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When does homologous recombination occur in eukaryotes?
In meiosis, homologous recombination occurs at the four-strand stage of meiotic prophase and involves only two non-sister strands of the four; it usually happens both in males (during spermatogenesis) and females (during oogenesis)
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What mechanism is a major part of DNA double stranded break repair mechanism in mitotic cells?
Recombination
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Most lethal form of DNA damage
DNA double-stranded break (DSB)
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How often do DSBs occur in mammalian cells?
2 to 5x a day!
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How are double stranded breaks repaired?
- Non-homologous end joining (NHEJ)
- Single-strand annealing (SSA)
- synthesis dependent strand annealing (SDSA)
- Double-strand break repair (DSBR)
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In DSBR, what follows broken end resection by exonucleases?
- formation of 3' overhangs
- one of the 3' ends invades homologous duplex, forming a D-loop
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How is the D-loop extended in DSBR?
- Extension of the 3'-OH by DNA synthesis enlarges the D-loop
- The displaced loop then pairs with the other side of the break, capturing the second double stranded break end
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How are Holliaday junctionscreated?
Following strand invasion, DNA synthesis fills in the gap and ligase seals the nicks, resulting in the formation of the double Holliday junctions
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How is homologous recombination in eukaryotes different in SDSA vs DSBR?
- Synthesis dependent strand annealing (SDSA) does not capture both strands as in DSBR.
- The strand in D-loop is displaced and anneals back to the end of the original strand
- patch recombinant
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What three proteins form the MRN complex (which acts as a sensor of DNA double-strand breaks)?
- MRE = meiotic recombination
- RAD = radiation sensitive
- NBS = Nijmegen breakage syndrome
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How is generic eukaryotic homologous recombination different than mammilian homologous recombination?
- Mostly we have more proteins to memorize
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Describe this image:
Enzymology of homologous recombination in mammals
The MRE11–RAD50–NBS1 (MRN) complex acts as a sensor of DNA double-strand breaks
MRN and CtIP 5’ endonuclease bind and process the ends (~end resection by RecBCD in bacteria); RNAPIII helps with 3’ end protection via RNA-DNA hybrids
Rad51, Rad52, and RPA associate with the resulting single-strand overhangs: Rad51 (~RecA) forms a nucleoprotein filament with the help of RPA and RAD52
RAD52, RAD54, BRCA1 and BRCA2 all participate in the strand invasion by the RAD51-coated single-strand tail
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What enzyme helps with protection of 3' ends during homologous recombination?
RNAPIII helps with 3' end protection via RNA-DNA hybrids
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What is MMEJ?
A subset of A-NHEJ relies on microhomologous sequences on either side of the DSB, thus is named as microhomology-mediated end joining (MMEJ) [10–12]. MMEJ is a mutagenic DSB repair mechanism, which always associates with deletions flanking the break sites and contributes to chromosome translocations and rearrangements.
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Single-strand annealing model
- DSB occurs
- following end processing, the single strands anneal by homology
- endonucleases remove single-strand tails and DNA ligase seals the nicks
- outcome: loss of one of the repeats of the sequence in between
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What DNA repair process is similar to microhomology mediated end joining?
Single strand annealing!
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Non-homologous end joining (bacteria):
What recognizes the double stranded breaks?
Ku protein homodimers
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Non-homologous end joining (bacteria):
Following Ku protein recognizing the breaks, what happens?
- Ku recruits LigD
- LigD has nuclease and polymerase activities and ligase activities
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Non-homologous end joining (mammals): what initially recognizes the double stranded breaks?
- Ku70/Ku80 heterodimer
- The Ku:DNA complexes are juxtaposed to bridge the ends
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Non-homologous end joining (mammals):
Following the Ku:DNA complexes bridging the ends, what then processes the ends?
The nuclease Artemis
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Non-homologous end joining (mammals):
Following end digestion by Artemis, how is the gap filled?
The gap is filled by processing enzymes Pol λ and Pol μ
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Non-homologous end joining (mammals):
Following filling the gap via polymerase enzymes, what ligates the ends?
- DNA Lig IV and XRCC4
- XRCC4= X-ray-repair cross-complementing protein 4
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Describe what is happening in this diagram:
Heterodimers of Ku70 and Ku80 bind DNA double-strand breaks and recruit DNA-PKcs (catalytic subunit of DNA-dependent protein kinase) to the site of DNA damage
This interaction induces a conformational change in DNA-PKcs and activates its protein-kinase activities
The DNA-PK holoenzyme (Ku70/80 plus DNA-PKcs) recruits Artemis, XRCC4 (X-ray-repair cross-complementing protein 4), DNA lig IV and DNA Pol μ to the site of DNA damage
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What is the outcome of NHEJ?
repair of dsDNA breaks, but incorporation of mutations (usually in the form of small indels)
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What repair pathway repairs a majority of dsDNA breaks induced by meganucleasess, TALENs, zinc finger nucleases and CRISPR?
NHEJ
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Defects in the NER mechanism are responsible for several genetic disorders, including:
- Xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin cancer incidence and premature aging
- Cockayne syndrome: hypersensitivity to UV and chemical agents
- Trichothiodystrophy: sensitive skin, brittle hair and nails
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What is a generic name that DNA repair disorders are often called?
"Segmental progerias"
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Diseases associated with reduced DNA repair function:
- fanconia amenia
- hereditary breast cancer
- hereditary colon cancer
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