RAD51C-XRCC3 structure and cancer patient mutations define DNA replication roles.

RAD51C is an enigmatic predisposition gene for breast, ovarian, and prostate cancer. Currently, missing structural and related functional understanding limits patient mutation interpretation to homology-directed repair (HDR) function analysis. Here we report the RAD51C-XRCC3 (CX3) X-ray co-crystal structure with bound ATP analog and define separable RAD51C replication stability roles informed by its three-dimensional structure, assembly, and unappreciated polymerization motif. Mapping of cancer patient mutations as a functional guide confirms ATP-binding matching RAD51 recombinase, yet highlights distinct CX3 interfaces. Analyses of CRISPR/Cas9-edited human cells with RAD51C mutations combined with single-molecule, single-cell and biophysics measurements uncover discrete CX3 regions for DNA replication fork protection, restart and reversal, accomplished by separable functions in DNA binding and implied 5' RAD51 filament capping. Collective findings establish CX3 as a cancer-relevant replication stress response complex, show how HDR-proficient variants could contribute to tumor development, and identify regions to aid functional testing and classification of cancer mutations.

Adsorption of cell-penetrating peptide Tat2 and polycation luviquat FC-370 to triticale microspore exine.

Microspores are the pre-gametophyte stage of pollen, and have proven to be a successful tissue culture material for the production of doubled haploid plants. Microspore culture has also been used as a platform for the production of transgenic plants. The use of cell-penetrating peptides (CPPs) as transfection agents in microspores has been previously demonstrated, but at low efficiencies. Here, the pH dependent adsorption of the cationic CPP Tat2 to the sporopollenin surface of the microspore (the exine) has been explored using electrophoretic light scattering (ELS). Furthermore the adsorption of a commercially available polycationic polymer; Luviquat FC-370 (polyquaternium D-16, PQ-D16) was similarly measured using ELS. It was found that a suspension media with a pH less than 7.0 showed an approximately ten fold decrease in the amount of Tat2 that was required before apparent surface neutralization. This data suggests that a change in the surface chemistry of the microspore occurs in acidic pH conditions, that modulates the binding affinity of the CPP Tat2 in a non-trivially complex manner.

Tetrabutylphosphonium Bromide Reduces Size and Polydispersity Index of Tat2:siRNA Nano-Complexes for Triticale RNAi.

Cell-penetrating peptides (CPPs) are short 8-30 amino-acid oligopeptides that act as effective transducers of macromolecular cargo, particularly nucleic acids. They have been implemented in delivering dsDNA, ssDNA, and dsRNA into animal and plant cells. CPPs and nucleic acids form nano-complexes that are often 100-300 nm in size but still effectively transit the cell membrane of animal cells, but are less effective with plant cells due to the plant cell wall. To overcome this obstacle, nano-complexes of the CPP Tat2 and various lengths of nucleic acid (21-mer siRNA duplex (dsRNA) to ~5.5 kb circular plasmid) were evaluated for size using dynamic light scattering (DLS), under conditions of increasing ionic strength (Ic) and addition of phase transfer catalyst salts (tetrabutylammonium bromide-TBAB and tetrabutylphosphonium bromide-TBPB) and sugars (maltose-mannitol solution). It was found that the combination of 21-mer siRNA:Tat2 complexes with TBPB produced small 10-20 nm diameter nano-complexes with a polydispersity index (PDI) of ~0.1. Furthermore, it was found that for each length of nucleic acid that a linear mathematical relationship existed between the theoretical volume of the nano-complex and the nucleic acid length. Next, nano-complex formulation was tested for its ability to carry small interfering RNA molecules into plant cells and to trigger silencing of phytoene desaturase (PDS) in Triticale leaves. RT-qPCR showed 75% suppression of PDS, demonstrating that TBPB acts as an adjuvant in effecting the entry and efficacy of siRNA in young Triticale plants.