The molecular events implicated in repair of CT99021 252917-06-9 strand breaks in DNA are becoming more clear, but an overall and quantitative picture of their repair in vivo which would contribute to understanding the systems biology of repair and the effects of inhibitors is not yet available. Current methods do not allow simultaneous and precise quantitation of repair of Perifosine single and double strand breaks. Repair of double strand breaks, which are believed to be the crucial lesions leading to cell death, is commonly assayed by restoration of the normal length of genomic DNA or restriction fragments using pulsed-field gel electrophoresis. Repair of single strand breaks, which may contribute to loss of viability by relaxing superhelical stress in genomic DNA loops and thus arresting transcription, cannot yet be quantitated specifically by methods with comparable precision. As a model system to approach this question we are studying the repair of strand breaks in vivo in a,170 kb circular minichromosome, the Epstein-Barr virus episome, which is maintained in the nuclei of Raji cells at 50�C100 copies localised at the periphery of interphase chromosomes. Two features of this minichromosome make it an attractive model for genomic chromatin: it can be considered as a defined region of chromatin in view of its canonical nucleosomal conformation and the well-studied sequence and properties of its DNA, and its closed circular topology and length resemble those of the constrained loops which genomic chromatin forms in vivo. After irradiating cells with 60Co c photons we assayed the repair of single strand breaks in the minichromosome by quantitating the loss of nuclease S1sensitive sites, and the repair of double strand breaks by PFGE assays of the reformation of supercoiled DNA from molecules which had been linearised. Circular molecules containing single strand breaks could not be quantitated directly, and instead their levels were calculated using a mathematical model developed to fit the experimental data. We exploited the possibility of quantitating repair in this system to examine the implication of particular enzymes, particularly topoisomerases I and II whose participation in repair has long been controversial, poly polymerase-1, Rad51, the catalytic subunit of DNA-protein kinase, and ATM kinase. New features of the repair of strand breaks in vivo and of their kinetics were revealed by mathematical modeling. The simultaneous repair of single and double strand breaks in a defined region of chromatin in vivo has not been studied previously using quantitative methods, to our knowledge. The methods used to detect strand breaks in earlier studies, filter elution or single-cell DNA electrophoresis, cannot provide absolute numbers of breaks and the reported rates were variable. We used two conditions to ensure that strand breaks were quantitated accurately: for PFGE, DNA was deproteinised at room temperature because extra strand breaks are created at higher temperatures, and hybridisation was carried out in dried gels because the transfer of large DNA fragments onto membranes is not quantitative. In another study published while this manuscript was in preparation, a significant amount of minichromosome DNA remained in the sample well of PFGE gels and was interpreted as nicked circles, but here little or no DNA remained in the wells and nicked circular DNA migrated slowly into the gel, possibly reflecting methodological differences.