Topic 7

Question 1

What are the two major purposes of homologous recombination?

Answer 1

1. Creation of new genetic diversity by exchanging genetic information between two DNA molecules with similar sequences
2. Recombinational DNA repair to fix single-stranded and double-strand breaks (DSB)

Question 2

True or false: DSBs are highly deleterious

Answer 2

True

Question 3

When do DSBs usually arise? (4)

Answer 3

1. DNA replication, when replication forks encounter a single-strand break in a template strand
2. Meiotic recombination
3. Exposure to UV light to γ radiation
4. Oxidative DNA damage during respiration

Question 4

What are the 4 possible effects of a damaged template on a replication fork?

Answer 4

1. Translesion synthesis can read through the lesions
2. Lesions prevent progress of the replisome, reseulting in a stalled replication fork
3. A single-strand break causes the replication fork to collapse, creating a DSB
4. A lesion is bypassed, leaving a singe-strand gap, and replication continues downstream

Question 5

Describe the repair of chromosomal DSB

Answer 5

1. The broken DNA ends are processed at the 5’ enduring strands to create 3’ overhangs at the site of the break
2. Recombinase catalyses the 3’-overhangs to form a D (Displacement)- loop structure by invading and recombining with the homologous chromosome to exchange short pieces of DNA
3. 2nd consecutive strand invasion (i.e. double crossover)
4. Using the undamaged homologous strands as template, the 3’ overhangs act as primers for DNA polymerase to extend and restore the lost information at the broken site

Question 6

What are the two pathways for completing DSB repair?

Answer 6

1. Synthesis-dependent annealing (SDSA) pathway
2. DSB repair pathway

Question 7

Describe the synthesis-dependent strand annealing (SDSA) pathway

Answer 7

The invading strands dissociate and anneal to each other followed by further replication and ligation to complete the process

Question 8

Describe the DSB repair pathway

Answer 8

Further extension occurs while strands are linked and creating 2 Holliday intermediates/junctions, which are resolved by Holliday intermediate resolvases
- X/X resolution (vertical cuts down junctions) of Holliday junction results in a “non cross over”
- X/Y resolution (1 vertical and 1 horizontal cut down junctions) of Holliday junction results in a “crossover”

Question 9

Describe what first occurs when a replication fork encounters a break in a template strand (4 steps)
- single-stranded break converts to DSB

Answer 9

1. A replication fork encounters a break in a template strand
2. Detachment of the machinery on one arm (the strand with the break) and converts to a double-strand break (because replication process on one arm is incomplete at site of single stranded break)
3. Replication fork is collapsed
4. Triggers a recombinational repair process

Question 10

Describe the steps of recombinational DSB repair at a collapsed replication fork to restore a viable replication fork (4 steps)

Answer 10

1. The broken DNA end is processed by nucleases to remove a segment of the 5’ end at the break to create a 3’ overhang, which will be used in a strand invasion reaction
2. Recombinase binds to the single-stranded 3’ overhang and promotes strand invasion to create a Holliday junction, so that the 3’ overhand is paired with its complementary strand and the other strand of the invaded duplex DNA is displaced
3. Branch migration (the junction point of a DNA crossover (called a Holliday junction) moves along the DNA strands.
   - The branch moves, may create a Holliday intermediate, but the net amount of duplex DNA does not change
4. Resolution of the Holliday intermediate, followed by ligation, restores a viable replication form

Question 11

Branch migration may trigger fork regression. What is fork regression?

Answer 11

Backward movement of the replication fork, allowing the lesion remaining reannealed with the parental strand

Question 12

Describe how fork regression restarts replication after the fork stalls using nucleotide excision repair

Answer 12

Nucleotide-excision repair pathway can repair the lesion and the replication can be restarted by digestion of the short arm and reloading of the replisome

Question 13

Describe how fork regression restarts replication after the fork stalls NOT using nucleotide excision repair

Answer 13

Replication of the short DNA arm followed by branch migration in the opposite direction allows the lesion to pair with the newly synthesized strand and replication can be started - lesion can be repaired later.

Question 14

What are the 5 stages of prophase I?

Answer 14

1. Leptotene
2. Zygotene
3. Pachytene
4. Diplotene
5. Diakinesis

Question 15

Describe leptotene stage of prophase I

Answer 15

Chromosomal condensation starts

Question 16

Describe zygotene stage of prophase I

Answer 16

Homologous chromosomes pair (i.e. synapsis) via the synaptonemal complex allowing crossing-over (i.e. recombination) to occur
- Synapsed chromosomes form a bivalent (refers to the pairing of the two homologous chromosomes) or tetrad (emphasizes that this structure contains four chromatids)

Question 17

Describe pachytene stage of prophase I

Answer 17

Synapsis is completed

Question 18

Describe the diplotene stage of prophase I

Answer 18

The synaptonemal complex disappears and homologous chromosomes start moving apart

Question 19

Chiasmata

Answer 19

The physical points of contact where homologous chromosomes exchange genetic material during crossing over in prophase I of meiosis.
- Chiasmata are the remaining points of attachment between homologous chromosomes until anaphase I

Question 20

What structure do diplotene bivalents form?

Answer 20

The “grasshopper”, which shows the chiasmata formed and accompanying inset (top and bottom)

Question 21

Double-strand breaks occur during which stage of prophase I?

Answer 21

Leptotene

Question 22

Strand invasion starts in which prophase I stage and carries into which stage?

Answer 22

Starts at end of leptotene, carries into zygotene

Question 23

Replicative extension starts in which prophase I stage and carries into which stage?

Answer 23

Starts in zygotene and ends in pachytene

Question 24

Double-crossover intermediates are present in which prophase I stage?

Answer 24

Pachytene

Question 25

When does the actual cross over occur during prophase I?

Answer 25

During the diffuse stage - a temporary period of chromatin relaxation that bridges the more active crossover and condensation stages of meiosis with the later stages of chromosome alignment and segregation.

Question 26

What is the breakage of chromosomes (i.e. recombination processes) visualized using?

Answer 26

Labelled H2A.X

Question 27

What protein catalyzes the formation of DSB? Explain how in general

Answer 27

- S. cerevisiae Spo11 is closely related to eukaryotic type II topoisomerases - Spo11 uses an active-site Tyr residue as a nucleophile in a transesterification reaction - Hydroxyl on Tyr attacks phosphate on 5' carbon, which occurs on the 5' carbon of both strands - Spo11 is linked to the broken strand by a 5' phosphotyrosyl linkage

Question 28

How many Spo11 proteins are required for one DSB?

Answer 28

2

Question 29

Describe what happens to the DNA after the Spo11 binds

Answer 29

1. Mre11-Rad50-Xrs2 complex binds to each Spo11 and cleaves the DNA on the 3' side of Spo11, releasing the linked Spo11 with a short segment of the attached 5' ended DNA strand (this creates two 3' overhangs) 2. The nuclease Sae2 degrades the same DNA strand a bit more 3. Sgs2-Dna2/Exo1 enzyme complex further degrades the 5' ends to create long 3' single-stranded extensions (overhangs) 4. Proteins, such as RPA and RecA-class recombinases, are loaded onto the 3' overhang strand to prepare the site for recombination (RPA binds to ssDNA to prevent formation of secondary DNA structures)

Question 30

What is gene conversion? When does it occur?

Answer 30

A non-reciprocal transfer of genetic information as an outcome of DNA repair, especially during meiosis - Gene conversion occurs during both crossover and non-crossover events from Holliday junction resolutions - Unlike typical recombination, where DNA is exchanged between homologous chromosomes, gene conversion involves unidirectional copying without swapping.

Question 31

True or false: MITOTIC recombination is rare.

Answer 31

True

Question 32

Exposure to what increases mitotic recombination?

Answer 32

Exposure to ionizing radiation, endonucleolytic action, and replication fork with a template strand break can lead to DSBs ini mitosis

Question 33

What stages of the cell cycle does mitotic recombination occur during?

Answer 33

S and G2 phases of the cell cycle

Question 34

What do mitotic DSBs trigger in the cell cycle?

Answer 34

Mitotic DSB triggers checkpoint that halts the progression of the cell cycle

Question 35

Describe the repair of mitotic DSB (5 steps)

Answer 35

1. Generation of 3' single-stranded overhangs by nuclease 2. RPA coating on single-stranded DNA 3. Recruitment of recombination mediator proteins, Rad 52 and BRCA2 4. Recruitment of Rad51 recombinase 5. DSB undergoes either the SDSA (synthesis-dependent strand annealing) or the DSBR pathway (double-stranded break pathway - involves resolution/dissolution of the double Holliday intermediates)

Question 36

Describe the SDSA (synthesis-dependent strand annealing) pathway

Answer 36

After synthesis of new strand facilitated by strand invasion, strand re-anneal into original strands and continue synthesis

Question 37

What are the two mating types in haploid S. cerevisiae? What do these mating types depend on?

Answer 37

a or α - depends on which genes are expressed at the MAT locus

Question 38

The genetic information for MATα is stored, but not expressed, in...

Answer 38

HMLα - Need to "migrate" to MAT locus to be expressed

Question 39

The genetic information for MATa is stored, but not expressed, in...

Answer 39

MMRa - Need to "migrate" to MAT locus to be expressed

Question 40

What allows S. cerevisiae to switch between mating type?

Answer 40

DSB created by HO nuclease followed by gene conversion allows switch between mating type.

Question 41

What pathway is used to switch between mating types in S. cerevisiae?

Answer 41

SDSA pathway

Question 42

Describe how recombinational DNA repair of DSB switches S.cerevisiae (6 steps)

Answer 42

1. HO nuclease introduces the DSB at the MAT locus 2. The free DNA ends are processed to generate 3' single-stranded overhangs 3. Rad51 binds to the overhangs and directs strand invasion 4. The original mating-type information on the MAT locus is removed by nuclease digestion 5. DNA polymerase extends the invading 3' end 6. Dissociation of the extended 3' end back to original strand and completion of the DNA synthesis (MAT locus is now the same type as the HML/MMR locus that was used as a template)

Question 43

True or false: Use of recombination to switch expression between different sets of genes is common in prokaryotes and eukaryotes

Answer 43

True

Question 44

What is the main way to repair DSBs while directed homologous recombination is not available?

Answer 44

Nonhomologous End Joining (NHEJ)

Question 45

Why is it important for differentiated mammalian cells to repair their DNA damages by NHEJ?

Answer 45

Differentiated mammalian cells divide rarely and typically spend time in the G1 and G0 phases

Question 46

True or false: NHEJ conserves the original DNA sequence

Answer 46

False - Unlike recombinational repair, NHEJ does not conserve the original DNA sequence (i.e. NHEJ can introduce mutations)

Question 47

In which stage of the cell cycle does NHEJ occur?

Answer 47

In G1 or G0

Question 48

Describe the steps of NHEJ (8 steps)

Answer 48

1. Recruitment of Ku70 and Ku80 heterodimer to the broken ends to act as a molecular scaffold 2. Ku70-80 interacts with kinase DNA-PK and nuclease Artemis -> synapsis (held together) of broken ends 3. Helicase separates the DNA 4. The kinase activity of DNA-PK activates the endonuclease activity of Artemis 5. Artemis causes cleavage of DNA segments, which forms sticky ends 6. Strands from the two different ends are annealed 7. Small DNA gaps are filled in by the eukaryotic Pols 8. The nicks are sealed by a protein complex consisting of XRCC4 (x-ray cross complementation group), XLF (XRCC4-like factor), and eukaryotic DNA ligase IV

Question 49

What two ways can CRISPR edit the genome?

Answer 49

1. Inducing homologous recombination using a sister chromatid or donor DNA to knock "IN" a gene 2. Inducing non-homologous end-joining (NHEJ) to insert/delete genes (knockOUT)