7.8
Homologous Recombination
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One…
An essential requirement of DNA replication is to maintain the integrity of the genetic material. For this reason double-strand breaks are preferentially repaired by homologous recombination and it is typically carried out after DNA synthesis when the two daughter DNA molecules are in proximity, and one can serve as a template for repair of the other.
In eukaryotes, a protein complex called MRN, composed of specialized nucleases degrade the damaged ends of the DNA while ends tethered to each other. The DNA is left with single-strand overhangs of 3-4 nucleotides with a 3’ OH end. This single-stranded section is stabilized by RPA proteins until Rad51 - homolog of prokaryotic protein RecA - is activated by ATP, and binds to the DNA forming a filament.
In the filament, the DNA exists as triplets of nucleotides where the DNA backbone is unwound between adjacent triplets. This DNA-protein filament binds to a duplex DNA from a sister chromatid by stretching the intact template DNA and destabilizing it, so that the two strands can be easily pulled apart and the broken strand can attempt to bind to the template, in a process known as strand invasion.
The invading strand searches for undamaged, homologous sequences in the genomic DNA by trying to form base pairs in a block of three nucleotides. If the basepairs mismatch, the invading DNA dissociates and looks for other homologous regions. If one triplet from the invading strand matches with the template, then the next three nucleotides are sampled.
If a sequence matches for a stretch of at least five triplets, a displacement loop structure is formed, with DNA polymerase using the invaded strand as a template.
Following this, a helicase displaces the now extended invading strand which basepairs with the uncoated damaged strand. Next, the second damaged strand anneals to the complementary strand of the template DNA for another round of DNA synthesis. Finally, the sister strands dissociate and a DNA ligase seals the nicks, restoring the repaired helices, and ensuring accurate repair of the intact chromosome.
Q1: Why is homologous recombination the preferred mechanism for repairing double-strand breaks?
Double-strand breaks are preferentially repaired by homologous recombination because it maintains genetic integrity by using an identical or nearly identical DNA sequence as a template for accurate repair. This mechanism is typically carried out after DNA synthesis when sister chromatids are in proximity, allowing one to serve as a reliable template for restoring the damaged strand.
Q2: What role do RPA and Rad51 proteins play in homologous recombination?
RPA proteins stabilize single-stranded DNA overhangs created after nuclease degradation of damaged ends. Rad51, the eukaryotic homolog of prokaryotic RecA, is then activated by ATP and binds to the DNA, forming a filament structure where the DNA backbone unwinds between adjacent triplets, enabling the search for homologous sequences.
Q3: How does strand invasion identify the correct homologous DNA sequence?
During strand invasion, the invading DNA strand searches for homologous sequences by attempting to form base pairs in blocks of three nucleotides. If base pairs mismatch, the strand dissociates and searches elsewhere. Once a triplet matches, the next three nucleotides are sampled, and if matching continues for at least five triplets, a displacement loop forms.
Q4: What happens after the displacement loop structure forms during homologous recombination?
After displacement loop formation, DNA polymerase uses the invaded strand as a template to synthesize new DNA. A helicase then displaces the extended invading strand, which base pairs with the uncoated damaged strand. The second damaged strand anneals to the complementary template strand for another round of DNA synthesis before DNA ligase seals the nicks.
Q5: Why is homologous recombination restricted to specific phases of the cell cycle?
Homologous recombination is restricted to the S and G2 phases of the cell cycle because DNA has already been replicated at these stages, making identical or similar sequences available on sister chromatids. This timing prevents recombination between non-identical sequences, ensuring accurate repair and avoiding loss of genes or chromosomal regions.
Q6: How is homologous recombination applied in CRISPR-Cas9 genome editing?
CRISPR-Cas9 creates targeted double-strand breaks to correct disease-causing mutations. Isolated DNA fragments are taken up by cells where homologous recombination mechanisms govern their accurate recombination with cellular DNA, replacing the targeted region. Fusing Cas9 with HR effector proteins like CtIP and Rad52 increases HR events and improves editing efficiency.
Q7: What is the MRN complex and what does it do at the start of homologous recombination?
The MRN complex is a protein complex composed of specialized nucleases that initiates homologous recombination by degrading the damaged ends of DNA while keeping them tethered together. This processing creates single-strand overhangs with 3' OH ends that are then stabilized by RPA proteins, preparing the DNA for Rad51 binding and filament formation.