Question 1

Why is homologous recombination the preferred mechanism for repairing double-strand breaks?

Accepted Answer

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.

Question 2

What role do RPA and Rad51 proteins play in homologous recombination?

Accepted Answer

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.

Question 3

How does strand invasion identify the correct homologous DNA sequence?

Accepted Answer

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.

Question 4

What happens after the displacement loop structure forms during homologous recombination?

Accepted Answer

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.

Question 5

Why is homologous recombination restricted to specific phases of the cell cycle?

Accepted Answer

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.

Question 6

How is homologous recombination applied in CRISPR-Cas9 genome editing?

Accepted Answer

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.

Question 7

What is the MRN complex and what does it do at the start of homologous recombination?

Accepted Answer

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.

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