The density of testicular cells in PBS was adjusted to approx imately cellsyml.Immediately after cytocentrifugation, the preparations were fixed in absolute methanol for min at C and then rinsed in icecold acetone for a few seconds.Gray scale images were pseudocolored and merged by using ONCOR IMAGE and ADOBE PHOTOSHOP software.Only signals that were clearly visible by eye through the microscope were scored to eliminate the possibility of false colocalizations caused by emission filter bleed through.In dsDNA the bromine atom is hidden in the phosphodiester backbone of the double helix and, therefore, is not accessible to the antibody.Ionizing radiation induces mostly single strand breaks and ox idized apurinicyapyrimidinic sites.Topoisomerase II binds covalently to dsDNA, cleaves both strands, and reseals the cleaved complex.When cells were exposed togirradiation or etoposide and analyzed at hr after DNA damage, the percentage of cells with ssDNA foci increased in a dosedependent manner. Cells with discrete ssDNA foci at hr did notfluoresce after FISEL and, therefore, were not apoptotic.Nuclei are counterstainedwith, diamidinophenylindole.To facilitate the demonstration of colocalizations, the green signals were shif ted purposely by one pixel to the right and by one pixel to the bottom.Thus, similar to FISEL abundance of ssDNA inside the nucleus can be used as a death marker. To demonstrate the specificity of our assay, control cells were exposed to a lethal dose of the transcriptional inhibitor actinomycin D or the protein synthesis inhibitor cyclohex imide.Therefore, we per formed combined immunof luorescent staining of RPA and ssDNA on girradiated fibroblasts.In cells with both ssDNA and RPA foci, approx imately of the foci were doublestaining. XPA patients have defects in the enzyme that is responsible for lesion recognition by nucleotide excision repair and, therefore, accumulate DNA damage.The unexcised DNA lesions in XPA cells stimulate intrachromosomal homologous recombination. Both the DSB and the ssDNA tail are obligatoryintermediates in meiotic recombination. PPL control cells exhibited a fold lower number of ssDNA fibers per slide.Although the degree of chromatin stretching is not uniform along the length of a DNA fiber, DNA in situ hybridization experiments with clones of known size on similar preparations have revealed DNA extensions var ying from to kbymm. Provided that ssDNA is not overstretched compared with dsDNA, the longest ssDNA filaments observed in both XPA and irradiated PPL cultures are estimated to be kb.However, these extremely long ssDNA filaments may only represent a minor part of the DNA damageinduced ssDNA.Because a stretched ssDNA tail of kb measures, mm, it would generate only a dotlike signal, which is almost indistinguishable from the relatively high background on fiber preparations.The resulting nucleoprotein filament is considered to be the key element for promoting the pairing and strand exchange between ssDNA and homologous dsDNA. Most models of recombination and repair involve a ssDNA intermediate.However, this is the first demonstration, in situ, of such an intermediate.This is likely to ref lect differences in the composition of recombination intermediates.Similar to meiotic recombination complexes, DNA damageinduced recombination intermediates may not have a set stoichiometr y.Obviously, such long stretches of ssDNA must be veryrare in normally growing cell cultures.