Extended Data Fig. 6: Dimer products are catenated sister DNAs.
From: Coordination of cohesin and DNA replication observed with purified proteins
a, Gel isolated dimer products and deproteinized whole reaction products were treated with the indicated enzymes. The band denoted by a red arrowhead was excised to isolate dimer products. A representative result from two independent experiments is shown. b, Cohesin does not obviously affect DNA catenation by Top2 under these reaction conditions. Cohesin was initially loaded onto nicked circular DNA in the presence of a loader at low ionic strength conditions, and then further incubated in salt-containing replication buffer for 55 min. After addition of 100 mM NaCl, the reaction was subjected to Top2 treatment. Reaction products were then separated by agarose gel electrophoresis, followed by SYBR Gold staining. A representative gel image is shown. c, Quantification of the experiments in b (n = 3 independent experiments; means ± s.d.). d, Dimer products consist of replicated DNAs. Top2 treatment could potentially generate dimer products by promoting catenation of the replicated monomer and unreplicated substrate DNA. To rule out this possibility, purified replication products obtained from the indicated conditions were subjected to DpnI restriction enzyme treatment. Circular DNA substrates used in this study were obtained from dam+ E. coli, and are thus highly sensitive to DpnI that digests the di-methylated GATC sequence (−Mcm10 samples). e, Whole DNA (Sybr Gold staining) and f, replicated DNA detection of the experiments depicted in d with the indicated proteins. g, Quantification of the experiments in e and f. Means from two independent experiments are shown. The amounts of dimer and monomer products were unchanged upon DpnI treatment (+Mcm10 samples). This further confirmed that the dimer products were catenated, replicated sister DNAs. The graphs represent means from two independent experiments. h, Schematic of the sister DNA decatenation after cohesin cleavage by TEV protease. i, Native gel analysis of the experiments depicted in h. j, Quantification of dimer products in i (n = 4 independent experiments; means ± s.d.). Note that Polδ was omitted from the experiments shown in Extended Data Fig. 6 to avoid the occurrence of strand displacement synthesis by Polδ, which is pronounced by Pif132.
