Repair of DNA double-strand-breaks (DSBs) by homologous recombination is crucial for cell proliferation and tumor suppression. However, despite its importance, the molecular intermediates of mitotic DSB-repair remain undefined. The double Holliday Junction (dHJ), presupposed to be the central intermediate for more than 25 years1, has only been identified during meiotic recombination2. Moreover, evidence has accumulated for alternative, dHJ-independent mechanisms3–6, raising the possibility that dHJs are not formed during DSB-repair in mitotically cycling cells. Here we identify intermediates of DSB-repair using a budding yeast assay system designed to mimic physiological DSB repair. This system utilizes diploid cells and provides the possibility for allelic recombination either between sister-chromatids or between homologs, as well as direct comparison with meiotic recombination at the same locus. In mitotically cycling cells, we detect inter-homolog Joint Molecule (JM) intermediates whose size and strand-composition are identical to the canonical dHJ structures observed in meiosis2. However, in contrast to meiosis, JMs between sister chromatids form in preference to those between homologs. Moreover, JMs appear to represent a minor pathway of DSB repair in mitotic cells, being detected at ~10-fold lower levels (per DSB) than during meiotic recombination. Thus, although dHJs are identified as intermediates of DSB-promoted recombination in both mitotic and meiotic cells, their formation is distinctly regulated according to the specific dictates of the two cellular programs.

Long-lived proteins have been implicated in age-associated decline in metazoa, but they have only been identified in extracellular matrices or postmitotic cells. However, the aging process also occurs in dividing cells undergoing repeated asymmetric divisions. It was not clear whether long-lived proteins exist in asymmetrically dividing cells or whether they are involved in aging. Here we identify long-lived proteins in dividing cells during aging using the budding yeast, Saccharomyces cerevisiae. Yeast mother cells undergo a limited number of asymmetric divisions that define replicative lifespan. We used stable-isotope pulse-chase and total proteome mass-spectrometry to identify proteins that were both long-lived and retained in aging mother cells after ∼18 cells divisions. We identified ∼135 proteins that we designate as long-lived asymmetrically retained proteins (LARPS). Surprisingly, the majority of LARPs appeared to be stable fragments of their original fulllength protein. However, 15% of LARPs were full-length proteins and we confirmed several candidates to be long-lived and retained in mother cells by time-lapse microscopy. Some LARPs localized to the plasma membrane and remained robustly in the mother cell upon cell division. Other full-length LARPs were assembled into large cytoplasmic structures that had a strong bias to remain in mother cells. We identified age-associated changes to LARPs that include an increase in their levels during aging because of their continued synthesis, which is not balanced by turnover. Additionally, several LARPs were posttranslationally modified during aging. We suggest that LARPs contribute to age-associated phenotypes and likely exist in other organisms.ACD | asymmetric cell division | RITE | recombination-induced tag exchange | replicative aging

Epithelial tubes are the infrastructure for organs and tissues, and tube morphogenesis requires precise orchestration of cell signaling, shape, migration, and adhesion. Follicle cells in the Drosophila ovary form a pair of epithelial tubes whose lumens act as molds for the eggshell respiratory filaments, or dorsal appendages (DAs). DA formation is a robust and accessible model for studying the patterning, formation, and expansion of epithelial tubes. Tramtrack69 (TTK69), a transcription factor that exhibits a variable embryonic DNA-binding preference, controls DA lumen volume and shape by promoting tube expansion; the tramtrack mutation twin peaks (ttktwk) reduces TTK69 levels late in oogenesis, inhibiting this expansion. Microarray analysis of wild-type and ttktwk ovaries, followed by in situ hybridization and RNAi of candidate genes, identified the Phospholipase B-like protein Lamina ancestor (LAMA), the scaffold protein Paxillin, the endocytotic regulator Shibire (Dynamin), and the homeodomain transcription factor Mirror, as TTK69 effectors of DA-tube expansion. These genes displayed enriched expression in DA-tube cells, except lama, which was expressed in all follicle cells. All four genes showed reduced expression in ttktwk mutants and exhibited RNAi phenotypes that were enhanced in a ttktwk/+ background, indicating ttktwk genetic interactions. Although previous studies show that Mirror patterns the follicular epithelium prior to DA tubulogenesis, we show that Mirror has an independent, novel role in tube expansion, involving positive regulation of Paxillin. Thus, characterization of ttktwk-differentially expressed genes expands the network of TTK69 effectors, identifies novel epithelial tube-expansion regulators, and significantly advances our understanding of this vital developmental process.

Significance Genes encoding ribosomal RNA (rDNA) are organized into a repetitive array in eukaryotic genomes. The copy number of these genes often varies and is responsive to genetics and environment. Here, we show that variation in copy number at the rDNA locus is capable of altering replicative lifespan in yeast. These results indicate that considering rDNA copy number, and conditions that could potentially change this dynamic chromosome locus, is critical for evaluating lifespan. We propose that this rDNA locus represents the kind of repeated element in eukaryotic genomes that escapes easy detection, yet has phenotypic consequences, in this case lifespan.