Figure 6.

ATP stimulates M/R/N cleavage of a 3′ overhang at the border of the duplex region. (A) Nuclease assays were performed with Mre11 (100 ng, lanes 2–5), M/R (150 ng, lanes 6–9) and M/R/N (150 ng, lanes 10–13) on a double-stranded DNA substrate with 3′ overhangs on each end. The 3′ end of the top strand was labeled with ³²P-labeled cordycepin, as diagrammed, which lengthens the 3′ overhang by one nucleotide. Reactions contained 1 mm MnCl₂ and 0.5 mm ATP as indicated, and aliquots were taken at 10- and 30-min time points. The locations of the cuts made by the proteins are shown as arrows in the diagram. (m) Size markers of 11 and 15 nucleotides are shown. (B) Nuclease assays were performed as in A with the same substrate, except that the top strand was labeled with ³²P at the 5′ end as shown in the diagram.