DNA repair and recombination

Question 1

What are the different types of damage that DNA is constantly subject to? (4)

Answer 1

DNA is subject to
hydrolytic depurination,
cytosine deamination,
guanidine oxidation,
and methylation of adenine

Question 2

What are mutations?

Answer 2

Mutations are permanent, transmissible changes to the genetic material of a cell or organism.

Question 3

What are mutagens?

Answer 3

Mutagens are chemical compounds, ultraviolet (UV) radiation, or ionizing radiation that increase the frequency of mutations.

Question 4

Mutations can occur… (3 ways)

Answer 4

spontaneously
by transposable elements
by errors during replication

Question 5

What are carcinogens?

Answer 5

Carcinogens are agents that cause cancer, and many of them are mutagens.

Question 6

What is the importance of DNA stability?

Answer 6

DNA stability is important because DNA is the information repository of the cell.

Question 7

Which DNA polymerases have a proofreading activity in eukaryotes?

Answer 7

DNA polymerases e and d, but not a, have a 3’ to 5’ exonuclease or “proofreading” activity.

Question 8

What is the mechanism of proof reading?

Answer 8

Incorporation of an incorrect base causes the polymerase to pause. The 3’ end of the new strand is free to move to the 3’ -> 5’ exonuclease site and the mispaired base is removed.

Question 9

What is the most common point mutation?

Answer 9

The most common point mutation is a single base change from C to T.

Question 10

How does the point mutation is change from C to T happen?

Answer 10

Deamination
(can also produce uracil from cytsine)
amino group not so stable and this reaction just happens.

Question 11

At what step must the base excision repair occur?

Answer 11

After the deamination and before the replication

Question 12

In a mismatched base pair,
which base is correct and which base is wrong?

Answer 12

T-G mismatches almost always result from deamination of C to U or 5mC to T, so T is wrong
and should be replaced by C.

Question 13

What are the steps of the base excision repair? (4)

Answer 13

1. The DNA glycosylase breaks the bond between T and the sugarphosphate backbone
2. The APEI endonuclease cuts the DNA strand where it is missing a base
3. AP lyase (part of DNA pol beta) removes deoxyribose phosphate
4. A special DNA polymerase (beta) inserts C using G as template, DNA ligase repairs
   sugar-phosphate backbone

Question 14

What is the difference between exonuclease vs endonuclease?

Answer 14

exo: cuts at the end of the strand
endo: cuts in the middle

Question 15

What types of error does the process of Mismatch Excision Repair repair?

Answer 15

Mismatch excision repair fixes errors introduced during replication, including :
-basepair mismatches
-insertions or deletions of one or a few nucleotides.

Question 16

During a Mismatch Excision Repair, which strand is the wrong one?

Answer 16

the newly synthesized strand is the wrong one, there are mechanisms to recognize which strand this is

Question 17

When does a Mismatch Excision Repair happen?

Answer 17

after DNA replication

Question 18

What are the steps for Mismatch Excision Repair? (4)

Answer 18

1. MSH2 and MSH6 recognize the mismatch and distinguish the newly synthesized (daughter) strand.
2. This triggers binding and activity of MLH1 endonuclease (dimerized with PMS2). MLH1 cuts the newly synthesized strand.
3. DNA helicase unwinds and DNA exonuclease digests several nucleotides of the daughter strand.
4. DNA polymerase (Pol d) fills in the missing nucleotides using the other strand as template and ligase repairs the sugar-phosphate backbone.

Question 19

What does Nucleotide Excision Repair fix?

Answer 19

DNA regions where chemically modified bases locally distort the double helix.

Question 20

What is the thymine-thymine dimer?

Answer 20

distortion of the helix caused by UV irradiation, where two adjacent thymine bases become chemically bonded to each other.

Question 21

Chemicals that can bind to DNA
________, including many carcinogens, also locally _______ the double _________.

Answer 21

bases
distort
helix

Question 22

What are the steps of Nucleotide Excision Repair?

Answer 22

1. XP-C and 23B proteins recognize the distorted double helix.
2. TFIIH, XP-G, and RPA unwind the helix to make a bubble of ~25 nucleotides.
3. XP-F and XP-G cut the damaged strand.
4. DNA polymerase fills in the missing nucleotides using the other strand as template and ligase repairs the sugarphosphate backbone.

Question 23

What are the names XP-n derive from ?

Answer 23

The names XP-n derive from xeroderma pigmentosum, a genetic disease that causes a high disposition to UV-induced cancer. Mutations that affect XP proteins cause this disease.

Question 24

What happens if a thymine-thymine dimer is not repaired and it enters a replication fork? (4)

Answer 24

• Normal replicative DNA polymerases (Pol d and Pol e) stall when they reach a thymine-thymine dimer.
• A special translesion polymerase (Pol eta n) can read through the thymine-thymine dimer, but this polymerase lacks proofreading activity.
• So the region in the vicinity of the thymine-thymine dimer will be likely to contain mutations caused by replication errors.
• Eventually Pol n will get replaced by one of the normal replicative DNA polymerases.

Question 25

What causes double-strand breaks in DNA ?

Answer 25

Radiation (X-rays, g-rays) and some anticancer drugs (bleomycin for example)

Question 26

What happens if a double-strand break is not repaired?

Answer 26

then the part of the chromosome distal to the break would be lost at the next cell division, probably causing lethality.

Question 27

What does Double-Strand Break Repair End Joining do?

Answer 27

rejoins the broken chromosome ends but base pairs are usually lost at the joining point, producing a small deletion

Question 28

What are the steps of Double-Strand Break Repair End Joining? (3)

Answer 28

1. Ku and DNA-PK bind the end of a double-strand break (DSB). 2. When two DSBs bound by these proteins come together, they recruit nucleases that remove several bases. 3. The two double-stranded molecules are then ligated together.

Question 29

Does the mechanism of Double-Strand Break Repair End Joining ensure that what is ligated together originated from adjacent parts of the same chromosome?

Answer 29

No, it does not ensure that what is ligated together originated from adjacent parts of the same chromosome, so it can fuse DSBs originating from different chromosomes or far apart on the same one, producing chromosomal rearrangements.

Question 30

What happens during the process of Double-strand Break Repair by Homologous Recombination?

Answer 30

* The damaged DNA sequence is replaced with a copy of an undamaged copy of the same sequence on the homologous chromosome in diploid organisms. * This involves an exchange of strands between separate DNA molecules, a process known as recombination.

Question 31

What are the steps of Double-strand Break Repair by Homologous Recombination ? (7)

Answer 31

1. Replication fork collapse 2. 5'-exonuclease acts on broken end. Other daughter strand ligated to repaired parental strand in unbroken chromosome. 3. RecA- or Rad51-mediated strand invasion 4. Branch migration 5. Cuts strands at crossover 6. Ligate ends 7. Rebuild replication fork and continue replication

Question 32

* A similar mechanism can repair a double-strand break in a chromosome and can also produce an _________ of segments between two double-stranded DNA molecules. * The __________ structure that involves all four strands can be resolved in two alternative ways. One regenerates the original ____________, while the other produces two _____________ strands and two recombinant strands containing DNA from both parents.

Answer 32

exchange Holliday chromosomes uniparental