is newly synthesized/erroneous?

Answer 22

Scan both strands in both directions to look for methylated base
* Unmethylated strand are newly synthesized (not methylated yet)

Question 23

In mismatch repair:

Describe the process of repair

Answer 23

1. Endonuclease nicks the backbone of newly synthesized strand across from methylated base
2. Exonuclease removes the bases from the nick to the mismatch site
3. Replicative DNA polymerase then fills gap created by exonuclease

Question 24

Is the identification of the new strand in mismatch repair the same in prokaryotes and eukaryotes?

Answer 24

No

Question 25

Direct repair

Answer 25

A few common types of damaged bases can be repaired by enzymes specific to those bases

Question 26

Base Excision Repair

Answer 26

Fixes abasic sites, single-strand breaks, and modified bases (including deaminated bases) that are not fixed by direct repair

Question 27

What are the two forms of base excision repair (BER)?

Answer 27

1. Short patch 2. Long patch

Question 28

What is involved in both forms of BER?

Answer 28

Endonuclease cuts the backbone at the site of damage if necessary

Question 29

# In BER: What does Short-Patch BER do?

Answer 29

Synthesis of only one new nucleotide

Question 30

# In BER: What does Long-Patch BER do?

Answer 30

2-10 new nucleotides are synthesized

Question 31

# In BER: What is the undamaged strand used for?

Answer 31

Used as template for new DNA synthesis in each type of BER

Question 32

# In BER: What enzymes are involved in Long-Patch BER?

Answer 32

* Endonuclease: Cleaves ssDNA on damaged strand that has been displaced by DNA synthesis * DNA ligase: Seals the nick that resulted from the endonuclease

Question 33

Nucleotide Excision Repair (NER)

Answer 33

Repairs pyrimidine dimers and base modifications that distort the helical structure of DNA

Question 34

How does NER work?

Answer 34

1. Helicase unwinds dsDNA in area of damage 2. Endonuclease cuts backbone of damaged strand on either side of site of damage 3. DNA polymerase and DNA ligase replaces it, using undamaged strand as a template

Question 35

Non-homologous end joining (NHEJ)

Answer 35

Fast but inaccurate * More common method used to repair a doublestrand break

Question 36

# In NHEJ: Describe the process

Answer 36

1. Ku protein recruits both ends together 2. Nuclease trims and adjusts the ends 3. Polymerase extends the ends 4. DNA ligase connects the two ends

Question 37

Homologous recombination

Answer 37

REQUIRES a second copy of the chromosome * Complementary DNA is used to synthesize new DNA to restore DNA * Slow but accurate

Question 38

# In homologous recombination: Describe the process

Answer 38

Recruit the DNA from the other copy of the chromosome complementary to the DNA surrounding the break * Used as a template to synthesize new DNA to restore the strand to original condition

Question 39

When is homologous recombination most convenient?

Answer 39

Most convenient shortly after replication when another copy of the chromosome is nearby