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. 2001 Mar 15;29(6):1317–1325. doi: 10.1093/nar/29.6.1317

Figure 6.

Figure 6

hMre11, but not hRad52-annealing activity is inhibited by precoating of substrate oligonucleotides with hRPA. (A) Complementary 61 nt ssDNAs (DG61 and DG62) were incubated separately at 1 nM with 3.4 nM hRPA for 15 min at 25°C. Reaction mixtures were combined and incubations were continued for 60 min at 16°C after addition of hMre11, hRad52 or hKu70/80 at 10 nM each, or without protein, as indicated. Annealing was analyzed by native PAGE after deproteinization, followed by autoradiography. Lane 1, no proteins added; lane 2, preincubation with hRPA only; lane 3, incubation with hMre11 only; lane 4, preincubation with hRPA, followed by addition of hMre11; lane 5, incubation with hKu70/80 only; lane 6, preincubation with hRPA, followed by addition of hKu70/80; lane 7, incubation with hRad52 only; lane 8, preincubation with hRPA, followed by addition of hRad52. (B) Complementary 61 nt ssDNAs (DG61 and DG62) were incubated separately at 1 nM with the indicated concentrations of hRPA in the top panel or 1.6 nM hRPA in the bottom panel for 15 min at 25°C. Reaction mixtures were combined and incubations were continued after addition of hMre11 or hRad52 at 10 nM in the top panel or at the indicated concentrations in the bottom panel, for 60 min at 16°C. Annealing was analyzed by native PAGE after deproteinization. (C) Quantification of the percentages dsDNA formed in (B). Values are expressed as percentage dsDNA for hMre11 and hRad52.