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Fig. 2. Orc1 and Sir2 specifically recognize elements on the telomeric and subtelomeric repeats. (A) Schematic representation of telomere and subtelomeric regions of P. falciparum chromosome ends. The probes used in the EMSA and ChIP assays are indicated. (B) Binding of nuclear proteins to telomeric and subtelomeric regions. ³²P-labelled telomere (left panel), TARE3 (middle panel) and TARE6 (right panel) probes were incubated with nuclear extracts. Competition shift assays were done using either 50-fold molar excess of unlabelled homologous (third lane) or heterologous (KAHRP and Sp1, fourth and fifth lanes) competitor. A free probe was run in the first lane in all cases. The telomere probe formed three protein-DNA complexes, whereas TARE3 and TARE6 formed only a single retarded complex. Arrowheads indicate DNA-protein complexes. (C) Binding of Orc1 and Sir2 to telomeric and subtelomeric repeats. A total of 30 µg of antiserum against Sir2 and Orc1, and respective non-immune sera, were pre-incubated for 15 minutes in the binding reaction, followed by the addition of the labelled telomere, TARE3 and TARE6 probes. Telomere and TARE3 probes produced supershifted complexes with both anti-Sir2 and -Orc1 antibodies (left and middle panels), whereas the TARE6 probe only formed a supershifted complex in the presence of Sir2 (right panel). NE, nuclear extracts; C, complex; SC, supershifted complex; KAHRP, upstream region of kahrp gene; Sp1, consensus Sp1-binding-site factor; PI, pre-immune serum.

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