DNA Repair

Question 1

When is base-excision repair used?
What are its steps?
Big change or little change?

Answer 1

Fixes chemical modifications to a single base. (Non-helix distorting)

1. DNA Glycosylase recognized altered base, and flips it out of the helix
2. An endonuclease and phosphodiesterase remove the sugar phosphate
3. DNA polymerase beta fills in the gap.
4. DNA ligase seals the nick to rejoin the strand
   - Little change, little cuts

Question 2

When is nucleotide excision repair used? Steps? (simplified)

Big change or little change?

Answer 2

When there are “bulky” helix-distorting lesions (Ex. Thymine dimers) “bulky” adducts

1. DNA damage recognized
2. DNA unwinds locally around lesion site
3. Incision by nuclease made on either side of lesion, and a single stranded DNA segment removed
4. DNA repair synthesis and strand ligation (pol and ligase)
   - Bigger change, cuts out more

Question 3

What is XP (xeroderma pigmentosa)? What is the mutation problem? Associated with what increase in risk?
Causes what symptoms?

Answer 3

Hyper sensitivity to light.
Mutation is lack of NER.
2000 fold increase in risk of skin cancer, as well as premature aging, eye problems, and neurological problems. Easily sunburned, lots of freckles.

Question 4

In mismatch repair, how does the cell know which is the daughter and which is the original strand?

Answer 4

It knows because the original is methylated, while the daughter strand is not yet methylated.

Question 5

Mismatch repair (MMR) proteins are frequently mutated in which type of cancer? What is the issue? 
How does this repair mechanism recognize, cleave, and fill in the gap after?

Answer 5

Hereditary Colon cancer (HNPCC).
Inactivation of a mismatch repair gene causes replication errors
Used in DNA repl. errors.
Recognizes using MSH2 gene encoded protein
Cleaves using (MLH1 gene) (also MutL, same thing) encoded protein
Fills in gap after using polymerase and ligase

Question 6

What is the difference between global genomic NER and transcription-coupled NER?

Answer 6

Global genomic- Damage recognized by RPA, XPA, XPC

Transition-coupled- Damage recognized due to blockage of transcription, via recruitment of CSB and CSA.

Question 7

In homologous recombination, what stops it if the homologs are not similar enough?

Answer 7

Mismatch repair machinery won’t allow it to happen.

Question 8

In mismatch repair in E. coli, how many MutS and MutL proteins are there? What are the homologs in eukaryotes, and how many types of these proteins are there, and what is their general name?

Answer 8

Only one each.

In humans/euk, there are at least least 5 MSH and MLH proteins each.

Question 9

MSH2 and MLH1 are what type of genes?

Answer 9

Tumor suppressors.

Question 10

What proteins are needed to recognize SS breaks?

Answer 10

SS breaks need PARP proteins. (Poly ADP, ribosyl polymerase)

Question 11

How are DS breaks fixed?

Answer 11

1. Homologous recombination (accurate)

2. Non-homologous end joining

Question 12

Explain when non-homologous end joining is used. Characteristics of it? What protein detects the break when using non-hom end joining?

Answer 12

Used in emergencies of DS breaks, it is quick but inaccurate. Joins together dissimilar sequences. Results in loss of nucleotides and in mutation. Most common type of DS break repair. Not very accurate. Used before and after DNA replication, unlike homologous recombination which is used during DNA replication (S/G2 phase).
DNA-PK recognizes the break.

Question 13

What percent of breast cancer is inherited?
What percent is linked to mutations? What genes specifically?
Both regulate homologous recombination, but which one also recognizes NHEJ?

Answer 13

5-10%
60-80%, BRCA1 and BRCA2
BRCA1 recognizes both types.

Question 14

A mutation in what gene causes ataxia-telangiectasia? Associated with increased risk of what type of cancer?

Answer 14

ATM gene.

Breast cancer.

Question 15

NHEJ order of things

Answer 15

Ku, binds termini.
DNA-Pk
endonuclease
ligation