ISALAN - dna recombination Flashcards
Double Stranded Breaks
Can occur during replication when DNA lesions/nicks prevent the processivity of DNA polymerase during DNA synthesis (ex. benzo[a]pyrene from burnt food, nuclease damage, ionizing radiation from x-ray damage causing hydrolysis of the phosphodiester backbones)
Double Stranded Break repair systems
- Non-homologous end joining
- Homologous recombination
Non-homologous End Joining
- Occurs due to microhomologies at the broken ends
- Ku proteins (Ku70 & Ku80) recognize the ds breaks on 2 DNA fragments
- It will then recruit DNA-Pkcs (DNA dependent protein kinase) and Artemis
- DNA-Pkcs is a catalytic subunit that phosphorylates the Ku proteins, resulting in a higher affinity to DNA so a tight complex is formed between 2 ends
- DNA-Pkcs also phosphorylates Artemis, causing it to remove ss extensions or hairpins
- Helicase unwinds the DNA ends, allowing for overlapping and hybridization of microhomologies; any overhangs are trimmed by Artemis – this is an error-prone process since insertion/ deletion can occur
- DNA is sealed by Ligase
Error-prone process of NHEJ can:
- Cause cancer: due to translocation between different chromosomes
Ex. BCR-ABL mutation. Translocation of BCR & ABL produces the BCR-ABL protein, which
is a type tyrosine kinase, causing certain blood cells to grow and divide out of control. - be exploited by modern genome editing techniques (ex. CRISPR/Cas9) CRISPR/Cas9 utilizes known microhomologies in the sequence to introduce targeted insertion/ deletions, causing target gene knockouts
- be exploited by the immune system for Vdj recombination (common in mammalian genomes) ds breaks are introduced to variable regions of antibodies, stimulating re-ligation by NHEJ to generate diversity between antibody variable regions
Bacterias were though to not have NHEJ since their ds breaks are mostly repaired by other
processes ex. Homologous recombination. However, recent studies have shown weak NHEJ activities in some bacteria which is a homologous process to the ones found in mammals.
Homologous Recombination
Utilizes a homologous DNA as a template to repair the region containing the lesion
* Relatively error-free process
* Homologous templates can be found easily at replication forks (even for haploid organisms – diploid organisms already have double the amount of chromosomes to use as template)
* Exploited by modern genome editing technology (ex. CRISPR/Cas9)
o Because homologous recombination can occur as long as there are homologous
sequences flanking the region of recombination (does not need to be 100%
identical)
o Therefore, homology-targeted repair can be used to introduce mutations to the
homologous DNA to insert genes/ modifications into a particular genomic locus
* Can occur in both ds breaks (from nucleases, X-ray damage) or ss nicks caused by bypassed lesions during replication. It just provides a general mechanism for repair when intramolecular template information has been lost due to DNA damage)
Homologous Recombination - Mechanism in E. coli:
Chi sequence: 5’-GTCGGTGG-3’
* present near the site which homologous recombination is more likely to occur than on average across the genome
- RecBCD recognizes and binds the ds break. The complex unwinds the DNA and degrades DNA in a reaction coupled with ATP hydrolysis. Helicase motors of RecC works in 3’-5’ direction while RecD works in 5’-3’ direction. RecC binds tightly to the Chi sequence, resulting in unequal degradation of 5’ and 3’ ends. Degradation of 5’ end increases while degradation of 3’ end decreases, forming a 3’ overhang.
- The 3’ overhang is coated by RecA. The ss RecA coated filament performs strand invasion on a homologous DNA, forming a D loop (bulge in DNA strand caused by unpaired bases)
- RecA utilizes its ATP-dependent activity to promote homology search along the homologous DNA. Once homology is found, RecA dissociates
- The homologous duplex is also nicked, and complementarity between bases promote strand exchange
- The ss 3’ overhangs then serve as primers for polymerase to bind and re-synthesize the DNA using the homologous duplex as a template.
- Ligase seals gaps on both ss. This completes the homologous recombination and forms a holliday junction, a branched structure that contains a 4- way intersection of DNA strands.
- RuvA stabilizes the formation of Holliday junction and contains a hydrophobic pin in the middle to help separate the strands during branch migration.
- RuvB motors bind to either side of RuvA and uses ATP to translocate the DNA and migrate the Holliday junction in a random direction.
- Resolution of Holliday junction via cleavage by RuvC nuclease
* Vertical cuts result in a splice resolution where there is rearrangement of genetic materials around the site of recombination while:
* Horizontal cuts result in a patched resolution where there is only a patch of hybrid DNA and there is no rearrangement of genetic material downstream. - The cuts/ nicks are then resealed by DNA ligase
RecA homology search and strand invasion
RecA is a bacterial recombinase
* Precise details are not completely understood
1. RecBCD acts as RecA loader, facilitating RecA nucleation on the ssDNA (nucleation occurs in 5’-3’ direction). This forms an
ordered helical filament <7000 subunits
2. Strand invasion is facilited by helical structure of RecA which allows formation of intermediate triplex structure with the homologous duplex.
3. D-loop forms as the triplex unwinds and one of the original DNA strands is displaced by the invading strand.
4. ATP hydrolysis is required for triplex unwinding to perform homology search across long DNA lengths.
5. Homology search stops when there is sufficient WCBP
Other types of homologous recombination:
- Double crossover (common during meiosis in eukaryotes ex. yeast
* Occurs due to 2 strand invasion events, forming 2 holliday junctions and branch migration of both crossover events
* Result in resolutions with a patch work of recombinants where information are shuffled in many places
Common in meiosis because it is efficient in shuffling information to promote variation in homologous chromosomes - Intramolecular crossovers, involve homologous/ repeated sequences in 1 DNA stretch.
Tandem repeats in 1 DNA can align to form a loop in which when recombined, the DNA sequences between the repeats are deleted.
Inverted repeats in 1 DNA will flip the loop region when they recombine, resulting in a flipped DNA orientation between the repeats (can result in rearrangement of promotor/ enhancer regions/ genes which will change overall gene expression pattern of the cell)
Homologous recombination will result in…
a random outcome, depending on the different mechanisms that acted on the recombinants after the recombination event.
* Ex.
1. Repaired by mismatch repair mechanisms: DNA sequence is restored to the parental sequence
2. Not repaired: Changes will persist following the recombination and will be inherited by one of the daughter progenies
Homologous recombination is involved in several important processes
DNA repair
* Cause gene shuffling (scrambling of maternal and paternal chromosomes) during meiosis which contributes to variation in offspring from sexual reproduction (result in non-parental combinations)
* Form physical links between homologous chromosomes to allow chromosome alignment during meiotic prophase
* Cause horizontal gene transfer from one organism to another, contributing to variation in bacterial evolution
* Exploited in biotechnology ex. homology directed repair utilized by CRISPR/Cas9 genome editing
Other types of recombination:
Site-specific recombination
* Requires shorter, more specific homologous DNA sequence than homologous recombination.
* Specific enzymes (recombinases) recognize those specific homologous sequences and solely drive the recombination
> The specific orientation of those sequences can result in different outcomes
- Inverted repeats will result inversion of sequences within 1 DNA or:
- Same orientation of repeats
* On 2 DNA strands can result in the insertion of one DNA strand into another ex. Invasion of viral genome into bacteria
* On 1 DNA strand can result in the deletion of a region of DNA sequence ex. excision of viral genome from bacterial genome
> the specificity of the orientation can be exploited by biotechnology to design repeat sites on genomes to generate a specific process/outcome ex. excision,
integration, etc.
The first recombinase to be characterized is the Lambda phage integrase (Int)
* Recognizes specific att sites which are homologous in both lambda phage virus circular genome and the E.coli genome. Integrase then recombine these specific sequences. The way in which the homologous sequence is oriented results in addition of the viral genome into the bacterial genome.
o Different from RecBCD pathway/ homologous recombination in such that:
- The mechanism of recombination (cutting, alignment, relegation of DNA) is done solely by the integrase instead of a whole series of proteins coming together. Some help provided by host cell’s extra machinery that are hijacked by the virus ex. IHF (integration host factor) and nucleases
- Does not use ATP – the enzyme has evolved a way of storing high E intermediates by having Tyr residues in their active site
Integrase mechanism
Works as a tetramer – 2 subunits bind to a specific sequence (recombination site) of both DNA strands
- 1 subunit on each DNA strand cleaves the DNA by utilizing the active site tyrosine. The tyrosine OH group forms phosphodiester bond with the DNA 3’ phosphate, cleaving the DNA and resulting in a high E intermediate.
- After isomerization, the high E intermediate allows the cleaved strands to join to new partners, forming Holliday junctions
- The other 2 subunits repeat the same steps to complete the recombination
Overall = 2 double crossover events
