v Dissecting the cellular response to DNA breaks using functional genomics Ásta Björk Jónsdóttir, Paul Russell, Alessandro Esposito, Tamara Gruener, Ashok Venkitaraman MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH Introduction Homologous recombination (HR) is an error-free DNA double- strand break (DSB) repair pathway. If breaks are not repaired cells can loose parts of chromosomes or gain chromosomal translocations 1). Germline mutations in HR proteins predispose carriers to breast, ovarian and other cancers 2) The aim of the project is to indentify novel HR related proteins and regulators Experimental layout High-content screening combines the use of high-throughput plate handling, automated microscopy, and subsequent automated image analysis Screen of selected siRNA libraries (kinome, ubiquitinome and druggable genome) was conducted in MCF10A cells. Assay plates were prepared in triplicate with Biomek NX and cells seeded with Well Mate Cells were treated with MMC 40hrs post transfection to induce DSB. Cells were fixed 8hrs post treatment and stained immunofluorescently with markers for DNA DSBs (  H2AX) and DSB repair foci (RAD51) using the Biomek NX Plates were scanned with Cellomics ArrayScan VTI 0.1µM siRNA per well Library plate 25nM siRNA per well Assay plates in triplicate MMC Fix and stain cells with α-RAD51 and α-γH2AX antibodies and Hoechst Image assay plates with the Cellomics ArrayScan VTI Reverse Transfectio n of MCF10A Data analysis cellHTS2 (R) RAD51 foci count %of cells with >1 spot DNARAD51 Figure 1 Results of primary screen cellHTS23), an R script based analysis tool, was used to analyze the raw intensity readings to get an annotated hit list The analysis included: a) per-plate normalization (plate median) b) scoring of replicate measurements (robust z-score) c) summarization of replicates (mean) Stratification of hits 40 candidates from the kinome- and the ubiquitinome screens, 164 from the druggable genome screen and other candidates of interest that have been reported to affect DSB repair were validated. Total of 255 genes were re-screened with new siRNA pools. Figure 3 Results of validation screen Cluster analysis was performed on validated hits for  H2AX and RAD51 to identify candidates that do not affect sensing of DSBs (  H2AX foci positive) and that are needed for RAD51 mediated repair (RAD51 foci negative) Conclusions DNA double-strand breaks (DSBs) are very toxic and make cells hypersensitive to radiotherapy We have indentified group of genes that inhibit the recruitment of RAD51 to the site of DNA damage. Functional studies will need to be conducted to understand the biological function they have in HR which may enable us to indentify potential targets for use in anticancer treatment. A)B) Figure 2 A) Z’ factor for assay robustness B) Z-score heat map for RAD51 foci analysis. Experiments were run in batches of 8 plates. Colour range goes from to v Volcano plot shows the negative decadic logarithm of the P value versus the mean z-score for RAD51 foci. In red are validated hits Figure 4 Cluster analysis for z- scores of  H2AX and RAD51 foci References 1) Regulation of DNA cross-link repair by the Fanconi anemia/BRCA pathway. Hyungjin Kim and Alan D. D'Andrea. Genes and Development, ) BRCA1 and BRCA2: different roles in a common pathway of genome protection. Rohini Roy, Jarin Chun & Simon N. Powell. Nature Reviews Cancer, ) Analysis of cell-based RNAi screens. Michael Boutros, Lígia P Brás and Wolfgang Huber. Genome Biology, 2006.