Figure 4.

M/R/N partially unwinds short DNA duplexes from a 3′ single-stranded overhang. (A) M/R/N (150 ng) was incubated in 5 mm MgCl₂ and 0.5 mm ATP as indicated with a DNA substrate containing a 17-bp duplex and either a 3′ or 5′ single-stranded overhang, with the 17-mer labeled at the 5′ end with ³²P. The reactions were incubated at 37°C for 30 min and were stopped with SDS and EDTA before separation in a native polyacrylamide gel. (Δ) Substrate that was heated to denature the duplex. The arrow indicates the position of the free labeled strand. (B) Mre11 (25 ng, lanes 2,3), M/R (60 ng, lanes 4,5), M/N (50 ng, lanes 6,7), and M/R/N (150 ng, lanes 8,9) were incubated with the same 3′ overhang substrate as in A in the presence of 0.5 mm ATP as indicated. The varying protein levels yield approximately the same amount of Mre11 (25 ng) in each reaction. (C) M/R/N (150 ng) was incubated as in A and B except with DNA substrates containing a 34-bp duplex region and either a 3′ or 5′ single-stranded overhang as shown.