Such repair synthesis was not observed with untreated control DNA.Furthermore, the repair reaction showed several char acteristics of nucleotide excision repair, such as slow kinetics and lack of PARP activation. When XP cellfree extracts were used in the assay with the oxidized and enzymetreated plasmid DNA, negligible amounts of repair rep lication were observed.Furthermore, the strongly reduced repair rep lication, compared with that seen with normal cell extracts, was restored by mixing two XP cell extracts derived from different XP complementation groups and by supplementation of purified XPA protein to XPA cellfree extracts, indicating that the reduced repair activity of oxidative damage was due to the known deficiency of nucleotide excision repair in XP.Since reactive oxidizing species are generated through normal cell metabolism, cellular DNA may undergo continuous oxidation at a low level. Hence, any oxidative DNA damage requiring nucleotide excision repair for correction would accumulate in XP cells, and such a buildup of nonrepaired lesions may be a cause of the neurological deterioration and increased frequency of endogenous tumor formation in XP patients.In cell survival assays, most XP cells show close to normal resis tance to ionizing radiation.Thus, XP cells, in general, appear to have normal repair activity for oxidative DNA damage.However, cell survival assays tend to show the effects of the most lethal and abundant DNA lesions, which mask the effects of other forms of DNA damage.Thus, the cellfree DNA repair assay used here has the advantage of disclosing the nature of lesions requiring nucleotide excision repair among other forms of more abundant damage.Cur rently, we are attempting to identify the nature of the lesion that cannot be repaired by XP cell extracts.Click on Request Permissions which will take you to the Copyright Clearance Centers Downloaded from cancerres.aacrjournals.org on September. American Association for Cancer Research. Antibromodeoxyuridine antibody recognizes the halogenated base analog incorporated into chromosomal DNA only when substituted DNA is in the single strand form.Eukar yotic cells are endowed with multiple pathways to repair damaged DNA.One of the major pathways is nucleotide excision repair, which can remove a broad range of DNA lesions.Nucleotide excision repair excises oligonucleotides of bp including the damaged DNA, filling in the singlestranded. This process is veryefficient and usually repairs most DNA lesions before the damaged region is replicated.DSB repair by homologous recombination starts with to exonucleolytic digestion of one DNA strand, which leads to the formation of overhanging ssDNA tails.The publication costs of this article were defrayed in part by page charge payment.The same cells show unscheduled DNA repair synthesis. In this paper we describe a verysimple and fast assay that allows visualization and quantification of ssDNA regions in individual cells.This assay can provide valuable information on DNA damage and mammalian DNA repair pathways.In control experiments, cultures were treated with mgyml cyclohex imide or ngyml actinomycin D for hr, which kill cells by inhibiting protein synthesis and transcription.The testicular tissue was cut into small pieces and the seminiferous tubules were emptied by squeezing with fine forceps.The resulting cell suspension was transferred to a ml centrifuge tube and allowed to settle for min.