Fig. 1

Phosphorylation of Sae2 regulates its capacity to promote the Mre11 endonuclease as well as its size distribution. a Recombinant Sae2 was expressed and purified either without (lane 2) or with (lane 3) phosphatase inhibitors (PIs). The phosphorylated pSae2 was mock-treated (lane 5) or treated with λ phosphatase (lane 6). b Phosphorylated (pSae2) and λ phosphatase-treated Sae2 (pSae2 λ) were used in nuclease assays with MRX and a DNA substrate with a single end blocked with streptavidin. Red triangles in the substrate cartoon indicate DNA cleavage positions. c–e Sae2 variant prepared without PIs (c, Sae2), with PIs (d, pSae2), and with PIs and then treated with λ phosphatase (e, pSae2 λ) were used in nuclease assays with MRX and a DNA substrate with both ends blocked with streptavidin. Red triangles in the substrate cartoon indicate DNA cleavage positions. f Quantitation of assays such as shown in c–e. Error bars, SEM, n ≥ 3. g DNA-binding analysis of pSae2 untreated or treated with λ phosphatase, using 100-bp-long dsDNA as a substrate. Quantitation of experiments such as shown in Supplementary Fig. 1a. Error bars, SEM, n = 3. h Size exclusion chromatography analysis of Sae2 variants prepared without PIs (Sae2, blue), with PIs (pSae2, red), and Sae2 prepared with PIs and then treated with λ phosphatase (pSae2 λ, green). The black rectangle indicates the position of the λ phosphatase peak. i, j Representative transmission electron microscopic images of Sae2 prepared without (Sae2, 800 nM, i) and with PIs (pSae2, 800 nM, j). Scale bar indicates 100 nm. k Representative atomic force microscopic image of pSae2 (50 nM). Examples of tetrameric proteins are circled. Scale bar indicates 100 nm. l Frequency distribution of pSae2 particle volumes based on experiments such as shown in k. Mean volume was calculated from nonlinear Gaussian fit (black curve). Error bars, SEM; 1543 particles were analyzed