Fig. 1.

Mismatch correction and nucleosome assembly in HeLa cell-free extracts. Mismatch-correction reactions with 3′ G-T or control 3′ A-T DNA and the indicated amounts of HeLa nuclear or cytosolic extract were performed and analyzed as described in Materials and Methods. (A) Recovered DNA products of the indicated mismatch correction reactions were cleaved with ClaI, separated under denaturing conditions in a 0.8% agarose gel, transferred to a nylon membrane, and hybridized with a ³²P-labeled probe complementary to the discontinuous strand position 2505–2526. The diagram on the left of the image shows the relative positions of the 3′ strand break, the G-T mispair (sharp bump), a ³²P-labeled probe (dashed asterisk), and the unique ClaI site in the 3′ G-T DNA. The asterisk specifies the position of the indirectly labeled ClaI fragment of the discontinuous strand detected in the control reaction carried out in the absence of any extract or purified proteins. In the extract reactions, the discontinuous strand is subjected to mismatch correction and/or ligation; ligation generates the 6.44-kb ligated product. The mismatch-correction reaction proceeds via mismatch-provoked degradation of the discontinuous strand that generates smaller fragments located in the area indicated by the bracket. Degradation of the discontinuous strand in a particular reaction expressed as a percentage of the total label and representing the mean of three experiments is indicated below the reaction lane. (B) Degradation of the discontinuous strand of 3′ G-T (▪ and □) or 3′ A-T (• and ◯) DNA as a function of the concentration of HeLa nuclear (□ and ◯) or cytosolic (▪ and •) extract. Data are from images like the one shown in A. (C) Dependence of mismatch correction of 3′ G-T DNA in HeLa nuclear (□) or cytosolic (▪) extract on the amount of extract used in the reactions. The data in B and C are presented as the means ± 1 SD, n = 3. (D) Assembly of nucleosomes on 3′ G-T or cc G-T DNA during mismatch correction in HeLa nuclear and cytosolic extracts. DNA products of the indicated mismatch correction reactions were subjected to micrococcal nuclease cleavage and separation in a 1.5% native gel, and Southern hybridization with a ³²P-labeled probe complementary to f1MR59 minus strand position 5733–5756 (35). ³²P hybridizations were visualized with a GE Storm PhosphorImager system. (E) Detection of CAF-I in HeLa nuclear and cytosolic extracts by Western analysis. Proteins of HeLa nuclear and cytosolic extracts (20 μg each) were separated in a denaturing SDS-gel, transferred to a PVDF membrane and incubated with rabbit CAF-I p150 antibodies (Santa Cruz), followed by detection of the immune complexes with an ECL Plus kit (GE HealthCare).