SUPPLEMENTARY DATA
Divergent β-hairpins determine double-strand versus single strand substrate recognition of human AlkB-homologues 2 and 3
Vivi Talstad Monsen, Ottar Sundheim, Per Arne Aas, Marianne P. Westbye, Mirta ML Sousa, Geir Slupphaug & *Hans E. Krokan Department of Cancer Research and Molecular Medicine, Faculty of Medicine, Norwegian University of Science and Technology, N-7489 Trondheim, Norway * Corresponding author: Hans E. Krokan, Telephone +47 72 57 30 74 Telefax: +47 72 57 64 00 E-mail:
[email protected]
Monsen et al.
Supplementary table and figures Table S1. Sequences of primers used to produce deletion mutants and hybrid proteins of ABH2 and ABH3.
ABH2-F1AB3
ABH3-F1AB2
ABH3 wt
ABH2 wt
Primer sequence 5'P-GTTCCCTGGAAACAGAGGACC-3' (forward) 5'P-TTGCTGATAAGTTATATCCTCTCTGATGCC-3' (reverse) 5'-CCCAGGAAGCAGGCAACGTATGGC-3' (forward) 5'-TTCTTTCTCCAACTCTTGGAAAATCTCATC-3' (reverse) 5’-GGAACAGCTTTGTCAAGATGTAGAATATTTTACAGGAGCA-3’ (forward) 5'-CATGCTGTAAGTCTTGGCACACTGTGCCACTTCCCGAATA-3' (reverse) 5'-TGCTCCTGTAAAATATTCTACATCTTGACAAAGCTGTTCC-3' (forward) 5'-TATTCGGGAAGTGGCACAGTGTGCCAAGACTTACAGCATG-3' (reverse) 5'-CCAAGACTTACAGCATGGTATGGAGAACTT-3' (forward) 5'-ATCTTGACAAAGCTGTTCCAATATCCAGTC-3' (reverse)
Marker
Primer 1 2 3 4 5 6 7 8 9 10
kDa
64 51 39 28
Figure S1. Purification of wild type ABH2 and ABH3 and hybrid proteins. Purified wild type and hybrid proteins of ABH2 and ABH3 were run on a 4-12% denaturing polyacrylamide gel and coomassie stained. SeeBlue® Plus Pre-Stained Standard (Invitrogen, lane 1), wt ABH2 (lane 2), wt ABH3 (lane 3), ABH3-F1AB2 (lane 4) and ABH2-F1AB3 (lane 5).
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ABH2
Enzyme
0 0.3 1
pmol
ABH3
3 10 0.3 1
ABH3-F1AB2 ABH2-F1AB3
3 10 0.3 1
3 10 0.3 1
3 10
3-meC in ssDNA 3-meC in dsDNA
Figure S2. Repair activity of wild type ABH2 and ABH3 and hybrid proteins. Repair of single- and double stranded DNA substrate containing 3-meC by wild type ABH2 (panel 2), wild type ABH3 (panel 3), ABH3 hybrid protein with ABH2 F1 (ABH3-F1AB2, panel 4) and ABH2 hybrid protein with ABH3 F1 (ABH2-F1AB3, panel 5). Increasing amounts of enzymes (0, 0.3, 1, 3, 10 pmol) were incubated with 1 pmol of 3-meC containing substrate for 20 min and demethylase activity (repair) measured as generation of cleavable substrate (lower of two bands) after 12% denaturing PAGE. Gels from typical repair assays are illustrated.
2OG turnover by ABH2 and ABH3 wt, and ABH2 and ABH3 with F1 deleted
dpm (released
14
CO2 )
1200
ABH2 ABH2Δ89-108 ABH3 ABH3Δ113-129
900 600 300 0 0
50 Enzyme (pmol)
100
Figure S3. Turnover of 2OG by wild type- and F1 deletion mutants of ABH2 and ABH3. Decarboxylation of 2OG by deletion mutants and wt ABH2 and ABH3 was determined by measuring the release of [14CO2]. 2OG (100 µM, containing 10% 1 -[14C]2OG (54.5 mCi mCi /mmol specific activity)) was assayed with 10, 50 and 100 pmol enzyme.
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F1 90 NP_001001655.1/Homo sapiens (89-135) XP_001104262./Macaca mulatta (89-135) XP_001500924./Equus caballus (88-134) NP_001019687.1/Bos taurus (88-134) XP_534719.2/Canis familiaris (87-133) NP_001119745./Rattus norwegicus (67-113) NP_778181.2/Mus musculus (67-113) XP_001508166./Ornithorynchus anatinus (97-143) XP_415188.2/Gallus gallus (68-114) XP_002194047./Taeniopygia guttata (68-114) XP_697761.1/Danio rerio (79-125) CAG08511.1/Tetraodon nigroviridis (78-124) XP_797704.1/Strongylcentrotus purpuratus (93-139) XP_001634156./Nematostella vectensis (42-88) XP_002221439./Brainchiostoma floridae (96-142) XP_973954.2/Tribolium castaneum (24-70) XP_001951457./Acyrthosiphon pisum (52-98)
VE VE VE VE VE VE VE VE VE VE VE LV LE I K L I VE VS
100 YF YF YF YF YF YF YF YF YF YF YF YA YF YD YN YL YF
TG AL TG AL TG AL TG AL TG AL TG AL TG AL TG DL E G EQ ED E L T G DN TG E E TG DL S - -V TG DL DG D L DG R L
A R VQ V F GK WH S V P T R V Q V F G KW H S V P A R VQ V F GK WH NV P A R VQ V F GK WH S V P A R VQ V F GK WH S V P A K VQ V F GK WH S V P A K VQ V F GK WH S V P S K VQ V F GK WH S I P T K L H V F GK WH N I P T K LQ V F GT WH K I P AK LQ V Y GK S Y N I P A T VQ V F GK V Y S I P A R I K V Y GK WH D I P S K V F V F GK Y H S V P A K V Q I F GQ F H N I P S K V R V F GK WHQ I P S Q V K V F GQ Y Y P I P
F1 120 NP_631917.1/Homo sapiens (113-154) XP_001113825./Macaca mulatta (113-154) NP_001032691.1/Bos taurus (113-154) XP_001489744./Equus caballus (113-154) XP_533147.2/Canis familiaris (148-189) NP_081220.1/Mus musculus (113-154) NP_001014202.1/Rattus norwegicus (113-154) XP_001367723./Monodelphis domestica (132-173) XP_421095.2/Gallus gallus (160-201) XP_002198596./Taeniopygia guttata (165-206) NP_001088803./Xenopus laevis (105-146) XP_002228829./Brainchiostoma floridae (116-158) XP_001510113./Ornithorhynchus anatinus (164-210) NP_001003511.1/Danio rerio (102-144) XP_001631257./Nematostella vectensis (103-143) XP_002119223./Ciona intestinalis (120-161) CAG01043.1/Tetraodon nigroviridis (59-100)
VP VP VP VP VP VP VP I P VP I P I P I P RV LP I P VQ LP
WKQ R T G WKQ R T G WKQ R T G WKQ R T G WKQ R T G WK Q R MG WK Q R MG WKQ R T G WGQ R T H WGQ R T H WR Q K T N WK Q R K G I E NR D S WSQK TN WE EK D I WEHRRN WSQK TN
ABH2 110
F2 120
130
R KQ A T Y G DA G L T Y T F S G L T L S P K P W - R K QA T Y GD AG L T Y T F S G L T L S P K P W - R KQ A T Y G D T G L T Y T F S G L T L S P K P W - R KQ A T Y G D T G L T Y T F S G L T L S P K P W - R KQ A T Y G NA G L T Y T F S G L T L S P K P W - R KQ A T Y G DA G L T Y T F S G L T L T P K P W - R KQ A T Y G DA G L T Y T F S G L T L T P K P W - R KQ A T Y G D S G L S Y T F S G L T L S P K P W - R KQ V T Y G Y P E L T Y T Y S G V T F S P K P W - R K K V T Y GD P G L S Y T Y S G V T F HP K P W - R KQ A T Y G D E G L MY S F S G V N L L A K P W - R KQ A T Y G DA G L T Y T Y S G I T RM A C P W - R KQ V A HG D T G L T Y K Y S G V T V P A K P W - R R Q T S F G D E G L K Y T F S G V T V T P Q HW K E R KQ V A F G DP G L S Y R F S G V E V P A R P W - R QQ A A Y G D Q G T V Y K F S G T S I P C K P W - R Q Q V A F G D A G L L Y K F S G T V V P AQ P W - -
ABH3 130
I R E D - - - - - I T YQ QP I R E D - - - - - I T YQ QP I R DD - - - - - V T Y QQ P I R E N - - - - - V T Y QQ P I R E D - - - - - VT Y KQ P I R E D - - - - - VT Y P Q P I R ED - - - - - I T Y PQP I R E D - - - - - T S Y LQ P TR QE - - - - - GS F E E P VR Q E - - - - - I S F E EP VG P D - - - - - G SY HE P I DR E - - - - GV E Y L QP A H A E K R A F G V I Y QQ P H R MM - - - - G D A Y E E P K I K G - - - - - E F HK Q P L K Y G - - - - - PNS ME P YR Q - - - - - GEA Y E EP
R R R R R R R R R R R R R R R R R
F2 140
L T L T LT LT LT LT L T LT LT L T LT L T LT L T L T LT L T
AWY G E AWY G E AW Y G E AW Y G E AW Y G E AW Y G E AWY G E AW Y G E SWYGE S WY G E CW Y G E AWF G E AW Y G E C WY G E AWF G E AW F S E C WY G E
I I I I I I V I I I T T T A T T P
LP YT Y LP YT Y LP YT Y LP YT Y LP YT Y LP YT Y LP YT Y LP YT Y LP YT Y LP YT Y VP YT Y LP Y S Y LP YT Y LP YT Y FP YT Y F SY SY LP YT Y
150 SR I SR I SR I SR I SR I SR I S RV SR I SR I SR I S HS SRL S RV SRS S GL SGV SRS
T M E P N P HW T M E P N P HW T M E P N P HW T M E P N P HW T M E P N P HW T M E P N P HW T M E P N P HW T M E P N P QW T M Q P N P NW T M Q P N P NW T M Q A N P HW T H E A NP H F T M E P N P QW T M E A N A QW T L R P - F QW VQ P P N P HW T M A A N AQ W
Figure S4. Alignment of F1 and F2 in orthologs of ABH2 and ABH3. Amino acid sequences of F1 and F2 segments in ABH2 (upper panel) and ABH3 (lower panel) of various species are displayed. The alignment is arranged in descending order of sequence similarity to the human protein. Species, accession number and amino acids included are indicated to the left of each sequence. Secondary structures are indicated by red cylinders (α-helix) and blue arrows (β-sheet). Individual residues within each motif are coloured according to ClustalW colour coding and the alignment was made using ClustalW and JalView (1).
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2OG decarboxylation 3-meC ssDNA demethylation
Repair relative to ABH2
1.4 1.2 1.0 0.8 0.6 0.4 0.2
3 AB 2F1
A
BH
L1 57 A
L1 29 A
L1 27 A
F1 24 A
10 8T
/F
V
10 2A
99 T V V
10 1A
99 E V
w tA B
H 2
0
Figure S5. Comparison of 2OG dearboxylation and 3-meC demethylation activity, by mutants of ABH2. Dearboxylation of 2OG was determined for mutants of ABH2 and compared to decarboxylation activity of wild type ABH2. Enzymes (100 pmol) were incubated with 100 μM 2OG containing 10% 1-[14C]2OG and the release of [14CO2] was measured, as described in materials and methods. Demethylation of 3-meC in ssDNA was determined for mutants of ABH2 and compared to repair activity of wild type ABH2. Demethylation was assayed as described in materials and methods, using 1 pmol of 3-meC containing ssDNA substrate and 1 pmol enzyme.
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ABH2 ABH3 ABH1 FTO
89-110 113-131 121-163 72-93
V V L HK
- - - HG
EY FT P WKQ Y SQ K CL FR
GA - - - -
L A R V QV F G KWH S V P - R T G I R ED I T Y QQP - P NV CN L D K H M S K E - D LV R I QG KD L L T P
- - ET - -
- - QD - -
- - - - - - LW EQ - - - -
- - SK - -
- - - - - - - - - - E F LRYK - - - - - -
- - - - - - EA TK - - - -
- - -R - - -R RRPR - - -V
Figure S6. Sequence alignment of the F1 loops in ABH2, ABH3, ABH1 and FTO. Secondary structure alignment of ABH2 (PDBid 3BUC), ABH3 (PDBid 2IUW) and FTO (PDBid 3LFM) was generated using TM-align (2) in STRAP (3). The amino acids in the F1 loop of ABH1 (NP_006011.2) were manually inserted according to structural prediction of ABH1 and sequence alignment reported in Westbye et al. (4). The final alignment was made in JalView (1) and colour coded according to the Percentage Identity colour scheme.
Supplementary references 1. 2. 3. 4.
Clamp, M., Cuff, J., Searle, S.M. and Barton, G.J. (2004) The Jalview Java alignment editor. Bioinformatics, 20, 426-427. Zhang, Y. and Skolnick, J. (2005) TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res., 33, 2302-2309. Gille, C. and Frommel, C. (2001) STRAP: editor for STRuctural Alignments of Proteins. Bioinformatics, 17, 377-378. Westbye, M.P., Feyzi, E., Aas, P.A., Vagbo, C.B., Talstad, V.A., Kavli, B., Hagen, L., Sundheim, O., Akbari, M., Liabakk, N.B., Slupphaug, G., Otterlei, M. and Krokan, H.E. (2008) Human AlkB homolog 1 is a mitochondrial protein that demethylates 3methylcytosine in DNA and RNA. J. Biol. Chem., 283, 25046-25056.
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