Supplementary Information for:
Structure and Specificity of FEN-1 from Methanopyrus kandleri
Santosh Shah, Pete Dunten, Amanda Stiteler, Chad K. Park and Nancy C. Horton
MATERIALS AND METHODS
Protein Purification
A synthetic codon optimized gene for Mk0566 (coding sequence based on Methanopyrus
kandleri genome sequence, Genbank number AE009439.1) was purchased (Biobasic, Inc.) and
cloned into the pMAL-c4X expression vector (New England Biolabs, Inc.) using the XbaI and
HindIII restriction sites. The protein is expressed as a fusion with an N-terminal E. coli maltose
binding protein (MBP) separated from the Mk0566 protein by a linker containing the cleavage site
for the protease Factor Xa. The cloning produced an additional seven amino acids N-terminal to
the first methionine residue of Mk0566 consisting of the sequence I-S-E-F-G-S-S, which remain
on the protein following cleavage with Factor Xa. The expression plasmid was transformed into
Top 10 E. coli cells (F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacΧ74 recA1 araD139
Δ(ara-leu) 7697 galU galK rpsL (StrR) endA1 nupG λ-)(Life Technologies) grown overnight in a
3 ml culture, and then mixed with glycerol, flash frozen, and stored at -80°C. Cells from the
glycerol stock were later streaked on an LB agar plate containing 50 µg/ml ampicillin and grown
overnight at 37ºC. A 3-ml culture was grown from a single colony and used to innoculate an 18-L
culture at 37°C. Expression of Mk0566 was induced with 0.4 mM IPTG when the OD600 of the
culture reached 0.5, followed by overnight incubation at 37ºC. The cells were harvested by
centrifugation using an Avanti J-20 centrifuge (Beckman Coulter) at 5000 rpm for 30 mins. The
cells were flash frozen in liquid nitrogen and then stored at -80°C until needed. Mk0566 was
1
purified by first resuspending the frozen cell pellets in amylose buffer A (50 mM Tris-HCl (pH
7.5@RT), 2 M NaCl, 2 mM 2-mercaptoethanol and 100 µM PMSF) and sonicated using Branson
Sonifier 450 (VWR Scientific). The lysate was centrifuged using a Sorvall Superspeed RC2-B
centrifuge at 16,000 rpm for 30 minutes, and then batch purified using amylose resin (New
England Biolabs, Inc.). The protein was eluted from the resin using amylose buffer B (50 mM
Tris-HCl (pH 7.5@RT), 50 mM NaCl, 30 mM maltose and 2 mM 2-mercaptoethanol). The maltose
binding protein (MBP) tag was removed by first incubating with Factor Xa protease (New England
Biolabs), in reaction buffer (20 mM Tris-HCl (pH 8.0@RT) with 100 mM NaCl and 2 mM CaCl2)
followed by a second round of amylose resin chromatography. The protein was further purified
using FPLC and Q-Sepharose fast flow followed by Butyl fast flow chromatography. The protein
was assessed at >99% pure by coomassie-blue stained SDS-PAGE. The purified protein was
dialyzed into storage buffer (20 mM Tris-HOAc (pH 8.0@RT), 50 mM KOAc, 1 mM EDTA, 1
mM DTT, 50% glycerol), aliquoted, and stored at -80°C.
DNA preparation
The oligonucleotides were prepared synthetically (Keck DNA Center, Yale University) and
purified
using
C18
reverse
phase
HPLC1.
The
concentration
was
measured
spectrophotometrically, with an extinction coefficient calculated from standard values for the
nucleotides2. The self-complementary DNA strands, or equimolar quantities of complementary
DNA, were annealed by heating to 90°C for 10 minutes at a concentration of 1 mM, followed by
slow-cooling to 4°C over 4-5 hours in a thermocycler.
The different DNA substrates used in cleavage assays are shown schematically in Figure S1, with
sequences given below:
NER1, 5’CTCCAAGCTCAGCCATCTCGGCTTGCAGGTGGCTCGTCCAGTACTAAGAC3’
2
NER2,5’GAGCGAGTCTCTCCCAGAGTACCTGCAAGCCGAGATGGCTGAGCTTGGAG3’
NER3, 5’GTCTTAGTACTGGACGAGCC3’
NER3+1nt3’, 5’GTCTTAGTACTGGACGAGCCC3’
NER4, 5’ACTCTGGGAGAGACTCGCTC3’
NER5,5’TCTTCTACAACTACTTCTCCAATATTCCAATCTTCTTCTCACGTGCACTCTTC
TTCTTCTCACAATATTCATTCCTCCTCCATACATCCA3’
NER6,5’TGGATGTATGGAGGAGGAATGAATATTGTGTCTTCTTCTTCTCACGTGCACT
CTTCTTCTTTGGAATATTGGAGAAGTAGTTGTAGAAGA3’
NER7,5’TGGATGTATGGAGGAGGAATGAATATTGTGAGAAGAAGAAGAGTGCACGT
GAGAAGAAGATTGGAATATTGGAGAAGTAGTTGTAGAAGA3’
DNA used in crystallization contained the following sequence (forming a truncated double flap
structure, with a 1 nucleotide 3’ flap, 2 nucleotide 5’flap, 8 bp downstream duplex and 9 bp
upstream duplex):
Mk1-1: 5’CTTGCAGGTGCTCGTCC3’
Mk2-1:5’GTACCTGCAAG3’
Mk3:5’GGACGAGCC3’
DNA Cleavage Activity Assays
Initial assays: oligonucleotides NER1, NER2, and NER5 were each 5’
32
P end-labeled at a
concentration of 5.5 µM in a 50 µl reaction using -32P labeled ATP (Amersham, Inc.), and T4
polynucleotide kinase (New England Biolabs, Inc.). The radiolabeled strands were then annealed
to unlabeled NER1, NER2, NER3, NER4, NER5, NER6, and NER7 in different combinations at
equimolar concentrations to produce a variety of unique DNA substrates (Table SI). Reactions
were 20 µl in total and contained 0.03 µM of DNA substrate with varying Mk0566 concentrations
3
ranging from 0 to 2.0 µM in Mk0566 reaction buffer (50 mM Tris-HCl (pH 7.5@RT), 20 mM
MgCl2, 10% glycerol, and 1 mM DTT). Following incubation at 57°C for 2 hours, each reaction
was terminated with 20 µl of a stop mix solution (80% formamide, 10 mM EDTA, 100 µg/ml
bromophenol blue, 2 mM NaOH) and then heated at 98°C for 5 minutes. Products were separated
on a 20% acrylamide (19:1 acrylamide:bisacrylamide) denaturing polyacrylamide gel (8 M urea)
and visualized on a phosphorimager (GE, Inc. or Biorad, Inc.).
Follow-up assays: 10 pmole of NER1 or NER2 was radiolabeled with
32
P using T4
polynucleotide kinase (New England Biolabs, Inc.) and -32P labeled ATP (Amersham, Inc.). Flap
(NER1, 2, and 3 or NER 1, 2, 3+1nt3’) or block flap/fork (NER 1, 2, 3, and 4 or NER 1, 2, 3+1nt3’,
and 4) were annealed using radiolabeled NER1 or NER2 with equimolar amounts of the other
unlabeled strands (NER1, NER2, NER3, or NER3+1nt) or 50 fold molar excess of unlabeled
NER4 as indicated. Assays were performed with 1 M MBP-Mk0566 and 5-7 nM DNA in reaction
buffer (10 mM Tris-HOAc, (pH 8.0@RT), 50 mM KOAc, 10 mM Mg(OAc)2, 1 mM DTT) at 57°C
for 5 hours. Each reaction was terminated with 20 µl of a stop mix solution (80% formamide, 10
mM EDTA, 100 µg/ml bromophenol blue) and then heated at 98°C for 5 minutes. Products were
separated on a 20% acrylamide (19:1 acrylamide:bisacrylamide) denaturing polyacrylamide gel (8
M urea) and visualized on a phosphorimager (BioRad, Inc.).
Sedimentation Velocity
Sedimentation velocity experiments were performed to determine the oligomeric form of
Mk0566 using a Beckman Coulter XL-I instrument with monochromator and interference
scanning optics (632 nm), automated scanning capability, and a Ti-50 rotor. Approximately 400
µl of sample containing 6 µM Mk0566 was loaded in one of the sectors of the two-sector
sedimentation velocity cells. The other sector was loaded with 425 µl of buffer (20 mM Tris-HOAc
4
(pH 8.0@RT), 150 mM NaCl, 10 mM Ca(OAc)2 and 1 mM DTT). The sample was allowed to
equilibrate at 4°C for at least 1 hour in the mounted rotor. The sample was then spun at 40,000
rpm (115,000xg) and absorbance scans were taken continuously at 280 nm for 10 hours. Data
obtained from the scans were fit to a sedimentation coefficient distribution, c(s), using SEDFIT3.
Fitted values of f/f0 were converted to diffusion coefficients and used in the Svedberg equation to
compute a molecular weight for each s value (Fig. S4). The viscosity of sample buffers was
measured by Cannon-Fenske viscometer and density was measured using the Mettler-Toledo
densitometer.
Sedimentation Equilibrium
Sedimentation equilibrium experiments were done as in the sedimentation velocity experiment
with some modifications: 110 µl of 6 µM Mk0566 were loaded into three chambers of a sixchamber cell, and buffer was loaded into the other three chambers. The samples were allowed to
equilibrate at 4°C for at least 1 hour in the mounted rotor. The samples were spun at three different
speeds: 12,600, 18,100 and 23,700 rpm for 72-96 hours (Fig. S5). The absorbance scans were
taken at 280 nm every 4 hours until there was no observable trend in the difference between
subsequent runs was observed. Data obtained from the scans were then fit to a single species using
SEDPHAT4.
Crystallization, Data Collection, Structure Solution, Refinement, and Analysis
The hanging drop vapor diffusion method5 was used to screen crystallization conditions. Drops
were composed of 1.5 µl Mk0566 protein at varied concentrations mixed with a 1.5 molar excess
of DNA and 1.5 µl of the precipitant solution. Crystals were obtained using 15% PEG 4K, 100
mM Tris-HCl (pH 8.5@RT), 150 mM NaCl, 10 mM CaCl2 as the precipitating solution. Crystals
reached full size in about 2 weeks at 17°C. The crystals were then exchanged into a cryoprotectant
5
solution (25% PEG 4K, 100 mM Tris-HCl (pH 8.5@RT), 300 mM NaCl, 10 mM CaCl2 and 30%
glycerol) and flash-frozen in liquid nitrogen. X-ray diffraction was measured using synchrotron
radiation at the Stanford Synchrotron Radiation Lightsource (SSRL) BL9-2. Data collection was
performed while maintaining the crystal at 100K. Image processing and data reduction were
performed with MOSFLM6 and SCALA7 respectively. The structure was solved by molecular
replacement using PHASER8. The MR model was based on structurally aligning 1RXW and 1A76
(FEN-1 from Archeoglobus fulgidus9 and Methanococcus jannaschii10, which share 58% and 48%
sequence identity with FEN-1 from Methanopyrus kandleri ), and then removing those parts of
1RXW where the chain traces differed. The resulting MR model was 1RXW 3-37, 57-80, 129185, 208-254, 272-324 (216 residues total). The PHASER Z-scores were RFZ 7.6, TFZ 16.8 for
the first molecule placed, and RFZ 8.1, TFZ 18.4 for the second. The initial R-factors reported by
REFMAC were R-work 49.1% and R-free 49.4%. Refinement was carried out using REFMAC
and the model was built using Coot11 and PDB_REDO starting with the 1RXW model. All
structure figures were prepared using PyMOL12. Structural comparisons and superpositions were
performed with the DALI server13. Sequence alignment and secondary structure depiction of
Figure 1B prepared with ESPript14.
Table SI. Activity of Mk0566 on DNA substrates
DNA
DNA Substrate
Results
substrate
1
2
(* indicates
32P
label)
No cleavage
No cleavage
6
3
No cleavage
4
No cleavage
5
Specific cleavage
of NER2
6
No cleavage of
NER1, evidence of
5’->3’ exonuclease
activity on NER2
7
No cleavage
8
No cleavage
9
Specific cleavage
of NER2
10
11
12
No cleavage of
NER1, evidence of
5’->3’ exonuclease
activity on NER2
No cleavage
No cleavage
13
No cleavage
14
No cleavage
7
Table SII. Comparison of the Mk0566 structure with other FEN-1 structures
Protein
PDB ID
%
ZRMSD
Number of
Identity score
residues/total
number
P. furiosus FEN-1
1B43
58
37.9
1.5 (chain
286/339
(chain
A)
A)
D. amylolyticus
3ORY
55
35.9
2.6
293/342
FEN-1
P. horikoshii FEN-1
1MC8
57
35.1
1.7
281/331
A. fulgidus FEN-1
1RXW/1RXV
58
34.4
1.5
266/305
Human FEN-1
3Q8M
44
34.1
1.8
284/335
M. jannashii FEN-1
1A77
47
33.6
1.8
273/315
Human FEN-1
1Ul1
45
33.3
1.9
272/313
S. solfataricus FEN2IZO
52
33.2
1.4
257/268
1
Human Exo I
3QE9
22
20.3
2.7
234/344
Taq DNA
1TAU
19
16.5
3.6
236/793
polymerase
8
Figure S1. Configurations of DNA substrates used in assays. A. Pseudo-Y, B. 5’flap, C. 3’flap,
D. double flap, E. fork, F. double stranded, and G. bubble DNA.
9
Figure S2. Evidence of 5’->3’ exonuclease activity. A. Reaction products with substrate 6 (Table
SI,
32
P labeled NER1 annealed to unlabeled NER2 and NER3 forming the 5’flap DNA of Fig.
S1B). Markers indicate positions of 20 nt, 50 nt, uncleaved double stranded DNA (UC ds), and
cleavage products. Lane 1: 32P labeled NER3, Lane 2: 32P labeled NER2, Lane 3: 32P labeled NER1
annealed to unlabeled NER2 and NER3, Lanes 4-10: reactions following 2 hr incubation with
Mk0566 at concentrations of 0.01, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0 M. B. As in A, but with
substrate 10, 32P labeled NER1 annealed to unlabeled NER2, NER3, and NER4 to form the blocked
flap or forked substrate of Fig. S1E.
Figure S3. Follow-up assays of DNA cleavage by Mk0566. Lane 1: NER1, Lane 2: NER 2, Lane
3: NER3, Lane 4: NER4, Lane 5: reaction of Mk0566 with substrate 6 (32P labeled NER1 with
unlabeled NER2 and NER3), Lane 6: reaction of Mk0566 with substrate 5 (32P labeled NER2 with
10
unlabeled NER1 and NER3), Lane 7: reaction of Mk0566 with substrate 10 (32P labeled NER1
with unlabeled NER2, NER3, and NER4), Lane 8: reaction of Mk0566 with substrate 9 (32P labeled
NER 2 annealed with NER1, NER3, and NER4), Lanes 9-12: as in Lanes 5-8 with the exception
that NER3 is replaced with NER3+1nt3’. Note: Lanes 1 and 2 have been adjusted to a brightness
and contrast to match lanes 3-12.
Figure S4: Sedimentation velocity of Mk0566. Calculated molecular weights (Materials and
Methods) shown above each peak.
1.4
1.2
1
0.8
0.6
12,600 rpm
0.4
0.2
18,100 rpm
23,700 rpm
0
6.4
6.45
6.5
6.55
6.6
6.65
Radius (cm)
11
Figure S5: Sedimentation equilibrium of Mk0566. Data from three different speeds is shown
(open circles, open squares, open triangles). Lines indicate calculated values from fits to single
species (39.5 kDa) as described in Materials and Methods.
Figure S6. Representative electron density map (2mFo-DFc at 1 sigma, blue, 1mFo-DFc at 3
sigma, magenta, and -3 sigma, yellow).
12
Figure S7. Comparison of Mk0566 with P. furiosus FEN-1 (PDB ID 1B43) and A. fulgidus
FEN-1 (PDB ID 1RXV). A. Superposition (using the Dali server13) of Mk0566(green) and P.
furiosus FEN-1 (blue) showing alterations in the region around residue Mk0566 265. B. As in A
showing similar structure around residues 189-202 (Mk0566 numbering) and the C terminal
helix. Chloride ions visible as green spheres. C. Superposition (using the Dali server13) of
Mk0566(green) and A. fulgidus FEN-1 (red) showing similar folding around residue Mk0566
265, although with coil rather than helix through residues 266-273 (Mk0566 numbering).
Figure S8: Comparison with DNA bound FEN-1 structures. A. Superposition (using Dali server13)
of human FEN-1 bound to DNA (PDB ID 3Q8M, protein cartoon in slate, DNA in blue) with
Mk0566 (green). Chloride ions of Mk0566 shown as red spheres. B. Superposition (using Dali
server13) of T4 RNaseH bound to DNA (PDB ID 2IHN, protein cartoon in slate, DNA in blue)
with Mk0566 (green). Chloride ions of Mk0566 shown as red spheres.
13
Figure S9. Cartoon of domains aligned between Mk0566 and human XPG, FEN-1, GEN1, and
Exo-I. The red rectangle refers to the conserved N domain, and blue, the conserved I domain.
Percentages refer to percent identity between the aligned regions, and E is the expect value.
Figure S10. Comparison of the Mk0566 structure with human FEN-1 bound to DNA (PDB ID
3Q8M) and P. furiosus FEN-1 (PDB ID 1B43). A. Superposition of human FEN-1 (magenta)
bound to DNA (wheat) with Mk0566 (green) near residue 265 (Mk0566 numbering). B.
Superposition of P. furiosus FEN-1 (red) and Mk0566 (green) near residue 265. DNA (wheat) is
derived from the human FEN-1 structure.
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