Search for: All records

Nuclear actin filaments (F-actin) form during S-phase and in response to replication stress to promote fork remodeling and repair. In mild replication stress conditions, nuclear actin polymerization is required to limit PrimPol recruitment to the fork while promoting fork reversal. Both short and long filaments form during this response, but their function in the nuclear dynamics of replication sites was unclear. Here, we show that replication centers associated with long nuclear actin filaments become more mobile than the rest of the forks, suggesting relocalization of replication sites as a response to prolonged fork stalling and/or fork breakage, even in response to mild replication stress. 

Pericentromeric heterochromatin mostly comprises repeated DNA sequences prone to ectopic recombination. In Drosophila cells, ‘safe’ homologous recombination repair requires relocalization of heterochromatic repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. DSBs are anchored to the nuclear periphery through the Nup107/160 nucleoporin complex. Previous studies suggested that the nuclear pore ‘basket’ protein Nup153 could also mediate anchoring, but Nup153 RNAi depletion also affects Nup107 association with the pores, preventing a direct assessment of Nup153 role. Using a separation of function mutant, here we show that Nup153 is not required for anchoring heterochromatic DSBs to the nuclear periphery. 

Pericentromeric heterochromatin is highly enriched for repetitive sequences prone to aberrant recombination. Previous studies showed that homologous recombination (HR) repair is uniquely regulated in this domain to enable ‘safe’ repair while preventing aberrant recombination. In Drosophila cells, DNA double-strand breaks (DSBs) relocalize to the nuclear periphery through nuclear actin-driven directed motions before recruiting the strand invasion protein Rad51 and completing HR repair. End-joining (EJ) repair also occurs with high frequency in heterochromatin of fly tissues, but how alternative EJ (alt-EJ) pathways operate in heterochromatin remains largely uncharacterized. Here, we induce DSBs in single euchromatic and heterochromatic sites using a new system that combines the DR-white reporter and I-SceI expression in spermatogonia of flies. Using this approach, we detect higher frequency of HR repair in heterochromatin, relative to euchromatin. Further, sequencing of mutagenic repair junctions reveals the preferential use of different EJ pathways across distinct euchromatic and heterochromatic sites. Interestingly, synthesis-dependent microhomology-mediated end joining (SD-MMEJ) appears differentially regulated in the two domains, with a preferential use of motifs close to the cut site in heterochromatin relative to euchromatin, resulting in smaller deletions. Together, these studies establish a new approach to study repair outcomes in fly tissues, and support the conclusion that heterochromatin uses more HR and less mutagenic EJ repair relative to euchromatin.