DNA Repair (Huang)

Question 0

When is direct reversal of damage used?

How is each accomplished?

Answer 0

1. Ligation of break in phosphodiester backbone = DNA ligase
2. Repair of O6-methylguanosine = MGMT
   (O6-methylguanosine methyltransferase)

Question 1

Types of DNA repair

Answer 1

1. Excision Repair
   i. Nucleotide excision repair (NER)
   ii. Base excision repair (BER)
   iii. Mismatch Repair (MMR)
2. Direct Reversal of Damage
3. Lesion Bypass
4. Double-Stranded DNA breaks
   i. Non-homologous end joining(NHEJ)
   ii. Homologous recombination

Question 2

In general terms, what steps are taken in excision repair?

Answer 2

1. excision at the mutation site
2. removal of mismatched or damaged nucleotide(s)
3. addition of new DNA to fill the gap (use other strand as template)
4. ligation of newly synthesized segment by DNA ligase

Question 3

What sort of lesions does NER work on?

Answer 3

Lesions that distort the DNA structure and block DNA or RNA polymerase

1. Thymine dimers (UV damage)
2. Adducts (carcinogens)

Question 4

How does NER begin?

Answer 4

distorted DNA recognized by multi protein complex with endonuclease activity

Question 5

Once lesion is recognized, how does NER proceed?

Answer 5

1. Endonuclease cuts damaged strand on BOTH sides of the lesion
2. Removal of the oligonucleotide containing the lesion
3. Helicase unwinds and releases the lesion-containing oligonucleotide
4. DNA polymerase fills in the gap using other intact strand as template
5. Ligase joins the strands

Question 6

What are two different recognition pathways in NER?

Answer 6

1. Global genome NER

2. Transcription-coupled NER

Question 7

How does Global genome NER work?

Answer 7

recognizes lesions / distorted DNA in any part of the genome

Question 8

How does Transcription-coupled NER work?

Answer 8

recognizes distorted DNA regions that are being actively transcribed

Question 9

Name 3 different diseases caused by mutations in genes that mediate NER.

Answer 9

1. Trichothiodystrophy (TTD)
2. Xeroderma Pigmentosum (XP)
3. Cockayne Syndrome (CS)

Question 10

Explain Xeroderma Pigmentosum

Answer 10

Mutation in DNA repair system (TFIIH complex)

Photosensitivity - unable to repair UV damage (thymine dimers; pyridine dimers)

Question 11

Give some symptoms of Cockayne Syndrome

Answer 11

Failure to grow / gain weight (short stature)
Microencephalopathy (cognition issues)
Photosensitivity
Premature aging

Question 12

Underlying genetic problem in Cockayne syndrome?

Answer 12

mutations in the DNA repair system; specifically NER system
sensitive to UV damage (cannot repair)
cells accumulate mutations - premature aging / cell death

autosomal recessive

Question 13

Give some symptoms of Trichothiodystrophy.

Answer 13

Brittle hair: lacks sulphur
Photosensitivity
Short stature
Intellectual disability

Question 14

Genetic factors of Trichothiodystrophy?

Answer 14

Repair by TFIIH complex is not functioning

Question 15

Symptoms of Xeroderma Pigmentosum?

Answer 15

Photosensitivity

Question 16

Genetic problem in Xeroderma Pigmentosum?

Answer 16

TFIIH not functioning correctly in DNA repair

Question 17

What is the main function of TFIIH?

Answer 17

subunit CDK7 phosphorylates C-terminal end of RNA poll II

subunits XPB and XPD have helicase activity to create the replication bubble

Question 18

What sort of lesions does Base Excision Repair (BER) work on?

Answer 18

lesions that are missed by NER

these lesions may not distort the DNA or block polymerase function

Question 19

What enzymes do the recognition for BER?

Answer 19

Glycosylases (familly of enzymes)

-each is specific to a particular altered base

Question 20

What does uracil glycosylase recognize?

Answer 20

U in place of C in DNA as a result of C deamination

(C deamination replaces C4 - NH2 with C4=O
amine to ketone)

for repair by BER

Question 21

What does 5-methylcytosine-DNA glycosylase recognize?

Answer 21

C5 methylated cytosine

for repair by BER

Question 22

How does a glycosylase function once it has recognized a lesion?

Answer 22

“Flips” the altered base out and excises the base
i.e. hydrolyzes the N-glycosidic bond (btwn sugar and base)

Removal of base produces an apurinic / apyrimidic (AP) site

Question 23

Once glycosylase has removed a base, what happens next?

Answer 23

AP-specific endonuclease (and AP lyase) removes the AP site
Gap is filled by DNA polymerase
Nick is sealed by DNA ligase

Question 24

When is MMR utilized?

Answer 24

Very shortly after replication when a single base mismatch occurs

Question 25

What proteins recognize the lesion in MMR?

Answer 25

Bactiria - MutS and MutL

Eukaryote - MSH and MLH (H = homologue)

Question 26

How do MutS/MutL or MSH/MLH recognize the new vs. old strand during MMR?

Answer 26

New strand is not yet methylated

Question 27

Once the lesion is recognized, how does MMR proceed?

Answer 27

1. Excision near the lesion site (one single cut) on the new strand
2. Exonuclease activity chews up the lesion-containing oligonucleotide
3. Helicase assists to open the DNA
4. Polymerase fills the gap using old strand as template
5. Ligase joins the two strands

Question 28

Give an example of a hereditary disease caused by mutation in MMR repair machinery

Answer 28

hereditary non-polyposis colorectal cancer (HNPCC)