Human DHX9 helicase unwinds triple-helical DNA structures

Area of Science:

• Genomics
• Molecular Biology
• Biochemistry

Background:

• Poly(purine.pyrimidine) sequences in the human genome can form non-canonical DNA structures like intramolecular triplexes (H-DNA).
• These H-DNA structures are implicated in regulating disease-linked genes and can cause DNA double-strand breaks and genomic rearrangements.
• Triplex-forming oligonucleotides (TFOs) are being explored for gene therapy applications.

Purpose of the Study:

• To investigate the role of the DHX9 helicase in maintaining genome stability.
• To determine if DHX9 interacts with and resolves triplex DNA structures in vitro.
• To characterize the substrate specificity and mechanism of DHX9 helicase activity on triplex DNA.

Main Methods:

• Co-immunoprecipitation assays to detect DHX9 interaction with triplex DNA in mammalian cells.
• In vitro helicase assays using purified human DHX9 and various duplex and triplex DNA substrates.
• Kinetic analysis of DHX9 helicase activity, including pre-steady-state and steady-state phases.

Main Results:

• DHX9 helicase co-immunoprecipitated with triplex DNA structures in cells, indicating an in vivo interaction.
• Purified DHX9 demonstrated ATP-dependent helicase activity, unwinding triplex DNA with a 3' --> 5' polarity.
• DHX9 preferentially unwound triplex DNA substrates containing a 3'-single-stranded overhang, displacing the third strand.
• Minimal helicase activity was observed on blunt triplexes, triplexes with 5'-overhangs, and various duplex DNA substrates.

Conclusions:

• DHX9 plays a role in genome stability by resolving potentially mutagenic triplex DNA structures.
• The enzyme specifically targets and unwinds triplex DNA with a 3'-overhang, removing the Hoogsteen-bonded third strand.
• These findings highlight DHX9 as a key factor in preventing genomic instability associated with non-canonical DNA structures.