Lecture 13

Question 1

What are the key features of all repair pathways? (3)

Answer 1

1. Multiple dynamic protein interactions
2. ordered hand-off of damaged DNA from one protein (complex) to another
3. DNA repair proteins are “modular” (multiple domains)

Question 2

What is a domain?

Answer 2

stable, independently folding region of a protein with a specific function, often associated with a DNA sequence it binds or a protein-protein interaction it mediates

Question 3

What does base excision repair do?

Answer 3

repairs DNA when a base of a nucleotide is damaged

Question 4

What is base excision repair mediated by?

Answer 4

4 types of enzymes…
1. DNA glycosylase
2. Endonuclease
3. DNA polymerase
4. DNA ligase

Question 5

What does DNA glycosylase do in base excision repair?

Answer 5

specific DNA glycosylase recognizes and excises the damaged base

Question 6

What does endonuclease do in base excision repair?

Answer 6

it removes 1-10 nucleotides around the abasic site

Question 7

What is an abasic site and how is it formed?

Answer 7

it is a location in the DNA strad where a purine or pyrimidine base has been removed leaving behind the sugar phosphate backbone
it is formed through spontaneous chemical damage or as it intermediates in cellular processes like base excision repair

Question 8

What does DNA polymerase do in base excision repair?

Answer 8

replaces missing nucleotides

Question 9

What does DNA ligase do in base excision repair?

Answer 9

seals the gap

Question 10

How do DNA repair proteins find the rare sites of damage in a vast expanse of undamaged DNA?

Answer 10

1. 3D diffusion
2. linear scanning along the DNA backbone

Question 11

What is the G-specific pocket?

Answer 11

active site pocket of DNA repair enzyme that is shaped to specifically recognize and bind a damaged or altered guanine base when that base is flipped out of the DNA helix

Question 12

What is a lesion recognition (oxoG) pocket?

Answer 12

a specialized region within a DNA repair enzyme, such as the hOGG1 protein, that is shaped to fit and bind specifically to the altered 8oxoG lesion… allows the enzyme to remove the damage from undamaged DNA

Question 13

What are the steps of base excision repair?

Answer 13

1. damage base removed (abasic site) (DNA glycosylase enzyme recognizes and cuts out the damaged base leaving behind an empty spot in the DNA called an abasic site)
2. 5’ or 3’ endonuclease cleavage (DNA backbone is cut at the abasic site now the sugar-phosphate backbone is open for repair)
3. nucleotide excision (the leftover sugar and phosphate without the base is removed so the DNA is clean and ready for a new nucleotide)
4. repair synthesis (DNA polymerase comes in and inserts the correct nucleotide by using the undamaged strand as a template)
5. ligation (DNA ligase seals the last nick in the backbone restoring a continuous double helix)

Question 14

What is mismatch repair?

Answer 14

Correction of mismatched base pairs

Question 15

What is the method of strand discrimination in mismatch repair?

Answer 15

ensures the correct strand is repaired

Question 16

What causes hereditary non-polyposis colon cancer?

Answer 16

inheritance of one inactive mismatch repair allele and somatic loss of wild type allele… accumulation of mismatches (point mutations) during DNA replication

Question 17

What type of damage does nucleotide excision repair fix?

Answer 17

bulky, helix-distorting DNA damage such as UV induced pyrimidine dimers (thymine dimers) and lesions from chemical agents

Question 18

What is the key difference between base excision repair and nucleotide excision repair?

Answer 18

NER fixes bulky helix distorting lesions that BER can’t handle

Question 19

What is the twist open mechanism?

Answer 19

an XPC protein with beta hairpin domain (homologous to yeast Rad4) is the first sensor of DNA distortion and when it encounters a T-T dimer, the beta hairpin wedges into the DNA, flipping out the damaged bases from the helix creating a “twist open” structure where the DNA helix locally unwinds and bends and that distortion signals to the rest of the NER machinery that there’s damage and then other proteins come to cut out a 24-32 nt stretch containing the lesion

Question 20

What are the recurrent themes in DNA repair?

Answer 20

hand off of damaged DNA from a complex with nuclease activity to a complex with polymerase activity to a complex with ligase activity

Question 21

What does ionizing radiation do?

Answer 21

damages DNA by directly or indirectly breaking its chemical bonds which generates free radicals

Question 22

How is ionizing radiation repaired?

Answer 22

Homologous recombination or non-homologous end joining

Question 23

What does homologous recombination do?

Answer 23

repairs double strand breaks by retrieving genetic information from an undamaged homologous chromosome

Question 24

What does non-homologous end joining do?

Answer 24

rejoins double strand breaks via direct ligation of the DNA ends without any requirement for sequence homology

Question 25

What double stranded break repair mechanism is primarily used in mammalian cells?

Answer 25

non-homologous end joining

Question 26

What are the steps of homologous recombination?

Answer 26

1. double stranded break 2. end processing and recognition 3. strand invasion and DNA synthesis

Question 27

What type of genes are BRCA1 and BRCA2?

Answer 27

tumor suppressor genes which are genes that inhibit cell growth and that cause cancer when they are not expressed

Question 28

What is the lifetime risk for breast and ovarian cancer for BRCA1 and BRCA2 mutations?

Answer 28

1: 50-87% 2: 15-44%

Question 29

What are the usual steps of non homologous end joining?

Answer 29

1. damage recognition 2. nuclease activity 3. DNA synthesis 4. ligation

Question 30

What does Holiday junction resolution mean?

Answer 30

the enzymatic process where specialized nucleases cleave the four way DNA structure of a Holliday junction, separating the entangled DNA strands to produce 2 distinct DNA molecules either as a crossover or non-crossover

Question 31

What is a Holliday junction?

Answer 31

a 4 way DNA structure formed during DNA repair, replication fork restart, and homologous recombination

Question 32

What is the key difference between homologous recombination and non-homologous end joining?

Answer 32

HR uses a homologous DNA template to ensure accurate, error free repair NHEJ directly ligates broken DNA ends without a template resulting in faster but error prone repair