Download Sup2 - Postech

Supplementary Data
Supplementary Methods
Structure determination and refinement
The following residues were not visible in electron density map and were presumably
disordered in the structures. Apo-PfNurA: A chain; residues 1–3, 224–227, 309–311,
and 441–451; B chain; 1–2, 211–215, 226–227, 308–309, and 442–451. PfNurAdAMP-Mn2+: A chain 1–5, 224–229, 416–418, and 440–451, B chain: 1–4, 209–214,
307–309, 327–329, 401–403, and 441–451. PfNurA-Mn2+: A chain 1–2, 224–227,
309–311, and 442–451; B chain 1–2, 211–214, 308–309, and 442–451.
Nuclease assays
Reaction mixtures containing various amounts of PfNurA (350 and 1750 nM) and 20
nM
32P-labeled
substrate DNA in reaction buffer (25 mM Tris-HCl pH 7.4, 60 mM
NaCl, 1 mM dithiothreitol, 5 mM MnCl2) were incubated for 120 min at 65°C, and the
reaction was stopped by adding the same volume of 2X reaction stop buffer (95%
formamide, 18 mM EDTA, 0.025% sodium dodecyl sulfate, 0.01% Bromophenol
blue), followed by 5 min of boiling at 100°C. For the PfHerA assays, various amounts
of PfNurA (35, 70, and 115 nM) and ATP (1 mM) were added to the reaction mixture.
Reaction products were resolved on 15% denaturing polyacrylamide gels containing
7 M urea in TBE buffer. Gels were run for 400 min at 13 Vcm -1. After electrophoresis,
the gels were fixed in fixing buffer (30% methanol, 5% acetic acid, and 5% glycerol),
dried, and subjected to autoradiography and phosphoimage analysis.
Substrates
Polyacrylamide gel electrophoresis-purified DNA substrates were purchased from
Bioneer (Seoul, South Korea). The dsDNA substrate consisted of 5′[32P]-labeled
TP580 annealed to TP124 phosphorothioate bonds, which are indicated by an “s”
between the nucleotides. The 3′ overhang substrate consisted of TP423 annealed to
TP424. TP424 was labeled with
32P
at the 5′ end. DNA substrates were prepared at
the 5′ end with Polynucleotide Kinase (Roche Applied Science, Rockford, IL, USA)
and [γ-32P]dATP (PerkinElmer, Waltham, MA, USA). The labeled oligos were boiled
for 5 min and then allowed to cool to anneal slowly.
Sequence of substrates:
TP 580
5’-CTGCAGGGTTTTTGTTCCAGTCTGTAGCACTGTGTAAGACAGGCCsAsGsAsTsG-3’
TP124
5’-CATCTGGCCTGTCTTACACAGTGCTACAGACTGGAACAAAAACCCTGCAG-3’
TP423
5’-CTGCAGGGTTTTTGTTCCAGTCTGTAGCACTGTGTAAGACAGGCCAGATC-3’
TP424
5’-CACAGTGCTACAGACTGGAACAAAAACCCTGCAGTACTCTACTCATCTC-3’
Analytical ultracentrifugation
The molecular mass of the PfNurA was analyzed by analytical ultracentrifugation
(Optima XL-A; Beckman, Fullerton, CA, USA) using the sedimentation equilibrium
technique. Sedimentation equilibrium data were evaluated using a nonlinear leastsquares curve-fitting algorithm (XL-A data analysis software). All samples were
analyzed in binding buffer containing 25 mM Tris-HCl (pH 7.4), 200 mM NaCl, 7 mM
β-mercaptoethanol, and 2 mM MnCl2. For the equilibrium analysis, scans at
equilibrium from multiple speeds (9,000, 10,000, 11,000, 13,000 and 16,000 rpm)
were collected at 15°C using an An60Ti rotor (Beckman) and by measuring
absorbance at 280 nm. The partial specific volume of the PfNurA was estimated from
the protein sequence to be 0.7494 cm3/g, using the SEDNTERP program, and a rho
value of 1.006 was used for the molecular mass calculation.
Mutant protein structural analysis
For gel filtration analysis, wild-type or dimeric interface mutants (2 mg/ml) was
loaded onto a Superdex 200 column (Amersham Biosciences Fairfield, CT, USA)
equilibrated with buffer containing 25 mM Tris-HCl (pH 7.4), 200 mM NaCl, 5%
glycerol, and 5 mM dithiothreitol.
Structural changes in the mutant PfNurA (4 uM) versus the wild-type PfNurA
were monitored with a Jasco J-715 CD spectrophotometer at wavelengths of 200–
260 nm. All samples were prepared in a buffer containing 25 mM Tris-HCl (pH 7.4),
150 mM NaCl, 7 mM β-mercaptoethanol, and 5% glycerol.
Supplementary Figure Legends
Figure S1. Analytical ultracentrifuge analyses reveal that PfNurA form a
dimeric interface structure. Equilibrium fit results of analytical ultracentrifuge for
the PfNurA. The lower panel depicts the fitted overlay (red line) to the experimental
data (blue circles). The upper panel depicts the residuals. The fitted parameter for
the weight-average molecular mass (Mw, app) was estimated to be 110.4 kDa.
Figure S2. Sequence alignment of PfNurA homologues.
Secondary structural elements are indicated above the sequence and three domains
are colored as in Fig. 2. Active sites are indicated in red boxes and strictly conserved
residues are highlighted in orange boxes. Similar residues are in green boxes. The
secondary structures of TmNurA (1zup) are shown below the sequence. Every tenth
residue is marked with a black circle. Abbreviations: Pf, Pyrococcus furiosus; Pa,
Pyrococcus abyssi; Af, Archaeoglobus fulgidus; Sto, Sulfolobus tokodaii; Tm,
Thermotoga maritima
Figure S3. Conformational differences between the two PfNurA subunits. The
M domains of the two PfNurA subunits exhibit significant structural differences and
one monomer should be rotated and translated by at least 20 Å to be approximately
overlayed with another PfNurA subunit.
Figure S4. A PfNurA-dsDNA binding model. Three different views of PfNurAdsDNA binding model. A dsDNA (blue) was modeled on the PfNurA dimer based on
the TtAgo-DNA (3hvr) complex. The modeled dsDNA collided with another PfNurA,
implying that a conformational change in this PfNurA or dsDNA region is necessary
to accommodate dsDNA.
Figure S5. Structural comparison between PIWI domains of PfNurA (left) and
RNase HII (right). The equivalent region in both structures is marked with a box.
Figure S6. Circular dichroism (CD) analysis of conformational differences
between wild type and mutant PfNurA. The spectra for active site mutants (E105A
and H411A) and DNA binding site mutants (K297E*, R323E* and K415E*) were
compared with that of a wild-type protein. A CD analysis showed that the various
mutations on the PfNurA used in this study did not affect the overall conformation of
the PfNurA except the R323E* and K415E* mutants. Although the K415E mutant
exhibited a slightly different CD spectra curve, this mutant showed the same
nuclease activity as wild-type PfNurA. The CD analysis was performed using a buffer
containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 7 mM β-mercaptoethanol, and
5% glycerol. Abbreviations: K297E*, K297E/Y380F/Y403F; R323E*, R323E/R435E;
K415E*, K415E/K419E.