Topic 7 (Homologous Recombination) Flashcards
What are the two major purposes of recombinational repair?
- Creation of new genetic diversity by exchanging genetic information between two homologous chromosomes
- recombinational repair to fix single strand and double strand breaks
When do DSB usually occur?
- DNA replication, when the replication fork encounters an SSB break in the template
- meiotic recombination
- exposure to UV or radiation
- oxidative DNA damage during replication
What are the four possible effects of a damaged template on the replication fork?
- translesion synthesis reads through the lesions (lesion is replicated)
- Lesions prevent progress of the replisome, resulting in a stalled fork
- SSB causes collapse of fork, creating a DSB
- lesion is bypassed, leaving a single-strand gap, and replication continues downstream
True/False? Lesion bypass can only occur on the lagging strand because its synthesis is already interrupted by Okazaki fragments
False. May occur on either
For the replication fork to collapse, which strand must have a single-strand break?
Leading
Explain the steps of homologous recombination repair of a DSB
- Broken ends are processed at the 5’ ends by 5’-3’ exonucleases, creating 3’ overhangs
- recombinase catalyzes the 3’ overhangs to form D (displacement)-loop structure by invading and recombining with the homologous chromosome to exchange short pieces of DNA
- second strand invasion (double crossover)
- strand extension by DNA Pol using the undamaged strands as templates and 3’ overhangs as primers
- completion of the pathway (two paths)
Following homologous recombination repair, what is the synthesis-dependent strand annealing (SDSA) pathway?
Invading strands dissociate and anneal to each other, followed by further replication and ligation (basically go back to the way they were pre-break)
Following homologous recombination repair, what is the double-strand break repair (DSBR) pathway?
Further extension occurs while the strands are linked, creating 2 Holliday junctions, which are resolved by Holliday intermediate resolvases in multiple ways
In the context of DSBR, what is dissolution? What is resolution?
Dissolution: non-crossover pathway
Resolution: crossover pathway
True/False? Both SDSA and DSBR can result in genetic exchange
False. Only DSBR
True/False? Both SDSA and DSBR can result in non-crossover
True
In the resolution of a Holliday junction, an X x X cut results in:
Non-crossover
In the resolution of a Holliday junction, a Y x Y cut results in:
Non-crossover
In the resolution of a Holliday junction, an X x Y cut results in:
Crossover
In the resolution of a Holliday junction, a Y x X cut results in:
Crossover
Describe the reconstruction process after a collapsed replication fork
- single-strand break encountered on leading strand by replisome
- SSB converted into DSB
- fork collapse
- 5’ end of the broken segment is processed to form a 3’ overhang
- recombinase binds to the 3’ overhang and promotes a strand invasion to create a Holliday junction, so that the overhang is paired with its complimentary strand and the other strand of the invaded duplex DNA is displaced
- branch migration
- resolution of the Holliday junction, followed by ligation, restores the fork
What does repair of a collapsed replication fork require?
Reattachment of the broken arm to recreate the fork
What is fork regression? What may it be triggered by?
Backward movement of the replication fork to before the lesion; branch migration
What may fork regression allow for?
Lesion remains within parental strand, and will be repaired later
Fork regression may result in these two pathways
- lesion is repaired (NER) and newly replicated strands are digested by a nuclease up until the replication fork, replisome reloaded
- replication of the short DNA arm followed by branch migration in the opposite direction (direction of replication fork), so the lesion is paired with the newly synthesized strand and can be repaired later (remains a lesion, not a mutation)
In which phase is the eukaryotic cell 4n (tetraploid)?
After S phase (meiosis I)
What are the stages within prophase I?
- leptotene
- zygotene
- pachytene
- diplotene
What is the leptotene phase?
Chromosomal condensation starts
What is the zygotene phase?
Homologous chromosomes pair via the synaptonemal complex
What is synapsis?
Homologous chromosomes pair, allowing for recombination to occur
When chromosomes form a tetrad, how are they connected?
Physically connected through the synaptonemal complex
What is the pachytene phase?
Synapsis is completed
What is the diplotene phase?
Synaptonemal complex disappears and homologous chromosomes start moving apart
What are chiasmata?
Physical points of attachment between homologous chromosomes (crossover sites)
In which prophase I stage do DSBs and 5’ digestion occur?
Leptotene
In which prophase I stage does strand invasion occur?
End of leptotene, beginning of zygotene
In which prophase I stage does replicative extension occur?
End of zygotene, beginning of pachytene
In which prophase I stage do double-crossover intermediates form?
Pachytene
Which protein is used to observe changes in chromosomal packing during the stages of prophase I?
H2A.X (used to see double-stranded breaks)
What is Spo11?
A bacterial enzyme closely related to eukaryotic type II topoisomerases
Describe the reaction that Spo11 catalyzes
Uses an active-site Tyr residue as a nucleophile in a transesterification reaction, which results in a DSB and Spo11 is linked to the broken strand via a 5’-phosphotyrosyl linkage
What big picture process is Spo11 used to initiate?
Recombination
Describe the steps following a DSB by Spo11
- Mre11-Rad50-Xrs2 complex binds to each Spo11 (5’ ends of DSB) and cleaves 3’ to Spo11, releasing Spo11 with a small DNA segment and leaving 3’ overhangs on the DNA
- Sae2 (5’-3’ exonuclease) degrades the 5’ ends a bit more
- Sgs2-Dna2/Exo1 enzyme complex further degrades the 5’ ends to create long 3’ overhangs
- RPA and RecA-class recombinases are loaded onto the overhang to prepare the site for recombination
What is gene conversion?
A non-reciprocal transfer of genetic information as an outcome of DNA repair, especially during meiosis
True/False? Mitotic recombination happens just as frequently as meiotic recombination
False. Rarer
Mitotic recombination can be caused by exposure to:
Ionizing radiation, endonucleolytic action, and replication fork with a SSB in the template can lead to DSB in mitosis
Mitotic recombination occurs in which phases?
S and G2
What happens to the mitotic cell cycle following a DSB?
DSB triggers a checkpoint that halts the progression of the cycle
Describe the process of mitotic recombination
- DSB
- generation of 3’ overhangs
- RPA coating on ssDNA
- recruitment of recombination mediator proteins, Rad52 and BRCA2
- recruitment of Rad51 recombinase
- SDSA/DSBR
True/False? All S. cerevisiae have both the HML(alpha) and HMRa genes, despite being haploid
True
True/False? Haploid S. cerevisiae can be either alpha or a type
True
True/False? Diploid S. cerevisiae can be either alpha or a type
False. Both alpha and a
For a sex phenotype to be expressed in S. cerevisiae, what must happen?
The gene for that phenotype must be located in the MAT locus
Describe mating type switch in S. cerevisiae
- HO nuclease makes a double-strand cut in the MAT locus
- 3’ overhangs generated
- Rad51 binds overhangs and directs strand invasion
- original mating type info is removed by nuclease digestion
- DNA Pol extends invading 3’ end
- Dissociation of extended 3’ end and completion of DNA synthesis
Which pathway is used in mating type switch in S. cerevisiae?
SDSA
What pathway is used when homologous recombination is not available?
NHEJ
True/False? NHEJ does not completely conserve the DNA sequence
True
Describe the steps of NHEJ
- DSB occurs in G0 or G1
- recruitment of Ku70 and Ku80 heterodimer to the broken ends to act as a scaffold
- Ku70/80 interacts with DNA-PK and Artemis to cause synapsis of broken DNA ends (held together)
- helicase separates DNA
- kinase activity of DNA-PK activates endonuclease activity of Artemis
- cleavage of DNA segments
- strands from the two different ends are annealed
- small gaps filled in by eukaryotic Pols
- nicks are sealed by XRCC4, XLF, and DNA ligase IV
True/False? DNA ligase IV is found in both prokaryotes and eukaryotes
False. Just eukaryotes
True/False? NHEJ is conserved between prokaryotes and eukaryotes
False. Just eukaryotes
Which DNA repair system is used for gene editing? Where did it originate from?
CRISPR-Cas9; prokaryotic immune system
How can CRISPR-Cas9 be used for gene editing (what kinds of genes may result from editing with this system)? Which pathways are used for each possibility?
Knock-ins (homologous recombination) or knockouts (NHEJ)
