The binding mode observed with MRCKb does indeed reflect of MRCKb

lls its level remained identical to that in control cells when topoisomerases or PARP were inhibited, but its stability in the presence of putative repair inhibitors could not be measured since they influenced its reformation by repair pathways. The level of linear minichromosome DNA in irradiated cells remained constant when NHEJ was inhibited, with a p-value for the difference in level between 0 h and 2 h of 0.55 for wortmannin and 0.88 for NU7441. To inhibit enzymes involved in repair of Motesanib strand breaks, we used chemical reagents whose specificity has been well established because in most cases siRNA methodology did not provide sufficient depletion of enzymes. In other studies depletion of PARP-1, DNA ligases, and topoisomerase II was also less than complete and in some cases lethal. Inhibitors of PARP-1 showed no effect on the repair of strand breaks in minichromosome DNA. The precise step in which PARP-1 intervenes in repair remains elusive; the current view is that it is not indispensable for repair of single strand breaks in genomic DNA and its role appears to be indirect, for example by binding to breaks and protecting them from further degradation. In another study using our experimental system published while this manuscript was in preparation, knockdown of PARP-1 did not significantly affect repair of single or double strand breaks. A possible role for topoisomerases I or II in DNA repair has been examined in several studies, but in some cases noncatalytic topoisomerase inhibitors were employed which themselves create strand breaks when DNA is deproteinised and therefore cannot provide evidence for a role of topoisomerases in repair. Topological considerations predict that if nucleosomes do not dissociate completely in the neighbourhood of a strand break, the negative superhelicity which results from DNA wrapping on their surface would be conserved in the nicked circular and linear forms. Thus after the repair of all breaks, the religated circular form would recover the negative superhelicity of the original circular minichromosome DNA. Our finding that the conversion of linear to supercoiled minichromosome DNA continues at the normal rate when topoisomerases I and II are 957054-30-7 distributor inhibited by catalytic inhibitors is consistent with this scenario. It appeared