Questions: Non-Homologous End Joining (NHEJ) and DSB Repair

Question 1

A cell sustains a double-strand break during G1 phase of the cell cycle. Which repair pathway predominantly handles this break, and why?

• A. Homologous recombination, because it is more accurate and preferred whenever possible
• B. NHEJ, because no sister chromatid template is available in G1, and NHEJ operates throughout the cell cycle
• C. Base excision repair, because it is the primary pathway for all single- and double-strand damage
• D. Mismatch repair, because G1 is the main window for post-replication proofreading

Answer: (B)

Homologous recombination requires a sister chromatid as a repair template and therefore operates primarily in S and G2 phases, after DNA replication when the sister is available. In G1, no sister chromatid exists, so NHEJ — which requires no template — is the dominant pathway. NHEJ is active throughout the entire cell cycle, making it the default repair mechanism for DSBs in most mammalian cells. Options C and D describe completely different repair pathways that do not address double-strand breaks.

Question 2

When CRISPR-Cas9 is used to knock out a gene, the most common outcome is a frameshift mutation that disrupts the reading frame. What directly produces this frameshift?

• A. Homologous recombination mistakenly repairs the Cas9-induced cut using a mismatched template
• B. The Cas9 nuclease itself removes several base pairs from the coding sequence as it cuts
• C. NHEJ repair of the double-strand break introduces small insertions or deletions (indels) at the cut site
• D. The guide RNA integrates into the coding sequence at the cleavage site

Answer: (C)

When Cas9 introduces a DSB, the cell's NHEJ pathway repairs it rapidly. During repair, the end-processing step (trimming damaged bases or filling gaps) introduces small indels — typically 1–20 bp. If these indels occur in a coding exon, they shift the reading frame for all downstream codons, usually introducing premature stop codons and destroying protein function. This is the standard CRISPR knockout strategy. Option B is wrong: Cas9 makes a blunt cut at a specific site without removing bases; the indels arise from NHEJ processing.

Question 3

NHEJ is essential for V(D)J recombination, the process by which the immune system generates the diversity of antibody and T-cell receptor sequences.

• T. True
• F. False

Answer: True

V(D)J recombination deliberately introduces DSBs at recombination signal sequences to rearrange gene segments. After RAG recombinase cuts the DNA, NHEJ is required to rejoin the coding ends. Crucially, the imprecision of NHEJ — the insertion or deletion of a few nucleotides at the junctions — is not a bug but a feature: it generates additional sequence diversity at the junctions (junctional diversity), enormously expanding the repertoire of possible receptor sequences. Patients with NHEJ defects show severe combined immunodeficiency precisely because V(D)J recombination cannot complete.

Question 4

NHEJ is a backup or last-resort DSB repair pathway, used mainly when homologous recombination is unavailable.

• T. True
• F. False

Answer: False

NHEJ is the dominant DSB repair pathway in mammalian cells, not a backup. It operates throughout the cell cycle, including G1 when HR cannot function. It is also faster than HR — completing within minutes. The characterization as 'backup' likely arises from its error-prone nature compared to HR, but error-prone does not mean secondary. In many contexts — telomere maintenance, V(D)J recombination, CRISPR knockouts — NHEJ is the primary and essential pathway, not an alternative.

Question 5

Why is NHEJ described as 'error-prone,' and under what circumstances does this imprecision become advantageous or even essential?

Model answer: NHEJ is error-prone because the end-processing step, which trims or fills broken ends to make them ligatable, often removes or adds a few base pairs. These indels alter the local DNA sequence at the repair junction. This imprecision is a disadvantage when NHEJ repairs a coding sequence — the indel may frameshift the reading frame and destroy gene function. However, the same imprecision is advantageous or essential in two contexts: (1) V(D)J recombination, where NHEJ-introduced junctional diversity vastly expands immune receptor repertoire diversity, and (2) CRISPR gene knockouts, where researchers deliberately exploit NHEJ indels to disrupt genes they want to silence.

The broader principle is that 'error-prone' must be evaluated relative to context. NHEJ's imprecision is a serious liability at critical coding sequences but a productive feature wherever sequence diversity is the goal. Speed and cell-cycle independence are NHEJ's other key properties: it can act immediately after a break in any phase of the cell cycle, which is often more important for cell survival than whether the repair is perfectly accurate.