Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

Simon et al. 10.1073/pnas.0706067105.

Supporting Figures

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SI Figure 7
SI Figure 8
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Fig. 7. Prediction of disordered regions in the sequence of UmuD'2 (A) and UmuD2 (B) by using PONDR protein disorder-prediction programs VLXT (solid black line), XL1-XT (dashed line), and CAN-XT (solid gray line). Residues with a PONDR score of >0.5 using any of these programs are predicted to be disordered. The dip in VL-XT score centered around residue 111 may represent a molecular recognition element; such signals are typical of protein regions that undergo a disorder-to-order transition upon ligand binding. NMR structure of UmuD'2 (C) and model of UmuD2 (D) colored according to predicted disorder. Regions of predicted disorder are shown in blue; those residues predicted to be ordered are colored yellow. N termini are at the top of the images.





Fig. 8. (A) UmuD2 is active for in vitro RecA:ssDNA-mediated cleavage to yield UmuD'2. RecA:ssDNA was formed from RecA, ssDNA, and ATP-g-S and added to 5 mM UmuD2. Aliquots were removed at t = 0 h (lane 1) and t = 1 h (lane 2), after which most UmuD2 had been converted to UmuD'2. (B) Both UmuD2 and UmuD'2 are active for heterodimer formation and ClpXP proteolysis. UmuD2 and UmuD'2 were coincubated for 30 min to allow formation of the UmuD'D heterodimer and added to ClpXP protease and ATP. Aliquots were removed at t = 0 h (lane 1) and t = 2 h (lane 2), after which most UmuD'2 had been degraded by ClpXP. Assay procedures are found in ref. 1.

1. Beuning PJ, Simon SM, Godoy VG, Jarosz DF, Walker GC (2006) Characterization of Escherichia coli translesion synthesis polymerases and their accessory factors. Methods Enzymol 408: 318-340.





Fig. 9. The CD spectrum of 5 mM UmuD'2 (A) or 5 mM UmuD2 (B) in the absence (dashed lines) or the presence (solid lines) of 2.5 M glucose.





Fig. 10. (A) Superdex 75 gel filtration of 5 mM UmuD'2 (black solid line) or 5 mM UmuD2 (black dashed line) as compared to size standards (gray solid line). Size standards are as follows: BSA (dimer of 132 kDa eluting at 34 ml and monomer of 66 kDa eluting at 41 ml), ovalbumin (43 kDa eluting at 46 ml), chymotrypsin (26 kDa eluting at 55 ml), and lysozyme (14 kDa eluting at 75 ml). (B) Lysozyme, chymotrypsin, and 5 mM UmuD'2 or UmuD2 were denatured in 6 M guanidinium for 2 h before loading and elution from a Superdex 75 gel-filtration column in buffer containing 6 M guanidinium hydrochloride. Note that all proteins eluted at a lower volume than their native counterparts in A. (C) Native gel electrophoresis of 500 nM (lanes 1-5) or 5 mM (lanes 7-11) UmuD2 (lanes 1 and 7), UmuD'2 (lanes 2 and 8), UmuD'D (lanes 3 and 9), cross-linked UmuD (F94C)2 (lanes 4 and 10), and mock-treated UmuD(F94C)2 (lanes 5 and 11).

This Article

  1. Published online before print January 23, 2008, doi: 10.1073/pnas.0706067105 PNAS January 29, 2008 vol. 105 no. 4 1152-1157
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