FIGURE 5.

A, length of duplex unwound by hChlR1 is stimulated by RPA. DNA helicase activity was assayed using 5 fmol of the partial duplex M13 substrate (39mer40dT-M13, 5 fmol) containing duplex regions varying in length between 40 and 600 bp and a 40-nt oligo(dT) tail at its 5′-end. The indicated levels of hChlR1 were preincubated with the substrate in the standard helicase reaction but in the presence of 100 μm ATP, at 25 °C for 10 min, after which human RPA (0.32 pmol) was added as indicated, and all reactions were adjusted to 1 mm ATP. After incubation at 37 °C for 30 min, reactions were stopped by the addition of 20 mm EDTA, 0.1% SDS, 0.125 mg/ml proteinase K, and the mixture were incubated for an additional 30 min at 37 °C and then subjected to electrophoretic separation through a neutral 1% agarose gel. Lane 1, ³²P-labeled 100-bp ladder DNA marker (denatured); lane 2, boiled substrate; lanes 3 and 10, reactions without hChlR1; lanes 4–6 and 7–9, increasing amounts of hChlR1 in the absence or presence of RPA. B, length of duplex unwound by hChlR1 is stimulated by Ctf18-RFC. The hChlR1-catalyzed displacement reaction was carried out as described in A, with hChlR1 preincubation and the subsequent addition of Ctf18-RFC, RFC, or PCNA, as indicated.