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Supplemental Material AdnAB: a Novel DSB-resecting Motor-Nuclease from Mycobacteria Krishna Murari Sinha, Mihaela-Carmen Unciuleac, Michael S. Glickman, and Stewart Shuman Supplemental Figures S1, S2, S3, S4, S5 Supplemental Table S1

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Figure S1. Mycobacterial AdnAB. The amino acid sequences of M. smegmatis (Ms) AdnA and

AdnB are aligned to each other. The N-terminal motor domains are aligned with the sequence of

the E. coli (Ec) UvrD helicase. The ATPase/helicase motifs are denoted in boxes and named

according to Lee and Yang (2006). The amino acids at the ATPase active site and DNA-binding

site of UvrD that are conserved in AdnA or AdnB are highlighted in yellow. The C-terminal

nuclease domains of AdnA and AdnB contain counterparts of the three active site motifs of the

RecB nuclease (shaded in gray). Amino acids in the nuclease motifs of AdnAB that were subjected

to alanine scanning are denoted by |. Positions of side chain identity/similarity in all of the aligned

proteins are indicated by •. Gaps in the alignments are denoted by –.

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Figure S2. pH profile of AdnAB dsDNA exonuclease. Reaction mixtures (10 µl) containing 20

mM Tris buffer (either Tris-acetate pH 4.5 to 7.0 or Tris-HCl pH 7.5 to 9.5), 2 mM MgCl2, 1 mM

ATP, 200 ng linear pUC19 (SmaI-digested), and AdnAB (10 ng AdnB) were incubated for 5 min at

37˚C. The reaction products were analyzed by electrophoresis through a 0.7% native agarose gel

and visualized by staining with ethidium bromide. The positions and sizes (kbp) of linear DNA

markers are indicated on the right.

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Figure S3. Stimulation of ATP hydrolysis by ssDNA. Reaction mixtures (10 µl) containing 20

mM Tris-HCl (pH 8.0), 1 mM [γ32P]ATP, 1 mM MgCl2, AdnAB (20 ng AdnB), and 24mer ssDNA

oligonucleotide (5’-GCCCTGCTGCCGACCAACGAAGGT) as specified were incubated for 10 min

at 37˚C. 32Pi release is plotted as function of the amount of ssDNA added. An apparent Km of 1 µM

24mer DNA was calculated in Prism by nonlinear regression curve fitting of the data.

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Figure S4. Divalent cation requirements for AdnAB ssDNase activity. (A) Metal specificity. Reaction mixtures (10 µl) containing 20 mM Tris-HCl (pH 8.0), 0.1 µM 5’ 32P-labeled 24-mer DNA, 1 mM ATP, AdnAB (7 ng AdnB), and 2 mM of the indicated divalent cation (as the chloride salt) were incubated for 5 min at 37°C. Divalent cation was omitted from a control reaction in lane –. (B) Metal mixing. Reaction mixtures (10 µl) containing 20 mM Tris-HCl (pH 8.0), 0.1 µM 5’ 32P-labeled 24-mer DNA, 1 mM ATP, AdnAB (7 ng AdnB), 2 mM MgCl2, and 2 mM of the indicated divalent cation were incubated for 5 min at 37°C. (C) Manganese dependence. Reaction mixtures (10 µl) containing 20 mM Tris-HCl (pH 8.0), 0.5 mM DTT, 0.1 µM 5’ 32P-labeled 24-mer DNA, either no nucleotide or 1 mM ATP, AdnAB (10 ng AdnB), and MnCl2 as specified were incubated for 5 min at 37˚C.

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Figure S5. Isolated AdnB and AdnA subunits. The AdnB and AdnA proteins were produced

singly in E. coli as His10Smt3 fusions and purified from soluble bacterial lysates by serial Ni-

agarose and DEAE-Sephacel chromatography, followed by Ulp1 cleavage, and isolation of the tag-

free Adn polypeptides after a second round of Ni-agarose chromatography. The recombinant

polypeptides were mixed with sedimentation standards catalase (native size 248 kDa; a

homotetramer of a 62 kDa polypeptide), BSA (66 kDa) and cytochrome c (12 kDa). The mixtures

were analyzed by zonal velocity sedimentation in a 15-30% glycerol gradient (see Experimental

Procedures). Aliquots (15 µl) of the odd-numbered gradient fractions were analyzed by SDS-

PAGE. Panel A shown the sedimentation profile of AdnB, which comprises a discrete monomeric

peak on the “heavy” side of BSA. Panel B shows the sedimentation profile of AdnA, which consists

of a mixture of monomeric and oligomeric species (in fractions embraced by the arrows). The

sedimentation profile of AdnB alone is shown in C (top panel), wherein aliquots (15 µl) of the odd-

numbered fractions were analyzed by SDS-PAGE. The ATPase activity profile in shown in the

bottom panel of C. Reaction mixtures (10 µl) containing 20 mM Tris-HCl (pH 8.0), 0.5 mM MnCl2,

0.1 mM [α32P]ATP, 1 µg salmon sperm DNA, and 2 µl of the indicated gradient fractions were

incubated for 60 min at 37˚C. The extents of 32P-ADP formation were determined by TLC.

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Table SI. AdnAB homologs in Actinomycetales

Species AdnA (aa) AdnB (aa) RecBCD Mycobacterium smegmatis 1045 1095 + Mycobacterium tuberculosis 1055 1101 + Mycobacterium sp. KMS 1051 1091 + Mycobacterium vanbaalenii 1038 1086 + Mycobacterium gilvum 1038 1091 + Mycobacterium avium 1044 1096 + Mycobacterium bovis BCG 1055 1101 + Mycobacterium ulcerans 1057 1101 + Mycobacterium abscessus 1058 1078 + Mycobacterium marinum 1057 1101 + Acidothermus cellulolyticus 1095 1164 – Arthrobacter sp. FB24 1150 1183 – Arthrobacter aurescens 1118 1167 – Brevibacterium linens 1086 1061 – Corynebacterium glutamicum 1016 1070 – Corynebacterium diphtheriae 1060 1076 – Corynebacterium efficiens 1024 1175 – Clavibacter michiganensis 1089 1091 – Frankia alni ACN14a 1170 1208 – Janibacter sp. HTCC2649 1047 1103 – Kineococcus radiotolerans 1098 1128 + Kocuria rhizophila 1241 1145 – Leifsonia xyli 1043 1125 – Micrococcus luteus 1145 1176 – Nocardia farcinica 1255 1184 + Propionibacterium acnes 1061 1072 + Rhodococcus sp. RHA1 1098 1115 + Saccharopolyspora erythraea 1051 1078 – Salinispora tropica 1144 1144 – Salinispora arenicola 1096 1162 – Streptomyces avermitilis 1129 1202 – Streptomyces coelicolor 1159 1222 – Thermobifida fusca 1044 1108 – Tropheryma whipplei 993 973 –