Brainscape's Knowledge GenomeTM

Browse over 1 million classes created by top students, professors, publishers, and experts.


See full index

CMMB 411- Doreen > Topic 7 > Flashcards

Topic 7 Flashcards

1
Q

What are the two major purposes of homologous recombination?

A
  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)
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

True or false: DSBs are highly deleterious

A

True

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

When do DSBs usually arise? (4)

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

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

A
  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 DSN
  4. A lesion is bypassed, leaving a singe-strand gap, and replication continues downstream
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

Describe the repair of chromosomal DSB

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are the two pathways for completing DSB repair?

A
  1. Synthesis-dependent annealing (SDSA) pathway
  2. DSP repair pathway
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Describe the synthesis-dependent strand annealing (SDSA) pathway

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Describe the DSB repair pathway

A

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”

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

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

A
  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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

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

A
  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 movement of the branch point in a branched DNA formed from two DNA molecules with identical sequences)
    - 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
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

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

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What are the 5 stages of prophase I?

A
  1. Leptotene
  2. Zygotene
  3. Pachytene
  4. Diplotene
  5. Diakinesis
How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

Describe leptotene stage of prophase I

A

Chromosomal condensation starts

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

Describe zygotene stage of prophase I

A

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)

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

Describe pachytene stage of prophase I

A

Synapsis is completed

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

Describe the diplotene stage of prophase I

A

The synaptonemal complex disappears and homologous chromosomes start moving apart

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

Chiasmata

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

What structure do diplotene bivalents form?

A

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

21
Q

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

A

Leptotene

22
Q

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

A

Starts in leptotene, carries into zygotene

23
Q

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

A

Starts in zygotene and ends in pachytene

24
Q

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

A

Pachytene

25
Q

When does the actual cross over occur during prophase I?

A

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.

26
Q

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

A

Labelled H2A.X

27
Q

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

A
  • 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
28
Q

How many Spo11 proteins are required for one DSB?

A

2

29
Q

Describe what happens to the DNA after the Spo11 binds

A
  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
  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)
30
Q

What is gene conversion? When does it occur?

A

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

31
Q

True or false: MITOTIC recombination is rare.

A

True

32
Q

Exposure to what increases mitotic recombination?

A

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

33
Q

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

A

S and G2 phases of the cell cycle

34
Q

What do mitotic DSBs trigger in the cell cycle?

A

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

35
Q

Describe the repair of mitotic DSB (5 steps)

A
  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)
36
Q

Describe the SDSA (synthesis-dependent strand annealing) pathway

A

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

37
Q

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

A

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

38
Q

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

A

HMLα
- Need to “migrate” to MAT locus to be expressed

39
Q

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

A

MMRa
- Need to “migrate” to MAT locus to be expressed

40
Q

What allows S. cerevisiae to switch between mating type?

A

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

41
Q

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

A

SDSA pathway

42
Q

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

A
  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 overhands 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)
43
Q

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

A

True

44
Q

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

A

Nonhomologous End Joining (NHEJ)

45
Q

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

A

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

46
Q

True or false: NHEJ conserves the original DNA sequence

A

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

47
Q

In which stage of the cell cycle does NHEJ occur?

A

In G1 or G0

48
Q

Describe the steps of NHEJ (8 steps)

A
  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
49
Q

What two ways can CRISPR edit the genome?

A
  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)

CMMB 411- Doreen (9 decks)

Brainscape helps you reach your goals faster, through stronger study habits.
© 2024 Bold Learning Solutions. Terms and Conditions