Supplementary information
Crystal structure of At5g06450, a putative DnaQ-like exonuclease domain-containing
protein from Arabidopsis thaliana, with novel homohexameric assembly
Running title: Crystal structure of AtDECP
David W. Smith,1* Mi Ra Han,2* Joon Sung Park,2 Kyung Rok Kim,2 Taeho Yeom,3 Ji Yeon
Lee,2 Do Jin Kim,2 Craig A. Bingman,1 Hyun-Jung Kim,4 Kyubong Jo,3 Byung Woo Han,2†
George N. Phillips, Jr.1,5 †
1
Center for Eukaryotic Structural Genomics, Department of Biochemistry, University of
Wisconsin-Madison, Madison, Wisconsin 53706, USA
2
Research Institute of Pharmaceutical Sciences, College of Pharmacy, Seoul National
University, Seoul 151-742, Korea
3
Department of Chemistry, Sogang University, Seoul 121-742, Korea
4
College of Pharmacy, Chung-Ang University, Seoul 156-756, Korea
5
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251, USA
*
David W. Smith and Mi Ran Han contributed equally to this work.
†
Correspondence to: Byung Woo Han, College of Pharmacy, Seoul National University,
Seoul 151-742, Korea. E-mail: bwhan@snu.ac.kr or George N. Phillips, Jr., Department of
Biochemistry and Cell Biology, Rice University, Houston, Texas 77251, USA. E-mail:
georgep@rice.edu
1
Size-exclusion chromatography profile analyses
When we purified the His6-tagged AtDECP, we observed both hexameric and monomeric
AtDECP fractions from the size-exclusion chromatography (HiLoadTM 16/600 Superdex200
pg, GE Healthcare) after Ni-NTA affinity chromatography. Two major fractions were eluted
at the elution volume 67 mL and 123 mL. Calculated from the gel filtration protein standard
(Bio-Rad), the fraction eluted at 67 mL corresponds to ~135 kDa, which is similar to the
expected size of the hexameric AtDECP (~25 kDa/monomer x 6 = ~150 kDa). Even though
the fraction eluted at 123 mL was calculated as much smaller protein than the AtDECP
monomer, it was validated that the fraction contained the intact AtDECP monomer on SDSPAGE [Fig. S1].
Figure S1. Gel filtration chromatography profile and SDS-PAGE results of His6-tagged
AtDECP after Ni-NTA affinity chromatography. The profile shows both hexameric and
monomeric peaks of AtDECP. Red and blue inverted triangles represent the hexameric and
monomeric AtDECP fractions, respectively.
Interestingly, the monomeric AtDECP faction disappeared after the subsequent ion
exchanged chromatography (HiTrap Q column, GE Healthcare) and most fractions were
eluted as hexameric AtDECP fractions, which show the in vitro transition from monomer to
hexamer AtDECP [Fig. S2].
2
Figure S2. Gel filtration chromatography profile and SDS-PAGE results of the His6tagged monomeric AtDECP after the anion exchange chromatography. The profile
shows a dominant hexameric AtDECP peak. Red inverted triangle represents the hexameric
AtDECP fraction.
In the case of the purification of the His6-maltose binding protein (MBP)-tagged AtDECP, we
observed the only hexameric fraction from the size-exclusion chromatography after the
removal of the His6-MBP tag using TEV proteases followed by second Ni-NTA affinity
chromatography [Fig. S3]
Figure S3. Gel filtration chromatography profile and SDS-PAGE results of the His6MBP-tagged AtDECP after the removal of His6-MBP tag using TEV proteases followed
by second Ni-NTA affinity chromatography. The profile shows a dominant hexameric
AtDECP peak. Red inverted triangle represents the hexameric AtDECP fraction.
3
Exonuclease activity assay of AtDECP
To measure the exonuclease activity of AtDECP, we constructed and purified both His6MBP-tagged and His6-tagged AtDECP proteins. The purified AtDECP was co-incubated with
oligonucleotides at 37 ℃ for 1 hour; AtDECP 21 μL and 33 μM 5’-overhang or gapped
dsDNA 9 μL (final concentration of dsDNA = 10 μM). Positive control is 0.27 μM Klenow
fragment (New England Biolabs) and is co-incubated with 10 μM dsDNA in the 1X reaction
buffer (New England Biolabs) containing 50 mM NaCl, 10 mM Tris-HCl pH 7.9, 10 mM
MgCl2, and 1 mM DTT at 37 ℃ for 30 min. The sequences of the co-incubated dsDNA are
shown below.
5'-overhang dsDNA for Figure S4;
5'- GTACGACTGCAGGGA - 3’
3'- CATGCTGACGTCCCTAGGAGCGCTTGTACCG -5'
Gapped dsDNA by one nucleotide for Figure S5;
5'- GTACGACTGCAGGGA CCTCGCGAACATGGC-3'
3'- CATGCTGACGTCCCTAGGAGCGCTTGTACCG-5'
Co-incubated samples were mixed with matrix solution onto Bruker MTP 384 target plate
polished steel MALDI sample support. The matrix solution contains 350 mM 3hydroxypicolinic acid (Tokyo Chemical Industry, Tokyo, Japan), 80 mM pyrazinecarboxylic
acid, and diammonium hydrogen citrate in 50% acetonitrile. MALDI-TOF MS analysis was
performed with a Bruker AutoflaxTM speed machine (Bruker Daltonics Inc., Billerica, USA)
in the linear positive ion mode using FlexControl 3.3 software for the automatic acquisition.
4
For the MALDI-TOF MS analysis with 3’-overhang dsDNA, AtDECP was co-incubated with
oligonucleotides at 37 ℃ for 30 min; AtDECP 3 μL, 33 μM 3’-overhang DNA 9 μL (final
concentration of dsDNA = 10 μM), and reaction buffer 18 μL containing 10 mM Tris-HCl
pH7.9, 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT. Positive control is T5 exonuclease
(New England Biolabs) 1 unit in the same reaction condition. The sequences of co-incubated
oligonucleotides are shown below.
3’-overhang dsDNA for Figure S6;
5' -CCTCGCGAACATGGC- 3'
3' -CATGCTGACGTCCCTGGAGCGCTTGTACCG- 5'
The Klenow fragment showed 3’-5’ exonuclease activity with the presence of 5’-overhang
and T5 exonuclease showed 5’-3’ exonuclease activity. However, in the case of AtDECP, the
smaller fragment peaks, from the cleaved oligonucleotides due to the exonuclease activities,
were not detected and the exonuclease activities of AtDECP could not be observed [Fig. S4S6].
5
Figure S4. MALDI-TOF MS detection of cleaved DNA from 5’-overhang dsDNA. ‘*’
represents the shorter strand of dsDNA and ‘•’ represents the longer strand of dsDNA. (A)
Hexameric AtDECP from the His6-MBP-tagged construct after the His6-MBP-tag removal
(concentration: 3.129 mg/ml, buffer condition: 50 mM KH2PO4 pH 7.0, 150 mM NaCl, 10
mM MgCl2, 5 mM DTT, and 5% glycerol). (B) Monomeric AtDECP from the His6-tagged
construct (concentration: 2.246 mg/ml, buffer condition: 10mM Tris-HCl pH 7.5, 100 mM
NaCl, 1 mM EDTA, 0.1 mM DTT, and 5% Glycerol) (C) Hexameric AtDECP from the His6tagged construct (concentration: 0.24 mg/ml, buffer condition: 10mM Tris-HCl pH 7.5, 100
mM NaCl, 1 mM EDTA, 0.1 mM DTT, and 5% Glycerol). (D) Positive control, Klenow
fragment (New England Biolabs).
6
Figure S5. MALDI-TOF MS detection of cleaved DNA from gapped dsDNA by one
nucleotide. ‘*’ represents two shorter strands of dsDNA and ‘•’ represents the longer strand
of dsDNA. (A) Hexameric AtDECP from the His6-MBP-tagged construct after the His6MBP-tag removal (concentration: 3.129 mg/ml, buffer condition: 50 mM KH2PO4 pH 7.0,
150 mM NaCl, 10 mM MgCl2, 5 mM DTT, and 5% glycerol). (B) Monomeric AtDECP from
the His6-tagged construct (concentration: 2.246 mg/ml, buffer condition: 10mM Tris-HCl pH
7.5, 100 mM NaCl, 1 mM EDTA, 0.1 mM DTT, and 5% Glycerol) (C) Hexameric AtDECP
from the His6-tagged construct (concentration: 0.24 mg/ml, buffer condition: 10mM Tris-HCl
pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.1 mM DTT, and 5% Glycerol).
7
Figure S6. MALDI-TOF MS detection of cleaved DNA from 3’-overhang dsDNA. ‘*’
represents the shorter strand of dsDNA and ‘•’ represents the longer strand of dsDNA. (A)
Hexameric AtDECP from the His6-tagged construct after the ion-exchange chromatography
(concentration: 0.148 mg/mL, buffer condition: 100 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1
mM DTT, and 5% glycerol) (B) Reconstituted hexameric AtDECP from the His6-tagged
construct after the ion-exchange chromatography (concentration: 0.500 mg/mL, buffer
condition: same as in A). (C) Hexameric AtDECP from the His6-tagged construct after the
initial Ni-NTA affinity chromatography (concentration 0.594 mg/ml, buffer condition: same
as in A). (D) Monomeric AtDECP from the His6-tagged construct after the initial Ni-NTA
affinity chromatography (concentration: 9.932 mg/ml, buffer condition: same as in A). (E)
Hexameric AtDECP from the His6-MBP-tagged construct after the His6-MBP-tag removal
(concentration: 1.301 mg/ml, buffer condition: 150 mM NaCl and 50 mM KH2PO4, pH 7.0).
(F) Positive control, 1 unit of T5 exonuclease (New England Biolabs).
8
dsDNA binding assay of AtDECP
We implemented the EMSA to check if the purified AtDECP could bind with dsDNA in
several different conditions. Three different types of dsDNA with 3’-overhang, 5’-overhang,
and blunt-end were manufactured using oligo synthesis service (Cosmogenetech) and each
overhang dsDNA contained five extra bases.
Five AtDECP samples same as in Figure S6 were co-incubated with 3’-overhang dsDNA at
37 ℃ for one hour. Co-incubated samples were mixed with 6x DNA loading dye and
electrophoretically eluted on 15% polyacrylamide gel. After the electrophoresis, the gel was
visualized using TAE buffer with EtBr. We could not observe a gel shift of dsDNA from the
interaction of AtDECP with dsDNA [Fig. S7].
The hexameric AtDECP sample, which was reconstituted from the His6-tagged construct
after the ion-exchange chromatography, was co-incubated with three different types of
dsDNA (5’-overhang, 3’- dsDNA, and blunt-ended) at 37 ℃ for one hour. Co-incubated
samples were mixed with 6x DNA loading dye and electrophoretically eluted on 15%
polyacrylamide gel. After the electrophoresis, the gel was visualized using TAE buffer with
EtBr. We could not observe a gel shift of dsDNA from the interaction of AtDECP with
dsDNA [Fig. S8].
9
Lane
1
Lane
2
Lane
3
Negative
control
Mg2+
D.W.
10x buffer
dsDNA
AtDECP
Total (μL)
28
2
30
+
25
3
2
30
Lane
4
Lane
5
A
27
2
1
30
Lane
6
Lane
7
B
+
24
3
2
1
30
27
2
1
30
Lane
8
Lane
9
C
+
24
3
2
1
30
27
2
1
30
Lane
10
Lane
11
D
+
24
3
2
1
30
27
2
1
30
Lane
12
E
+
24
3
2
1
30
27
2
1
30
+
24
3
2
1
30
Figure S7. Gel shift assay of dsDNA with AtDECP : (A) Hexameric AtDECP from the
His6-tagged construct after the ion-exchange chromatography (concentration: 0.148 mg/mL,
buffer condition: 100 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1 mM DTT, and 5% glycerol) (B)
Reconstituted hexameric AtDECP from the His6-tagged construct after the ion-exchange
chromatography (concentration: 0.500 mg/mL, buffer condition: same as in A). (C)
Hexameric AtDECP from the His6-tagged construct after initial Ni-NTA (concentration 0.594
mg/ml, buffer condition: same as in A). (D) Monomeric AtDECP from the His6-tagged
construct after initial Ni-NTA (concentration: 9.932 mg/ml, buffer condition: same as in A).
(E) Hexameric AtDECP from the His6-MBP-tagged construct after the His6-MBP-tag
removal (concentration: 1.301 mg/ml, buffer condition: 150 mM NaCl and 50 mM KH2PO4,
pH 7.0). 10x buffer condition: 100 mM Tris, pH 7.5, 500 mM KCl, 10 mM DTT, 50 mM
MgCl2.
10
Lane
1
Lane
2
Lane
3
Lane
4
Lane
5
Lane
6
Lane
7
Lane
8
Negative control
Lane
9
Lane
10
Lane
11
Lane
12
Protein
dsDNA/
Mn2+
5’/
-
5’/
+
3’/
-
3’/
+
Gap/
-
Gap/
+
5’/
-
5’/
+
3’/
-
3’/
+
Gap/
-
Gap/
+
DW
10x buffer
dsDNA
AtDECP
25
3
2
-
25
3
2
-
25
3
2
-
25
3
2
-
25
3
2
-
25
3
2
-
23
3
2
2
23
3
2
2
23
3
2
2
23
3
2
2
23
3
2
2
23
3
2
2
Total (μL)
30
30
30
30
30
30
30
30
30
30
30
30
Figure S8. Gel shift assay of three different types of dsDNA with the hexameric
AtDECP. The protein used in this assay was the reconstituted hexameric AtDECP from the
His6-tagged construct after the ion-exchange chromatography (concentration: 0.500 mg/mL,
buffer condition: 100 mM NaCl, 10 mM Tris-HCl pH 7.5, 0.1 mM DTT, and 5% glycerol).
10x buffer condition without Mn2+: 100 mM Tris, pH 7.5, 500 mM KCl, 10 mM DTT. 10x
buffer condition with Mn2+: 100 mM Tris, pH 7.5, 500 mM KCl, 10 mM DTT, 50 mM
MnCl2.
11