Replication protein A (RPA) is an essential trimeric protein complex that binds to single-stranded DNA (ssDNA) in eukaryotic cells and is involved in various aspects of cellular DNA metabolism including replication and repair. to DNA damage sites depends on a process termed DNA-end resection. Consequently RPA recruitment to sub-nuclear regions bearing DSBs has been used as readout for resection and for ongoing HR. Quantification of RPA recruitment by immunofluorescence-based microscopy techniques is time consuming and requires extensive image analysis of relatively small populations of cells. Here we present a high-throughput flow-cytometry technique that allows the usage of RPA staining to measure cell proliferation and DNA-damage fix by HR within an unparalleled impartial and quantitative way. ? 2012 International Culture for BI605906 Advancement of Cytometry axis represents the strength of RPA2 indicators (logarithmic size). In the control test (No Ab) just the supplementary antibody was utilized. B: Removal of samples ahead of fixation differentiates … Even though the RPA complex is certainly ubiquitously expressed through the entire cell routine its binding to ssDNA is basically limited to cells going through DNA replication (8). Unlike many nucleoplasmic proteins elements tightly destined to chromatin and/or DNA have a tendency to end up being resistant to removal with detergents or raising salt concentrations features which have been the foundation for mobile fractionation (or “chromatin fractionation”) tests (21 22 To assess whether we’re able to distinguish between free of charge and DNA-bound RPA by movement cytometry we treated cells with detergent ahead of fixation (discover Materials and strategies and Ref. 23). As proven in Body 1B (still left panel) removal of soluble RPA2 before fixation led to the looks of two different but overlapping cell populations with relation of RPA2 staining. Notably in comparison to total DNA articles by staining with DAPI the RPA-positive cell inhabitants seemed to represent cells in S stage (Fig. 1B correct -panel). To even more directly check out this connection we pulse-labeled cells using the nucleotide analogue EdU BI605906 extracted them and performed dual staining through the use of click chemistry to identify EdU (24) as well as anti-RPA2 antibodies (discover Materials and strategies). Analyses from the ensuing samples established that a lot of cells staining positive for RPA had been also EdU positive (Fig. 1C). Used together these outcomes demonstrated that RPA staining after removal can be found in movement cytometry in an effort to identify cells going through DNA replication. DNA Damage Causes Elevated Strength of RPA Indicators Agents that trigger DNA harm or DNA replication tension are recognized to make local deposition of RPA into focal buildings that may be easily noticed by immunofluorescence analyses of set cells (14). To check whether DNA harm could also modification the design of RPA2 staining noticed by movement cytometry we treated U2Operating-system cells with camptothecin (CPT) an inhibitor of DNA topoisomerase I (TopI) that triggers the forming of TopI-DNA covalent adducts that are after that changed into DSBs in S-phase if they are came across by energetic replication forks (25). As proven BI605906 in Body 2A whenever we examined cells by movement cytometry CPT treatment resulted in a clear upsurge in RPA2 indication strength within S-phase cells (for a good example of the gating system see Supporting Details Fig. S1). Quantification uncovered that as the general percentage of cells exhibiting RPA2 staining didn’t significantly transformation upon CPT treatment (Fig. 2B still left -panel) the strength of RPA2 indication increased around 2-flip (Fig. 2B middle -panel; for an alternative solution method to measure distinctions in RPA2 staining find Supporting Details Fig. S2). To even more clearly reveal the distinctions in RPA2 staining between neglected and treated cells we described a gate at the bigger strength degree of RPA staining for some cells (>95%) in neglected conditions (dashed square in Fig. 2A; observe Supporting Information Fig. S1) and used this as the basis for further quantifications. Strikingly when this new gate was applied to define DNA-damage induced RPA positivity the difference between untreated BI605906 and CPT-treated samples was now very dramatic (Fig. 2B right panel). Physique 2 Mouse monoclonal to CD3/CD19/CD45 (FITC/PE/PE-Cy5). A: DNA damage increases the intensity of RPA2 signals. Cells were treated with 1 μM of camptothecin (CPT) for 1 h before harvesting. The dashed square marks the gate (showing the percentage of cells in it) utilized for quantification in the right … One of the earliest markers for DDR activation is usually phosphorylation of histone variant H2A.X on Ser-139 to yield the phosphorylated species termed γH2AX (6). Given that CPT treatment preferentially.