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Abstract Alternative end joining (alt-EJ) mechanisms, such as polymerase theta-mediated end joining, are increasingly recognized as important contributors to inaccurate double-strand break repair. We previously proposed an alt-EJ model whereby short DNA repeats near a double-strand break anneal to form secondary structures that prime limited DNA synthesis. The nascent DNA then pairs with microhomologous sequences on the other break end. This synthesis-dependent microhomology-mediated end joining (SD-MMEJ) explains many of the alt-EJ repair products recovered following I-SceI nuclease cutting in Drosophila. However, sequence-specific factors that influence SD-MMEJ repair remain to be fully characterized. Here, we expand the utility of the SD-MMEJ model through computational analysis of repair products at Cas9-induced double-strand breaks for 1100 different sequence contexts. We find evidence at single nucleotide resolution for sequence characteristics that drive successful SD-MMEJ repair. These include optimal primer repeat length, distance of repeats from the break, flexibility of DNA sequence between primer repeats, and positioning of microhomology templates relative to preferred primer repeats. In addition, we show that DNA polymerase theta is necessary for most SD-MMEJ repair at Cas9 breaks. The analysis described here includes a computational pipeline that can be utilized to characterize preferred mechanisms of alt-EJ repair in any sequence context. 

The wide-scale use of liposomal delivery systems is challenged by difficulties in obtaining potent liposomal suspensions. Passive and active loading strategies have been proposed to formulate drug encapsulated liposomes but are limited by low efficiencies (passive) or high drug specificities (active). Here, we present an efficient and universal loading strategy for synthesizing therapeutic liposomes. Integrating a thermal equilibration technique with our unique liposome synthesis approach, co-loaded targeting nanovesicles can be engineered in a scalable manner with potencies 200-fold higher than typical passive encapsulation techniques. We demonstrate this capability through simultaneous co-loading of hydrophilic and hydrophobic small molecules and targeted delivery of liposomal Doxorubicin to metastatic breast cancer cell line MDA-MB-231. Molecular dynamic simulations are used to explain interactions between Doxorubicin and liposome membrane during thermal equilibration. By addressing the existing challenges, we have developed an unparalleled approach that will facilitate the formulation of novel theranostic and pharmaceutical strategies. 

Recurrent mutations are frequently associated with transcription factor (TF) binding sites (TFBS) in melanoma, but the mechanism driving mutagenesis at TFBS is unclear. Here, we use a method called CPD-seq to map the distribution of UV-induced cyclobutane pyrimidine dimers (CPDs) across the human genome at single nucleotide resolution. Our results indicate that CPD lesions are elevated at active TFBS, an effect that is primarily due to E26 transformation-specific (ETS) TFs. We show that ETS TFs induce a unique signature of CPD hotspots that are highly correlated with recurrent mutations in melanomas, despite high repair activity at these sites. ETS1 protein renders its DNA binding targets extremely susceptible to UV damage in vitro, due to binding-induced perturbations in the DNA structure that favor CPD formation. These findings define a mechanism responsible for recurrent mutations in melanoma and reveal that DNA binding by ETS TFs is inherently mutagenic in UV-exposed cells.