Temporal proximity proteomics reveals ANP32A roles in replication fork progression and end joining

Abstract

Acidic nuclear phosphoprotein 32 family member A (ANP32A) regulates chromatin and histone homeostasis, but its contribution to DNA replication and repair remains unclear. Time-resolved AirID proximity labeling with data-independent acquisition mass spectrometry revealed phased remodeling of the ANP32A proximal proteome during hydroxyurea-induced replication stress. Early stress enriched DNA replication and repair factors, including FEN1, XRCC5/Ku80, and XRCC6/Ku70, whereas prolonged exposure shifted the network towards checkpoint and cell-cycle regulators. Orthogonal proximity ligation assays revealed sustained ANP32A-FEN1 proximity and transiently increased ANP32A-XRCC5 proximity. ANP32A was dynamically recruited to γH2AX-marked lesions following laser microirradiation and showed increased proximity to γH2AX after hydroxyurea treatment. ANP32A loss impaired proliferation and G1/S progression and moderately reduced replication fork velocity. However, it did not promote nascent strand degradation at stalled forks or measurably alter homologous recombination reporter activity. In contrast, ANP32A loss reduced non-homologous end-joining reporter activity, consistent with its stress-induced proximity to Ku proteins. These findings identify ANP32A as a chromatin-associated factor supporting replication fork progression and end joining while dispensable for stalled-fork protection, linking ANP32A-dependent chromatin regulation to genome maintenance. HIGHLIGHTS Replication stress dynamically remodels the ANP32A proximal proteome ANP32A loss impairs G1/S progression and slows replication forks ANP32A is recruited to γH2AX-marked DNA lesions during replication stress ANP32A promotes end joining but is dispensable for stalled fork protection IN BRIEF Jurkovic et al. show that replication stress dynamically remodels the ANP32A proximal proteome. ANP32A supports G1/S progression, replication fork progression, and end joining but is dispensable for stalled fork protection, linking ANP32A-dependent chromatin regulation to genome maintenance.