Persistent DNA damage signaling and DNA polymerase theta promote broken chromosome segregation.

Cycling cells must respond to DNA double-strand breaks (DSBs) to avoid genome instability. Missegregation of chromosomes with DSBs during mitosis results in micronuclei, aberrant structures linked to disease. How cells respond to DSBs during mitosis is incompletely understood. We previously showed that Drosophilamelanogaster papillar cells lack DSB checkpoints (as observed in many cancer cells). Here, we show that papillar cells still recruit early acting repair machinery (Mre11 and RPA3) and the Fanconi anemia (FA) protein Fancd2 to DSBs. These proteins persist as foci on DSBs as cells enter mitosis. Repair foci are resolved in a stepwise manner during mitosis. DSB repair kinetics depends on both monoubiquitination of Fancd2 and the alternative end-joining protein DNA polymerase θ. Disruption of either or both of these factors causes micronuclei after DNA damage, which disrupts intestinal organogenesis. This study reveals a mechanism for how cells with inactive DSB checkpoints can respond to DNA damage that persists into mitosis.

Proliferation of Double-Strand Break-Resistant Polyploid Cells Requires Drosophila FANCD2.

Conserved DNA-damage responses (DDRs) sense genome damage and prevent mitosis of broken chromosomes. How cells lacking DDRs cope with broken chromosomes during mitosis is poorly understood. DDRs are frequently inactivated in cells with extra genomes (polyploidy), suggesting that study of polyploidy can reveal how cells with impaired DDRs/genome damage continue dividing. Here, we show that continued division and normal organ development occurs in polyploid, DDR-impaired Drosophila papillar cells. As papillar cells become polyploid, they naturally accumulate broken acentric chromosomes but do not apoptose/arrest the cell cycle. To survive mitosis with acentric chromosomes, papillar cells require Fanconi anemia proteins FANCD2 and FANCI, as well as Blm helicase, but not canonical DDR signaling. FANCD2 acts independently of previous S phases to promote alignment and segregation of acentric DNA produced by double-strand breaks, thus avoiding micronuclei and organ malformation. Because polyploidy and impaired DDRs can promote cancer, our findings provide insight into disease-relevant DNA-damage tolerance mechanisms.

Thermodynamic analysis of self-assembly in coiled-coil biomaterials.

Coiled-coil protein structural motifs have proven amenable to the design of structurally well-defined biomaterials. Mesoscale structural properties can be fairly well predicted based on rules governing the chemical interactions between the helices that define this structural motif. We explore the role of the hydrophobic core residues on the self-assembly of a coiled-coil polymer through a mutational analysis coupled with a salting-out procedure. Because the resultant polymers remain in solution, a thermodynamic approach is applied to characterize the polymer assembly using conventional equations from polymer theory to extract nucleation and elongation parameters. The stabilities and lengths of the polymers are measured using circular dichroism spectropolarimetry, sizing methods including dynamic light scattering and analytical ultracentrifugation, and atomic force microscopy to assess mesoscale morphology. Upon mutating isoleucines at two core positions to serines, we find that polymer stability is decreased while the degree of polymerization is about the same. Differences in results from circular dichroism and dynamic light scattering experiments suggest the presence of a stable intermediate state, and a scheme is proposed for how this intermediate might relate to the monomer and polymer states.