Recombinant Human DNA replication ATP-dependent helicase/nuclease DNA2 (DNA2), partial

What are the primary biochemical functions of Recombinant Human DNA2 in DNA replication and repair?

DNA2 exhibits three core activities: 5’-to-3’ helicase, structure-specific nuclease, and DNA-dependent ATPase. Its primary functions include:

• Okazaki fragment maturation: DNA2 processes RNA-DNA flaps during lagging-strand synthesis, working synergistically with FEN1 to remove displaced flaps. In vitro assays using fluorescently labeled flap substrates show that DNA2 cleaves long flaps (>20 nt) that are refractory to FEN1, while shorter flaps are processed by FEN1 alone .

• Replication fork restart: DNA2 resolves reversed replication forks by degrading regressed arms, enabling replication restart. This is demonstrated via electron microscopy of reconstituted fork structures and viability assays in cells treated with hydroxyurea .

• Double-strand break (DSB) resection: DNA2 collaborates with RecQ helicases (BLM/WRN) to generate 3’ ssDNA overhangs for homologous recombination. Quantitative resection assays using linear dsDNA substrates show that DNA2’s nuclease activity degrades ssDNA regions unwound by BLM/WRN .

Methodological Note: To dissect these functions, researchers employ:

• Kinetic assays with recombinant DNA2 and radiolabeled DNA substrates (e.g., 5’-flaps, Y-structures)

• siRNA/CRISPR knockout models combined with DNA fiber analysis to quantify replication fork progression

• Chromatin immunoprecipitation (ChIP) to map DNA2 localization at stalled forks or DSBs

How do the nuclease and helicase domains of DNA2 contribute to its role in Okazaki fragment maturation?

The nuclease domain (D277A mutation) is essential for flap cleavage, while the helicase domain (K654R mutation) enhances processivity on complex structures. Key findings include:

• Nuclease activity: In dna2Δ yeast, overexpression of nuclease-dead DNA2 (D277A) fails to rescue lethality, confirming the indispensability of cleavage activity .

• Helicase activity: Helicase-dead DNA2 (K654R) retains ~70% flap processing efficiency in vitro but shows delayed fork restart in vivo, suggesting the helicase aids in substrate unwinding prior to cleavage .

Experimental Validation:

• Gel-based cleavage assays: Compare wild-type, D277A, and K654R DNA2 using 5’-flap substrates with/without RPA. RPA directs DNA2’s nuclease to ssDNA-dsDNA junctions, increasing cleavage specificity .

• Single-molecule imaging: Track DNA2’s movement on forked DNA substrates to quantify unwinding vs. cleavage events.

What experimental approaches are used to validate the essentiality of DNA2 in mammalian cell viability?

DNA2 is conditionally essential due to redundancy with EXO1 in DSB resection. Key methodologies include:

• RNAi/CRISPR knockdowns: DNA2 depletion in EXO1-proficient cells causes G2/M arrest, while dual DNA2/EXO1 knockdown induces synthetic lethality .

• Complementation assays: Expressing wild-type or mutant DNA2 (e.g., D277A, K654R) in knockout cells. Helicase-dead DNA2 rescues viability in EXO1-positive but not EXO1-negative backgrounds .

Data Interpretation Caveat: Viability outcomes depend on cell type (cancer vs. primary) and replication stress levels. For example, cancer cells overexpressing DNA2 tolerate its depletion better due to redundant helicases .

How do DNA2 and EXO1 exhibit functional redundancy in DSB resection, and how can this redundancy be experimentally dissected?

DNA2 and EXO1 provide parallel resection pathways, with DNA2 dominating in replication-associated DSBs. To isolate their roles:

• Genetic ablation: In DNA2−/−/EXO1−/− cells, DSB resection is abolished, measured via RPA ChIP or ssDNA quantification .

• Substrate-specific assays:

  • DNA2 preferentially resects protein-bound DSBs (e.g., CTCF-coated sites), while EXO1 acts on "clean" breaks .

  • In vitro reconstitution using chromatinized DNA shows DNA2’s dependence on BLM/WRN for resection, unlike EXO1 .

Contradiction Alert: While DNA2 is dispensable for viability in EXO1+ cells , its helicase activity becomes critical under replication stress, complicating redundancy models .

What mechanisms underlie the contradictory findings regarding DNA2’s helicase activity in DNA end resection?

Discrepancies arise from model systems and assay conditions:

Resolution Strategy:

• Perform species-specific complementation: Express human DNA2 helicase mutants in yeast to test functional conservation.

• Use ATPase inhibitors (e.g., ATPγS) to acutely block helicase activity during resection time courses.

How does DNA2 prevent genomic instability through flap removal, and what assays quantify this role?

DNA2 nuclease prevents large insertions at DSBs by excising displaced flaps. Key evidence includes:

• CRISPR/Cas9-induced DSB models: In dna2Δ yeast, 15% of DSB repair events incorporate 0.1–1.5 kb foreign DNA fragments, visualized via pulsed-field gel electrophoresis and sequencing .

• qPCR-based detection of extrachromosomal DNA: DNA2-deficient cells accumulate ssDNA fragments homologous to rDNA and telomeres .

Methodological Insight: Combine long-read sequencing (PacBio) with DNA combing to map insertion origins and replication fork dynamics in dna2Δ mutants.

What in vitro assays demonstrate the cooperative dynamics between DNA2 and RecQ helicases during replication fork restart?

A hierarchical resection mechanism is observed:

• BLM/WRN unwinding: RecQ helicases unwind dsDNA regions ahead of DNA2.

• DNA2 engagement: DNA2 degrades the displaced strand, measured via loss of fluorescence in FRET-labeled substrates .

Protocol:

• Electrophoretic mobility shift assays (EMSAs): Show DNA2 binds ssDNA-dsDNA junctions 10-fold tighter than BLM alone.

• Real-time resection assays: Use magnetic tweezers to apply torsion, mimicking replication stress. DNA2-BLM complexes resolve supercoiled DNA 3× faster than BLM alone .

How does DNA2 balance resection to prevent over-replication toxicity in Fanconi anemia (FA) pathways?

DNA2 over-resection exacerbates replication stress in FANCD2-deficient cells. Key approaches:

• Psoralen crosslink repair assay: In FANCD2−/− cells, DNA2 knockdown reduces hyper-resection by 60%, measured via Southern blotting of repair intermediates .

• GFP reporter systems: DNA2 inhibition increases precise repair of ICLs from 22% to 68% in FA models .

Therapeutic Implication: DNA2 inhibitors (e.g., small-molecule ATPase blockers) may synergize with cisplatin in FA-deficient cancers .

What role does DNA2 play in removing ssDNA-RPA filaments during meiosis?

In meiosis, DNA2 prevents RPA accumulation on de novo ssDNA, which otherwise triggers aberrant recombination. Critical data:

• RPA ChIP-seq: dna2-md yeast show expanded RPA signals spanning 5–15 kb from DSB sites vs. 1–3 kb in wild type .

• Genetic interaction: dna2-md rad52Δ double mutants exhibit 90% spore inviability, indicating synthetic lethality .

Technical Note: Use meiosis-specific degrons to deplete DNA2 synchronously with DSB formation, ensuring temporal resolution.