Nuclear actin has been implicated in DNA repair, but exactly how actin dynamics and actin-binding proteins drive processing and repair of DNA double-strand breaks (DSBs) remains unclear.

As part of our ongoing effort to decipher emerging roles of the DYRK1A kinase in mammalian DNA damage responses, we profiled the DYRK1A phospho-proteome, and have uncovered cytoskeleton organisation factors as its putative targets, including actin nucleator Spir1. In support of the DYRK1A kinase in orchestrating nuclear actin dynamics, we found that DYRK1A enforces filamentous actin (F-actin) enrichment at laser-induced DNA damage tracks, in part via promoting Spir1 phosphorylation and turnover from the damaged chromatin. Indeed, phospho-mimetic mutation on the Spir1 polypeptide attenuated its docking at DSBs and dampened local F-actin assembly. Together with our observation wherein DYRK1A supports DSB mobility, these preliminary findings highlight DYRK1A as a candidate master regulator of nuclear actin dynamics on DSB-flanking chromatin, and that this may be effected, at least in part, via targeting Spir1.

To define how nuclear actin dynamics supports DSB processing and repair, we will delineate the molecular regulation of the DYRK1A-Spir1 axis in actin nucleation on the damaged chromatin, and will elucidate how DYRK1A-dependent Spir1 phosphorylation orchestrates actin assembly and disassembly in vitro and in vivo. Finally, we will study how the DYRK1A phospho-proteome drives DSB clustering and repair, and will investigate how perturbing the dynamics of the nuclear cytoskeletal network alters DSB repair pathway choice, genome stability, and cell sensitivity to genotoxic stress.

Mechanistic understanding of nuclear actin in DNA repair has been limited in the past largely due to the lack of research tools that allow separation of its housekeeping function in the cytoplasm. With the advent in imaging and synthetic biology tools, we will combine cell biology, biochemical and biophysical methods to systematically and comprehensively define how F-actin dynamics underlies DSB repair and genome integrity protection. Our proposed experimentations thus represent a timely initiative to define how the cytoskeletal network actively participates in mammalian cellular responses to DNA damage. Understanding how DSBs are processed and repaired will also guide development and optimisation of gene-editing tools for genome engineering and gene therapy.