8.14:
Homologous Recombination
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.
8.14:
Homologous Recombination
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the likelihood of an identical or similar DNA sequence on a sister chromatid is high. Thus, the timing of repair prevents recombination between non-identical sequences. This is a critical feature of HR, particularly during the recombination of parental DNA sequences in an offspring, where faulty HR can lead to loss of the entire gene and the surrounding chromosomal region.
The accurate repair ensured by HR has been applied in gene-editing techniques. HR is the earliest method that has been used to edit genomes in living cells. The CRISPR-Cas9 system is used to create targeted double-strand breaks to correct disease-causing mutations in the genome. The isolated fragments are taken up by cells, where they can recombine with cellular DNA and replace the targeted region of the genome. HR mechanisms govern the repair of the breaks and their accurate recombination with the cellular genome. To help the HR proteins localize precisely at double-strand breaks, Cas9 proteins are fused with HR effector proteins that can recruit repair proteins at the damaged sites. Studies have shown that fusing Cas9 with proteins such as CtIP, Rad52, and Mre11 can increase HR events in the cell by two-folds while discouraging Non-homologous end joining. Such applications of HR in genome editing can revolutionize gene therapy and provide treatment for genetic diseases that are currently considered incurable.
Suggested Reading
- Tran, Ngoc Tung, Sanum Bashir, Xun Li, Jana Rossius, Van Trung Chu, Klaus Rajewsky, and Ralf Kühn. "Enhancement of precise gene editing by the association of Cas9 with homologous recombination factors." Frontiers in genetics 10 (2019): 365.
- Barrangou, Rodolphe, and Jennifer A. Doudna. "Applications of CRISPR technologies in research and beyond." Nature biotechnology 34, no. 9 (2016): 933.
- Li, Xuan, and Wolf-Dietrich Heyer. "Homologous recombination in DNA repair and DNA damage tolerance." Cell research 18, no. 1 (2008): 99.