Elucidating HLTF-Mediated DNA Fork Remodeling via Native Mass Spectrometry

Replication fork reversal (RFR) is a crucial DNA damage tolerance mechanism that protects genome stability by remodeling DNA fork structures. The helicase-like transcription factor (HLTF) is one of the key components in the RFR process and is responsible for the conversion of a stalled replication fork into a four-way reversed fork, mediated by its ATPase activity. In contrast to a wealth of biochemical evidence depicting the biological activities of HLTF, very little information is available on how it molecularly and functionally interplays with DNA molecules. In this study, we employed native mass spectrometry (MS) to probe the stoichiometry of HLTF-DNA complexes and to elucidate their functional association with DNA fork remodeling. We revealed that HLTF exists as an inactive monomer with low accessibility to substrate ATP yet retains DNA fork binding activity. Intriguingly, in the presence of a DNA fork, monomeric HLTF forms a hetero protein-DNA complex that enhances its ATP accessibility, suggesting allosteric structural modulation through DNA fork interaction. Using both homologous and heterologous DNA forks, we further uncovered ATP-induced dimerization of HLTF and demonstrated its critical role in triggering the DNA unwinding activity of HLTF and subsequent DNA fork regression. Together, our findings provide unique insight into the molecular processes of a DNA remodeler and underscore the utility of native MS in probing the macro-assembly of protein-DNA complexes.