Elucidating HLTF-Mediated DNA Fork Remodeling via Native Mass Spectrometry

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Replication fork reversal (RFR) is vital for genome stability.
• Helicase-like transcription factor (HLTF) drives RFR by remodeling DNA forks.
• HLTF's molecular interactions with DNA are poorly understood.

Purpose of the Study:

• To investigate the molecular mechanisms of HLTF-DNA complex formation.
• To elucidate the functional role of HLTF in DNA fork remodeling.
• To explore the utility of native mass spectrometry (MS) in studying protein-DNA interactions.

Main Methods:

• Native mass spectrometry (MS) was used to analyze HLTF-DNA complexes.
• Homologous and heterologous DNA forks were employed to study HLTF interactions.
• Biochemical assays were performed to assess HLTF activity.

Main Results:

• HLTF functions as an inactive monomer with DNA binding but low ATP accessibility.
• DNA fork interaction allosterically enhances HLTF's ATP accessibility.
• ATP binding induces HLTF dimerization, triggering DNA unwinding and fork regression.

Conclusions:

• HLTF undergoes allosteric modulation and dimerization upon DNA fork interaction.
• ATP-dependent dimerization is critical for HLTF's DNA remodeling activity.
• Native MS is a powerful tool for studying large protein-DNA complexes.