An open-source platform to quantify subnuclear foci and protein colocalization in response to replication stress.

Nuclear reorganization, including the localization of proteins into discrete subnuclear foci, is a hallmark of the cellular response to DNA damage and replication stress. These foci are thought to represent transient environments or repair factories, in which the lesion is sequestered with molecules and co-factors that catalyze repair. For example, nuclear foci contain signaling proteins that recruit transducer proteins. One important class of transducers is the structure-selective endonucleases, such as SLX1-SLX4, MUS81-EME1, and XPF-ERCC1, which remove branched DNA structures that form during repair. The relocalization of structure-selective endonucleases into subnuclear foci provides a visual read-out for the presence of direct DNA damage, replication barriers, or DNA entanglements and can be monitored using fluorescence microscopy. By simultaneously probing for two or more fluorescent signals, fluorescence microscopy can also provide insights into the proximal association of proteins within a local environment. Here, we report an open-source and semi-automated method to detect and quantify subnuclear foci, as well as foci colocalization and the accompanying pixel-based colocalization metrics. We use this pipeline to show that pre-mitotic nuclei contain a basal threshold of foci marked by SLX1-SLX4, MUS81, or XPF. Some of these foci colocalize with FANCD2 and have a high degree of correlation and co-occurrence. We also show that pre-mitotic cells experiencing replication stress contain elevated levels of foci containing SLX1-SLX4 or XPF, but not MUS81. These results point towards a role for SLX1-SLX4 and XPF-ERCC1 in the early cellular response to replication stress. Nevertheless, most of the foci that form in response to replication stress contain either FANCD2 or one of the three endonucleases. Altogether, our work highlights the compositional heterogeneity of subnuclear foci that form in response to replication stress. We also describe a user-friendly pipeline that can be used to characterize these dynamic structures.

DNA repair by Rad52 liquid droplets.

Cellular processes are influenced by liquid phase separation, but its role in DNA repair is unclear. Here, we show that in Saccharomyces cerevisiae, liquid droplets made up of DNA repair proteins cooperate with different types of DNA damage-inducible intranuclear microtubule filaments (DIMs) to promote the clustering of DNA damage sites and maintain genome stability. Rad52 DNA repair proteins at different DNA damage sites assemble in liquid droplets that fuse into a repair centre droplet via the action of petite DIMs (pti-DIMs). This larger droplet concentrates tubulin and projects short aster-like DIMs (aster-DIMs), which tether the repair centre to longer DIMs mediating the mobilization of damaged DNA to the nuclear periphery for repair. Our findings indicate that cooperation between Rad52 liquid droplets and various types of nuclear filaments promotes the assembly and function of the DNA repair centre.

The Association between Alcohol Consumption and Breast Density: A Systematic Review and Meta-analysis.

BACKGROUND: Percent breast density (PBD) is a strong risk factor for breast cancer that is influenced by several other risk factors for the disease. Alcohol consumption is associated with an increased risk of breast cancer with an uncertain association with PBD. We have carried out a systematic review and meta-analysis to examine the association of alcohol consumption with PBD. METHODS: We searched nine databases to identify all relevant studies on the association between alcohol intake and breast density. Two independent investigators evaluated and selected 20 studies that were included in our analyses. We divided the studies into three groups according to the methods used to measure and analyze the association of breast density with alcohol consumption. RESULTS: Meta-analysis of the 11 studies that used quantitative methods to measure and analyze PBD as a continuous variable found a statistically significant difference in PBD when comparing the highest with the lowest alcohol level [ß = 0.84; 95% confidence interval (CI), 0.12-1.56]. Three studies that used quantitative methods to measure PBD and categories of PBD for analysis had a summary OR = 1.81 (95% CI, 1.07-3.04). Five studies that used categories to classify PBD and analyze their association with alcohol intake had a summary OR = 1.78 (95% CI, 0.90-3.51). CONCLUSIONS: These results suggest that there is a positive association between alcohol intake and PBD. IMPACT: Alcohol may increase the risk of breast cancer associated with PBD. Cancer Epidemiol Biomarkers Prev; 26(2); 170-8. ©2016 AACR.