Homologous recombination plays a key role in the repair of double-strand breaks (DSBs), and thereby significantly contributes to cellular tolerance to radiotherapy and some chemotherapy. DSB repair by homologous recombination is initiated by 5’ to 3’ strand resection (DSB resection), with nucleases generating the 3’ single-strand DNA (3’ssDNA) at DSB sites. Genetic studies of Saccharomyces cerevisiae demonstrate a two-step DSB resection, wherein CtIP and Mre11 nucleases carry out short-range DSB resection followed by long-range DSB resection done by Dna2 and Exo1 nucleases. Recent studies indicate that CtIP contributes to DSB resection through its non-catalytic role but not as a nuclease. However, it remains elusive how CtIP contributes to DSB resection. To explore the non-catalytic role, we examined the dynamics of Dna2 by developing an immuno-cytochemical method to detect ionizing-radiation (IR)-induced Dna2-subnuclear-focus formation at DSB sites in chicken DT40 and human cell lines. Ionizing-radiation induced Dna2 foci only in wild-type cells, but not in Dna2 depleted cells, with the number of foci reaching its maximum at 30 minutes and being hardly detectable at 120 minutes after IR. Induced foci were detectable in cells in the G2 phase but not in the G1 phase. These observations suggest that Dna2 foci represent the recruitment of Dna2 to DSB sites for DSB resection. Importantly, the depletion of CtIP inhibited the recruitment of Dna2 to DSB sites in both human cells and chicken DT40 cells. Likewise, a defect in breast cancer 1 (BRCA1), which physically interacts with CtIP and contributes to DSB resection, also inhibited the recruitment of Dna2. Moreover, CtIP physically associates with Dna2, and the association is enhanced by IR. We conclude that BRCA1 and CtIP contribute to DSB resection by recruiting Dna2 to damage sites, thus ensuring the robust DSB resection necessary for efficient homologous recombination.

No abstract

Aim:To study the relationship between age-related maculopathy (ARM) and exposure to sunlight using an objective method.Methods:In a case–control study of Japanese men aged ⩾50 years (67 controls without ophthalmic disease and 148 with ARM), those with ARM were separated into groups of early (n = 75) and late (n = 73) ARM. Facial wrinkle length and area of hyperpigmentation, which are considered to be associated with exposure to sun, were measured using imaging with computer-based image analysis. Skin tone was also measured on the upper inner arm, which is not exposed to sun. Early and late ARM association with skin measurements was then evaluated.Results:Significantly more facial wrinkling (p = 0.047, odds ratio 3.8; 95% CI 1.01 to 13.97) and less facial hyperpigmentation (p = 0.035, odds ratio 0.3; 95% CI 0.08 to 0.92) was present in late ARM cases. The relationship between skin tone and ARM risk was not statistically significant.Conclusions:This objective method showed that lifetime exposure to sunlight is an important factor in the progression of late ARM. An individual’s reaction to sunlight exposure may have a role in ARM progression in addition to total lifetime exposure to sunlight.

The numbers of thymic epithelial cells (TECs) and thymocytes steadily increase during embryogenesis. To examine this dynamic, we generated several TEC-specific transgenic mouse lines, which express fluorescent proteins in the nucleus, the cytosol and in the membranes under the control of the Foxn1 promoter. These tools enabled us to determine TEC numbers in tissue sections by confocal fluorescent microscopy, and in the intact organ by light-sheet microscopy. Compared to histological procedures, flow cytometric analysis of thymic cellularity is shown to underestimate the numbers of TECs by one order of magnitude; using enzymatic digestion of thymic tissue, the loss of cortical TECs (cTECs) is several fold greater than that of medullary TECs (mTECs), although different cTEC subsets appear to be still present in the final preparation. Novel reporter lines driven by Psmb11 and Prss16 promoters revealed the trajectory of differentiation of cTEC-like cells, and, owing to the additional facility of conditional cell ablation, allowed us to follow the recovery of such cells after their depletion during embryogenesis. Multiparametric histological analyses indicate that the new transgenic reporter lines not only reveal the unique morphologies of different TEC subsets, but are also conducive to the analysis of the complex cellular interactions in the thymus.

The difference of clinical features between the groups might reflect different disease stages, although not all of the features observed in the small-short group appeared to represent the early stages of those recorded in the large-long group. Thus, the variation in histopathologic features among previous reports might be partly attributable to differences in disease stage.