Homologous recombination: overview Flashcards
(44 cards)
1
Q
What are DSBs? (3)
A
- Double strand breaks generated when 2 complementary strands of DNA are broken simultaneously at adjacent sites
- 2 DNA fragments become physically dissociated from each other
- Transcription and replication can no longer happen as a result
2
Q
What is the consequence of DSBs? (2)
A
- If not repaired accurately, can lead to various genomic rearrangements such as DNA deletion, translocations and fusions
- Genomic rearrangements are common in cancer cells that have defects in homologous recombination (HR) as they can’t repair spontaneous DSBs accurately
3
Q
What are the 2 types of DSBs generated by random DNA damage events?
A
- Two ended DSBs
- One ended DSBs
4
Q
What are the 2 events which result in DSBs?
A
- Random DNA damage events (two/one ended DSBs)
- Programmed DSB formation
5
Q
What are two ended DSBs? (2)
A
- Classic break in both strands of the duplex
- Caused by ionising radiation, radio-mimetic chemicals (e.g. bleomycin), mechanical stress on chromosomes
6
Q
What are one ended DSBs? (2)
A
- SSBs or alkylation damage may cause replication fork to collapse resulting in a one ended DSB
- Depletion of nucleotides, alkylation and UV damage can stall replication forks
7
Q
When are programmed DSBs required? (2)
A
- In V(D)J recombination in developing lymphoid cells
- In meiosis to initiate homologous recombination during crossing over to create genetic diversity
8
Q
How are one ended DSBs formed by a nick in one DNA strand? (3)
A
- As the replication fork encounters a nick in one DNA strand, the lagging strand joins up with the broken segment, filling the gap on one strand
- This leaves the leading strand as a one ended DSB
- Replication can’t proceed without repair
9
Q
How are one ended DSBs formed by a lesion in one DNA strand? (3)
A
- DNA lesion (adduct) blocks the progress of the replication fork
- The lagging strand can’t continue to be made causing fork reversal and a cruciform ‘chicken foot’ structure with the leading strand pairing with the lagging strand instead of the template strands
- The cruciform structure is cleaved by holliday junction resolvase resulting in a one ended DSB which requires repair
10
Q
What are endogenous causes of DSBs? (3)
A
- ROS
- Metabolic intermediates
- Oncogene expression
11
Q
What are exogenous causes of DSBs? (3)
A
- Radiation
- Chemotherapy
- Environmental toxins
12
Q
When might mechanical stress cause DSBs?
A
During cell migration
13
Q
What are the 4 pathways for DSB repair?
A
- Classical non-homologous end joining (c-NHEJ)
- Homologous recombination (HR)
- Alternative end joining (aEJ)
- Single-strand annealing (SSA)
14
Q
What is c-NHEJ? (4)
A
- Direct ligation of 2 DNA fragments
- Restricted to G1, S and G2 phase
- Doesn’t require broken ends to contain homology or any resection of the break site
- Rapid but end processing is typically error prone and may lead to chromosome rearrangements such as deletions and translocations
15
Q
What is HR? (5)
A
- Usually error free
- Limited to S and G2 phase
- Requires extensive end processing of the DNA breaks (5’-end resection)
- Requires a homologous DNA template sequence
- Slow but accurate
16
Q
What is aEJ? (3)
A
- Requires resection and microhomology (4-20bp) to base pair within the duplex, forming flaps which are removed and religated to repair
- Limited to S and G2 phases
- Error prone
17
Q
What is SSA? (3)
A
- Requires resection and more extensive homology than aEJ
- Limited to S and G2 phase
- Error prone
18
Q
What factors determine which DSB repair pathway is going to be used? (2)
A
- Extent of 5’-end resection
- Level of homology
19
Q
What are the steps of HR? (5)
A
- DNA end resection
- Strand invasion
- Strand extension and second end capture
- DNA ligation with original fragments and double Holliday junction formation
- Resolution of the double Holliday junction resulting in non-crossover or crossover products
20
Q
What are the main principles of DNA replication of the leading strand? (2)
A
- DNA replication is 5’ to 3’
- ssDNA must be exposed for extension
21
Q
What is DNA end resection in HR? (2)
A
- Produces a 3’ ssDNA tail from a DSB
- Requires nucleolytic degradation of the 5’ terminated strands called 5’-3’ end resection
22
Q
What is strand invasion in HR? (2)
A
- The generated 3’ ssDNA (invading strand) searches for a homologous region on the 5’-3’ template DNA strand through base pairing
- The 3’ ssDNA displaces the 3’-5’ like template strand and forms base pairs resulting in a D-loop DNA structure
23
Q
What is strand extension and second end capture in HR? (2)
A
- Extension of the invading strand in the D loop is called first end synthesis and is similar to leading strand synthesis in DNA replication
- The displaced template 3’-5’ strand pairs with the other 3’ ssDNA end in the broken duplex (second end capture) which is then extended by DNA synthesis
24
Q
What is the role of strand invasion? (2)
A
- To establish the replication fork-like structure and prepare for DNA synthesis
- D loop structure is similar to a leading strand replication fork but lacking a lagging strand
25
What is DNA ligation and double Holliday junction formation in HR?
After synthesis, the ends of the 2 DNA fragments are ligated by a DNA ligase and a double Holliday junction is formed
26
What is a Holliday junction?
A branched nucleic acid structure that contains four double-stranded arms joined together
27
How are single Holliday junctions resolved? (2)
- 2/4 of the strands in the structure are nicked by a resolvase enzyme and then religated
- Whether the structure cuts horizontally or vertically determines the outcome of the DNA products
28
How does nicking the single Holliday junction horizontally impact the resulting DNA duplexes? (2)
- Parental DNA segments remain covalently linked and are not exchanged resulting in non-recombinant products
- Resulting duplex contains some regions of heteroduplex
29
How does nicking the Holliday junction vertically impact the resulting DNA duplexes? (2)
- Parental segments are exchanged resulting in recombinant products
- Resulting duplex contains some regions of heteroduplex
30
What are the ways in which a double Holliday junction can be resolved in HR? (2)
- Nicks in the same DNA strands (i.e. the 3'-5' strand of each duplex which are actively tangled in each junction) results in repair without crossover
- Nicks in different DNA strands (i.e. one junction is nicked horizontally at the tangled stands and the other vertically at the non-tangled strands) results in crossover products
31
What is chromosomal crossover?
The exchange of genetic material between the damaged and template chromosomes that results in a homologous recombinant
32
What is the alternative to double Holliday junction resolution in HR?
Dissolution of double Holliday junctions
33
What is dissolution of double Holliday junctions? (3)
- The 2 Holliday junctions migrate towards each other
- Hemi-catenation occurs where the 2 strands from separate duplexes are wound around each other
- Enzymes decatenate the DNA resulting in separation of the duplexes and repair without crossover
34
What are the potential sources of homologous template in HR? (3)
- Usually uses sister chromatids which are formed during DNA replication which is why HR takes place in late S/G2 phase of the cell cycle
- Can use homologous chromosomes e.g. crossing over during meiosis
- Rarely HR can happen with a non-homologous chromosome resulting in translocations e.g. cancer
35
What are sister chromatids vs homologous chromosomes? (2)
- Sister chromatids are identical copies formed during DNA replication, are joined by the centromere and are separated in anaphase and then called chromosomes
- Homologous chromosomes have the same genes but different alleles as one is maternal and the other paternal
36
What are the genetic consequences of HR? (3)
- Crossover with gene conversion (lost gene is replaced with the allele from the homologous chromosome AND the resulting products are recombinant)
- Non-crossover with gene conversion (lost gene is replaced with the allele from the homologous chromosome but the products are non-recombinant)
- Non-crossover
37
What is gene conversion? (3)
- An event in which a gene in a heterozygous diploid appears to have taken on the identity of its allele
- If the alleles were different this causes loss of heterozygosity (LOH)
- Distinguished from crossing-over by its nonreciprocal nature
38
What is usually the result of HR in mitotic cells?
Natural bias towards dissolution and non-crossover to limit genomic instability
39
How do mitotic cells limit the effects of gene conversion? (2)
- Use sister chromatids as the template which means HR is restricted to S and G2 phase of the cell cycle
- The cohesin complex maintains tight association of sister chromatids, therefore improving genome stability maintenance
40
What are the subpathways of HR? (4)
- 2 ended HR
- Synthesis dependent strand annealing
- Break induced repair
- All result in non-crossover events
41
What is synthesis-dependent strand annealing? (4)
- Same resection and strand invasion as normal HR
- NO second end capture, the invading strand is extended and anneals to the other resected end of the DSB
- Gap on the other strand is filled and ligated using its original strand (newly repaired) as the template
- Result is non-crossover gene conversion
42
What is break induced repair? (6)
- Repair of one ended DSBs caused by replication fork collapse
- 5'-3' end resection of break site and ligation of lagging strand with template
- Template switching occurs followed by 3' strand invasion forming a Holliday junction
- Lagging strand is reprimed and the replication fork is re-established
- Single Holliday junction is resolved horizontally or vertically
- Results in gene conversion with no crossing over
43
What is the impact of using homologous chromosomes as the template for HR in mitosis vs meiosis? (2)
- High frequency in meiosis to generate genetic diversity
- Low frequency in mitosis to prevent loss of heterozygosity which would lead to cancer/disease
44
What is the impact of using sequence from a different chromosome for the HR template in mitosis or meiosis?
Chromosomal translocation which can cause cancer, infertility and inherited disorders
