Lecture RR2. DNA repair and recombination

Question 1

What are mutations?

Answer 1

permanent, transmissible changes to the genetic material of a cell (or organism)

mutations can occur spontaneously, by transposable elements, and by errors during replication

Question 2

What are mutagens?

Answer 2

chemical compounds, ultraviolet (UC) radiation or ionizing radiation (e.g x-rays and atomic particles) that increase the frequency of mutations.

Question 3

what is a carcinogen?

Answer 3

carcinogen is an agent that causes cancer

Many carcinogens are mutagens

Question 4

What are the 2 systems used to repair the mismatch mistakes of the polymerases?

Answer 4

1. proofreading exonuclease
2. mismatch repair

Question 5

how does the proofreading activity work in DNA polymerases?

Answer 5

1. the erroneous nucleotide in the growing chain in detected
2. There is a conformational change bringing the 3’ end to the exo site
3. few nucleotides will be degraded and removed from the growing chain
4. The growing chain goes back to the active site and continues polymerization

Question 6

How does Mismatch repair work?

Answer 6

1. the error is incorporated to the new strand
2. MSH2 and MSH6 bind to daughter strand (they detect the mismatch)
3. triggers binding and activity of MLH1 endonuclease (dimerized w/ PMS2)
4. DNA helicase unwinds and DNA exonuclease digests segment of daughter strand
5. Gap repair by pol delta and DNA ligase

Question 7

to what step of the Mismatch Repair do these enzymes belong to?
a) MLH1 endonuclease
b) MSH1 and MSH6
c) pol delta and DNA ligase
d)PMS2
e) DNA helicase and DNA exonuclease

Answer 7

a) Triggers binding activity dimerized with PMS2
b) bind to the daughter strand and detect mismatch
c) Gap repair
d) helps binding activity dimerization with MLH1
e) Unwinding and digestion of segment

Question 8

What is the deamination of cytosine?

Answer 8

It is a spontaneous reaction where the amine group is removed and is replaced by an oxygen instead
turning the cytosine into a uracil
It changes the sequence in the DNA and there is not supposed to be any uracil in DNA

Question 9

what small modification helps with gene regulation, transposon (jumping genes) silencing and Chromatin regulating?

Answer 9

Cytosine methylation

(DNA methyltransferases)

Question 10

What does methylated cytosine turn into when it is deaminated?

Answer 10

Thymine

Question 11

what is the difference between Mismatch Repair and Base Excision Repair (BER)?

Answer 11

MR happens during replication, when the polymerase makes a mistake

BER happens in an already formed DNA

Question 12

When a mismatch goes undetected by BER and is replicated, what is the difference between the mutant DNA and the wild-type DNA.

Answer 12

The mutant DNA is the double strand that is made from the strand with the mismatch

The wild-type DNA is the one made from the strand that did not have the mismatch

Question 13

what is the role of DNA glycosylase

Answer 13

hydrolyzes the bond between the mispaired base and the sugar-phosphate backbone

DNA glycosylases provide specificity in repair. There are multiple different types of DNA glycosylases in our cells

Question 14

what is the role of Apurinic/apyrimidinic endonuclease 1 (APE1)

Answer 14

It cuts the DNA backbone during BER

Question 15

what is the AP lyase’s role?

Answer 15

associated with pol delta, it removes the deoxyribose phosphate

Question 16

What are the steps to Base Excision Repair (BER)? (4 steps)

Answer 16

1. DNA glycosylase: hydrolyzes the bond between the mispaired base and the sugar-phosphate backbone (it is the one who spots)
2. Apurinic/apyrimidinic endonuclease 1 (APE1): cuts the DNA backbone
3. AP lyase associated with pol beta, it removes the deoxyribose phosphate
4. DNA polymerase beta fills the gap and DNA ligase seals the nick in the sugar-phosphate backbone

Question 17

What is the difference between Base Excision Repair (BER) and Nucleotide Excision Repair (NER)?

Answer 17

With BER it is only one small modification whereas NER is the repair of a bulky legions

Question 18

Explain the formation of a thymine-thymine dimer:

Answer 18

when exposed to UV irridation, two consecutive thymine residues have the possibility to form a bond with each other
it changes the structure of the double-stranded DNA, it forms a kink

Question 19

Give 3 examples of causes to bulky legions:

Answer 19

1. Thymine-Thymine dimer
2. Aflatoxin B
   3.Benzo(a)pyrene

Question 20

What is aflatoxin B?

Answer 20

it is produced by a fungi (fungi aspergillus) which grows on food, cereal in particular.
if we eat this molecule, it reacts with some of the bases in our DNA (enzymatic modification) . It’s a big molecule that adds itself to the base

Question 21

where is Benzo(a)pyrene found ?

Answer 21

in the smoke of organic materials, (cigarette smoke or charbroiled food)

Question 22

What are the steps to (NER) Nucleotide Excision Repair? (4 steps)

Answer 22

1. Initial damage recognition. the complex formed by protein 23B and XP-C.
2. Opening of the DNA double-helix. the protein complex recruits the transcription factor TFIIH, which catalyzes the unwinding of the DNA region around the lesion. This in turn recruits RPA and XP-G, which also has an unwinding activity
3. XP-F and XP-G endonuclease. XP-F is recruited, both XP-F and XP-G have endonuclease activities, They generate cuts 24-32 bases apart, releasing the fragment carrying the lesion which will then be degraded.
4. DNA polymerase and DNA ligase. The ssDNA gap is then filled in by a DNA polymerase and joined to create a continuous strand by the DNA ligase

Question 23

what is a genomic rearrangement/ translocation

Answer 23

when two different chromosomes are joined together erroneously

Question 24

what are the names of the 2 ways to repair a double-strand break?

Answer 24

1. Non-homologeous end-joining
2. Homologous recombination

Question 25

TARDIGRADES!!

Answer 25

TARDIGRADESSS!!!

Question 26

What happens if a DNA strand is undergoing replication while it still has a single-strand break?

Answer 26

The break will be converted to a double-stranded break

Question 27

what is (HR) homologous recombination?

Answer 27

• High-fidelity mechanism to repair DNA breaks
• used during S and G2 phases ( those are when we have 2 copies of our genome)
• requires an undamaged copy of the chromosome to be repaired
• The process requires processing of breaks to expose ssDNA sequence and conduct a search for identical sequences. Use them as template for DNA synthesis and repair the break

Question 28

What are the steps to (HR) homologous recombination?

Answer 28

0. double-stranded break
1. ends digested by 5’ endonuclease leaving 3’ single-stranded ends
2. RecA (bacteria) or Rad51(eukaryotes)-mediated strand invasion of homologous chromosome. They bind to the single-strand DNA, they extend it and with the help of other proteins, they go to the nucleus and find the homologous sequence
3. The 3’ end of the invading strand is extended by DNA polymerase until the displaced single-strand base pairs with the other 3’ single strand generated initially
4. 3’ end is extended by DNA polymerase.
5. 5’ ends are ligated to form Holiday structures
6. Holiday structures are resolved

Question 29

What is Non-Homologous End Joining?

Answer 29

• Error-prone and can introduce mutations
• Used during the G1 phase of the cell (you don’t have a second copy of the genome)
• a complex formed by DNA-dependent protein kinase (DNA-PK) and Ku80/Ku70 heterodimer binds to the ends of the break (they recognize the break)
• ends are digested by nucleases

Question 30

give an example where double-stranded breaks are useful

Answer 30

during meiosis DNA recombination occurs between chromosomes from mom and dad to mingle their sequences

Question 31

How are the holiday structures drawn?

Question 32

What is Non-homologous End Joining know for?

Answer 32

It is error prone