We are trying to determine the cause of the phenotype we have observed, but other than RAD54, we do not really have good ideas of where and what to look at.
I'm not by all means an expert in the subject, but as far as I know, RAD51 participates in homology searching during the homologous recombination repair of double-strand breaks. So you might want to look for co-localisation of yH2AX and RAD51 (if you haven't already). Also, perhaps you'll find such co-localisation in S- and G2-arrested cells, since homologous recombination is not a major DSB repair mechanism in G1.
Thanks for the suggestion Gerson. We are following HR repair of DSBs and find that we have a defect in RAD51 resolution. The foci form properly and with normal kinetics, but then they persist far longer than controls.
Depends on your experimental setup. ATP-gamma-S, yes, sure. Else, Rad54 mutants or Helicase loader mutants or other mutants, e.g. resolvase mutants or...some sort of BRCA2-Rad51 or other protein-Rad51 cross-links? What are your cell types/treatment/reproducibility? Did you use formaldehyde in your assay? Geez, there could be many reasons Rad51 gets stuck on DNA in an assay. Just go through your protocol step by step and eliminate the obvious and run all controls. Also, please let me know when your data got published, I want to read the manuscript.
Stefan, we are treating primary keratinocytes cells with IR and determining if HPV oncoproteins interfer with HR repair. Our control (vector only) cells have relatively normal repair, but Rad51 gets stuck in foci (by IF) in some of our HPV cells.
Have you measured y-H2AX foci as well? Do they persist alongside Rad51?
Gerson, H2ax foci persist as well as other markers for DSBs. Just trying to understand the mechanism behind this persistance.
Ionizing radiation causes single and double-stranded DNA breaks, which are generally repaired by Non-Homologous End-Joining (NHEJ) and Homologous Recombination (HR). During DNA double-strand break repair, RAD51 loads onto resected DNA, which can be visualized as RAD51 foci. How is your phenotype affected in BRCA2 cells? Removal of RAD51 may correlate with increased RPA foci. In budding yeast Srs2 helicase removes Rad51, and human homologues exist. In cells the frequency of different DNA double-strand break repair usage can be measured using Maria Jasin GFP construct assays with the reporter cell lines, U2OS DR-GFP (HR) or H1299 dA3 (NHEJ), respectively. Persistence of H2AX foci would indicate unrepaired breaks, which could also be checked by chromosomal break analysis.
Jason, thanks for the suggestions thsoe are some down stream targets that I play to look at. to answer your question, i am expressing only some HPV proteins (not the whole genome) and I am doing this via viral transduction.
John, I also appreciate your comments. it is definitely a defect in HRI see increased H2AX foci persistance as well as decreased GFP positive cells using Maria Jasin's assay in U2OS cells.
I thank both of you for your comments and suggestions.
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