Lecture 9 - Homology directed DSB repair and homologous recombination. Flashcards
What is the difference between non-homologous DSB repair and homology directed base repair.
DSB (double strand breaks) need to be repaired, but there is no undamaged strand to act as a template unlike in NER, BER, MMR.
Non-homologous end joining rejoins the ends. Predominant in non-dividing cells.
Homology-directed DSB repair uses homologous DNA as a template
* Sister chromatid (identical) late S-phase, G2
* Homologous chromosome G1 - but template may not be exactly the same as the original (loss of heterozygosity)
The homology-directed repair pathway is adapted for recombination- crossing over of chromosomes in meiosis, bacterial conjugation, transduction and transformation, and occasionally in mitosis.
Give an overview of homolgy directed repair of DSBs.
Homology-directed repair of DSBs
1. Generation of ssDNA at the site of DSB (Parasynapsis)
2. Pairing by one of the single-stranded ends invading an intact homologous duplex to form a heteroduplex (Synapsis)
3. Repair of the damaged duplex by DNA synthesis using strands from intact duplex as template (Post synapsis)
4. Separation of two duplexes (post synapsis)
Accurate repair of double-stranded DNA breaks involves pairing of DNA duplexes.
* Single stranded DNA is generated at break sites by helicases and exonucleases.
* One of the 3’ ends invades the intact duplex and base pairs to make a heteroduplex and form a D-loop.
* Pairing in the D-loop is specific ~90-100% accuracy over a 20-30 bp stretch
* After formation of D-loop repair can proceed in several different ways.
Describe homology directed repari via synthesis-dependent strand annealing (SDSA).
- DNA synthesis initiated from 3’OH of invading strand in D-loop - using undamaged strand as template
- DNA synthesis continues until the new DNA can pair with the other 3’ overhang
- As synthesis proceeds, newly synthesised strand is released from template strand and D-loop moves along template strand.
- Following recapture of the end of the newly synthesised strand the damaged DNA now has a region of ssDNA rather than a double strand gap.
- Gap is filled using newly synthesised strands are the result of new DNA synthesis (conservative)
- No joining of DNA from undamaged duplex to repaired duplex
What are the key protiens mediating homolgy directed repair in E.coli.
Generation of ssDNA tails at DSBs by the RecBCD complex - presynaptic step.
* Formation of long single stranded tails is known as the presynaptic step, and involves helicases and nucleases
* The RecBCD complex in E. coli has both helicase and nuclease functions.
* RecBCD attaches at double strand breaks and moves along DNA, unwinding and degrading it.
* RecBCD activity is modulated when it encounters a Chi sequence (GCTGGTTG)
* At Chi sequence nuclease activity decreases and preferentially degrades the 5’ strand - leaving a 3’ single-strand tail.
* RecBCD mutants very sensitive to DNA damaging agent.
Describe the mechanisms of DNA straind pairing and exchange.
Mechanism of DNA strand pairing and Exchange
* Key step in strand-exchange process is the identification of homologous DNA between and invading strand and DNA duplex.
* DNA strand pairing is promoted by specialised recombinases - Strand exchanges recombinases bacterial RecA and eukaryotic Rad51
Mutants in strand exchange recombinases are very sensitive to DNA damage.
Homologous pairing catalyses by the recombinase nucleoprotein filament
* RecA is loaded on to the single-stranded DNA, forming the presynaptic filament
* The helical nucleoprotein filament distorts the DNA, extending it by up to 50%
* RecA nucleoprotein filament promotes pairing with homologous DNA
Recombinase proteins catalyse strand exchange after homologous pairing is established.
* RecA/Rad51 is sufficient for strand exchange in vitro.
* In vivo, other proteins are involved e.g. SSB/RPA to prevent secondary DNA structures impending the reaction.
* BRCA1 and BRCA2 are accessory proteins. Defects in these predispose individuals to breast cancer.
Why does homology directed repair result in gene conversion.
- When sister chromatids are not available (as in G1), homologous chromosomes can be the template
- Homologous chromosomes are usually slightly different in sequence
- When a break is repaired from a homologous chromosome, the repairing now has the sequence of the homolog, not the original sequence - this is gene conversion.
- Gene conversion has occurred - there are two b alleles instead of one b and one B
- Loss of heterozygosity - no longer have B allele, homozygous for b.
What is homologous recombination?
- Homologous recombination uses the same cellular machinery that carries out homology-directed repair to generate new combinations of genetic information.
- Homologous recombination can occur between any DNA molecules with extensive regions of identical sequence or very similar sequence.
○ Homology-directed repair results in gene conversions
○ Homologues recombination can result in reciprocal exchange of large segments of DNA between homologues duplexes.
- Homologous recombination can occur between any DNA molecules with extensive regions of identical sequence or very similar sequence.
When and how does homologous recombination occour in eukaryotes and bacteria?
Eukaryotes - recombination during prophase of meiosis I
Homologous recombination during meiosis I plays key roles in:
1. Providing a pairing mechanisms for homologous chromosomes, and physical connection between recombining chromosomes ensuring that homologous chromosomes are separated in meiosis I.
2. Generating diversity by reciprocal exchange of random segmented of maternal and paternal (non-sister) chromatids - produces unique gametes.
Meiotic recombination is initiated at DSBs. A specific enzyme, Spo11, introduces double-strand breaks to initiate meiotic recombination as chromosomes start to pair.
Bacteria - Homologous recombination following gene transfer - integration of donor DNA
Conjugation and transduction.
Donor DNA integrated must be integrated into recipient chromosome by homologous recombination to be stably inherited.
How is homologous recombination initiated?
Homologous recombination is initiated by a DSB
* Initial steps of homologous recombination are same as those used for homology directed repair of DSB.
* DNA molecules paired, 3’ tail invade the homologous duplex.
* 3’ end of invading strand primes new DNA synthesis.
* Displaced D-loop strand ‘captured’ by second 3’ end of the DSB
* New DNA synthesis from 3’ end- displaced D-loop strand template
* Two intact dsDNA regions joined by two holliday junctions are formed.
What are holliday junctions?
Homologous recombination occurs by the formation of Holliday junctions between homologous DNA molecules.
* Holliday junctions are four-armed cross-stranded structure in which the participating DNA molecules are physically linked by a region of heteroduplex DNA and exchanged strands.
* Holliday junctions can be moved along the recombining DNA by branch migration, extending the heteroduplex region linking the participating molecules.
How does branch migration change the position of holliday junctions. and how does cleavage affect the products?
Branch migration changes the position of Holliday junctions
* In bacteria branch migration is driven by the RuvAB complex.
* All 4 arms of the Holliday junction are bound by a tetramer of RuvA which opens the junction to the square planar configuration.
* RuvB is a hexametric helicases, one hexamer binds to each of two opposite arms of the junction
* RuvB moves the Holliday junction along the DNA, breaking and re-forming base pairs in one direction
* Depending on the junction movement, more (top) or less (bottom) heteroduplex DNA is present
Cleavage of a Holliday junction can result in either of two products.
* RuvAB binding to Holliday junctions results in unfolding junctions into a planar, unfolded cross-like structure
* Holliday junctions are resolved by introducing symmetrically placed nicks, in either plane across the junction
* RuvAB recruits RuvC Holliday junction resolvase to cleave junction, separating participating DNA molecules
* Direction of cleavage directs whether there is no exchange of DNA or exchange and production of a recombinant
What is the difference between homology directed repair and homologous recombination.
Homology-directed repair VS homologous recombination
* In homology-directed repair - the second end of the double strand break captures the newly synthesised DNA primed from the first end and the tow duplexes do not become linked.
* In homologous recombination the second end of the double strand break captures the displaced strand of the D-loop - the two duplexes are linked.
Resolution of a recombination intermediate containing two Holliday junctions can yield multiple products
* If the two junctions are resolve in the same direction, there is no recombination
* If the two junctions are resolved in different directions, recombinant molecules are generated.
