any diseases have been linked to SVs, most often defined as genomic changes at least 50 bp in size, but SVs are challenging to detect accurately. Conditions linked to SVs include autism 1 , schizophrenia, cardiovascular disease 2 , Huntington's disease and several other disorders 3. Far fewer SVs exist in germline genomes relative to small variants, but SVs affect more base pairs, and each SV might be more likely to affect phenotype 4-6. Although next-generation sequencing technologies can detect many SVs, each technology and analysis method has different strengths and weaknesses. To enable the community to

Exposure of cells to DNA-damaging agents results in a rapid increase in the formation of subnuclear complexes containing Rad51. To date, it has not been determined to what extent DNA damage-induced cytoplasmic to nuclear transport of Rad51 may contribute to this process. We have analyzed subcellular fractions of HeLa and HCT116 cells and found a significant increase in nuclear Rad51 levels following exposure to a modest dose of ionizing radiation (2 grays). We also observed a DNA damageinduced increase in nuclear Rad51 in the Brca2-defective cell line Capan-1. To address a possible Brca2-independent mechanism for Rad51 nuclear transport, we analyzed subcellular fractions for two other Rad51-interacting proteins, Rad51C and Xrcc3. Rad51C has a functional nuclear localization signal, and although we found that the subcellular distribution of Xrcc3 was not significantly affected by DNA damage, there was a damageinduced increase in nuclear Rad51C. Furthermore, RNA interference-mediated depletion of Rad51C in HeLa and Capan-1 cells resulted in lower steady-state levels of nuclear Rad51 as well as a diminished DNA damage-induced increase. Our results provide important insight into the cellular regulation of Rad51 nuclear entry and a role for Rad51C in this process.Cellular surveillance of genome integrity and repair of DNA damage are essential processes that ensure proper development and survival of all organisms. DNA double-strand breaks (DSBs) 2 are a particularly deleterious form of genome damage and occur following exposure of cells to exogenous mutagens as well as during normal metabolic processes, e.g. antigen receptor gene rearrangement, restart of stalled replication forks, formation of meiotic DNA crossovers, etc. (1, 2). Mammalian cells use two distinct mechanisms for repair of DSBs, non-homologous end joining and homologous recombination (HR). Processes requiring imprecise DNA repair, such as the creation of antibody diversity, exploit the error-prone non-homologous end joining mechanism. In contrast, HR is an error-free DNA repair pathway and is critical for avoidance of unwanted genetic changes during the meiotic exchange of information between paternal and maternal alleles and for error-free repair of broken chromosomes (3). Rad51 is the central enzymatic component of HR. Upon its regulated recruitment to sites of DNA breaks, Rad51 forms a nucleoprotein filament by polymerizing onto single-stranded DNA at the processed break. This filament catalyzes DNA strand exchange with an undamaged sister chromatid or homologous chromosome, which serves as a template for the restoration of missing genetic information (3, 4).Visible nuclear Rad51 clusters, or foci, form during S-phase and appear to localize to sites of replicating DNA (5-7). A dramatic increase in the number and size of nuclear Rad51 foci is a hallmark of the early cellular response to genomic insult (7-10). The appearance of DNA damage-induced nuclear Rad51 foci is blocked in cells with deficiencies in several HR-related proteins, including Brca2 ...

The insulin-regulated glucose transporter, GluT4, is a key molecule in peripheral insulin signaling. Although GluT4 is abundantly expressed in neurons of specific brain regions such as the hippocampus, the functional role of neuronal GluT4 is unclear. Here, we used pharmacological inhibition of GluT4-mediated glucose uptake to determine whether GluT4 mediates insulin-mediated glucose uptake in the hippocampus. Consistent with previous reports, we found that glucose utilization increased in the dorsal hippocampus of male rats during spontaneous alternation (SA), a hippocampally-mediated spatial working memory task. We previously showed that insulin signaling within the hippocampus is required for processing this task, and that administration of exogenous insulin enhances performance. At baseline levels of hippocampal insulin, inhibition of GluT4-mediated glucose uptake did not affect SA performance. However, inhibition of an upstream regulator of GluT4, Akt, did impair SA performance. Conversely, when a memory-enhancing dose of insulin was delivered to the hippocampus prior to SA-testing, inhibition of GluT4-mediated glucose transport prevented cognitive enhancement. These data suggest that baseline hippocampal cognitive processing does not require functional hippocampal GluT4, but that cognitive enhancement by supra-baseline insulin does. Consistent with these findings, we found that in neuronal cell culture, insulin increases glucose utilization in a GluT4-dependent manner. Collectively, these data demonstrate a key role for GluT4 in transducing the procognitive effects of elevated hippocampal insulin.

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free repair of DNA double strand breaks. Rad51 is the central catalyst of HR in all eukaryotes, and to this point studies of human Rad51 have focused exclusively on events occurring within the nucleus. However, substantial amounts of HR proteins exist in the cytoplasm, yet the function of these protein pools has not been addressed. Here, we provide the first demonstration that Rad51 and the related HR proteins Rad51C and Xrcc3 exist in human mitochondria. We show stress-induced increases in both the mitochondrial levels of each protein and, importantly, the physical interaction between Rad51 and mitochondrial DNA (mtDNA). Depletion of Rad51, Rad51C, or Xrcc3 results in a dramatic decrease in mtDNA copy number as well as the complete suppression of a characteristic oxidative stress-induced copy number increase. Our results identify human mtDNA as a novel Rad51 substrate and reveal an important role for HR proteins in the maintenance of the human mitochondrial genome.