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Supplemental Information
Figure S1 Sensitivity of strains bearing a deletion of dinB to NFZ and 4-NQO. a,
NFZ sensitivity. A lexA(Def) background was used to minimize differences in
SOS-induction between each agent. b, 4-NQO sensitivity. Error bars, the s.d.
from three experiments. Microsoft Powerpoint file (1.4 MB).
Figure S2 SDS-PAGE of purified native DinB. Approximately 30 mg of enzyme
are obtained per liter from our purification protocol. Lane 1 contains molecular
weight markers, and lanes 2-6 contain 1.9, 9.5, 19, 38, 57 g of purified DinB.
The specific activity of the enzyme on undamaged template is ca. 1,000 pmol
min-1 mg-1. Microsoft Powerpoint file (0.5 MB).
Figure S3 Structures of lesions used in this study. a, N2-furfuryl-dG. b, N2-B[a]PdG. c, THF, an apurinic site analog. The proposed formula for the modified
base containing the N2-dG adduct produced by nitrofurazone is C12H11N8O3.
Microsoft Powerpoint file (58 KB).
Figure S4 Schematic of running start, standing start, and mismatch extension
assays. Template is shown in blue and radiolabeled primer is shown in red. In
running start assays, DinB encounters the lesion after initial polymerization on
an undamaged portion of the template (1 base pair for all experiments in this
publication). In contrast, in standing start experiments DinB initially polymerizes
opposite the lesion. For fidelity measurements, standing start experiments were
performed with a single dNTP at a time. Mismatch extension experiments are
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performed as the standing start experiments, but with primers that have
different bases (X) at their 3’ ends opposite to the lesion. The ability of DinB to
extend from the correctly paired vs. mismatched base is then determined by
assaying the addition of the next correct base (dA) on each substrate. Microsoft
Powerpoint file (25 KB).
Figure S5 A homology model of DinB, constructed in part with SWISS-Model,
reveals an N2-dG lesion binding pocket and suggests a mechanism for rate
enhancement on a damaged template. The templating base is shown in yellow,
the incoming nucleotide in blue, the 3’ base of the primer in green, and the
‘steric gate’ residue in red. a, The conformation of the incoming nucleotide is not
favorable to phosphodiester bond formation. b, A space-filling representation of
the model viewed from the minor groove shows a pocket that is correctly
positioned to accommodate an N2-dG adduct and that the ‘steric gate’ residue is
at the back of this cavity. c, A schematic representation of a model for rate
enhancement suggesting that binding of the N2-furfuryl-dG adduct stimulates a
conformational change in the active site, propagated through the ‘steric gate’
residue, that positions the incoming nucleotide for efficient phosphodiester bond
formation, providing rate enhancement on a damaged template. d, A spacefilling representation of the lesion-bound model shows that the pocket is
occupied by the furfuryl moiety. Microsoft Powerpoint file (1.4 MB).
Figure S6 SDS-PAGE of purified DinB(F13V). Approximately 27 mg of enzyme
are obtained per liter in this purification. Lane 1 contains molecular weight
markers, and lanes 2-8 contain 0.9, 2.3, 4.5, 9, 13.5, 18, 27 g of purified
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DinB(F13V). The specific activity of the enzyme on undamaged template is ca.
1,150 pmol min-1 mg-1. Microsoft Powerpoint file (374 KB).
Figure S7 Products of running start primer extension reactions (30 min) with 1,
10, and 50 nM DinB and DinB(F13V) on undamaged and tetrahydrofuran
bearing templates reveal that the F13V mutation does not render DinB able to
bypass this lesion. Primers are 14.8%, 70.1%, 73.2%, 3.9%, 6.2%, 6.9%, 7.5%,
24.5%, 57.8%, 2.6%, 6.2%, and 10.2% extended. Microsoft Powerpoint file (3.8
MB).
Figure S8 Dominant negativity of pdinB(F13V). The dominant negative effect of
pdinB(F13V) on survival is not significantly altered in an rnhB mutant, indicating
that the mechanism of the dominant negative effect is not merely through
improper incorporation of rNTPs by DinB(F13V) in vivo. Microsoft Powerpoint
file (411 KB).
Figure S9 MALDI-TOF mass spectrometry of the N2-furfuryl-dG adduct.
Oligonucleotides were synthesized as described in the supplementary material
and were purified by reversed-phase HPLC using an analytical column
(Microsorb-MV 100-5 C18 250 x 4.6 mm, Varian) at a flow of 1 mL/min and a
gradient of 0 to 30% B over 60 min (A: 0.1 M triethylammonium acetate; B:
100% acetonitrile) or by gel purification through 30 cm of 20% denaturing
polyacrylamide, elution by diffusion into water, and desalting with a Centricon 3.
The HPLC retention time was 29.1 min for the furfuryl oligonucleotide, and
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MALDI-TOF (shown in figure) revealed an observed mass of 4986.26 (4986.27
calculated). MALDI-TOF of the gel purified oligonucleotide revealed an
observed mass of 4986.12 as well as a small peak corresponding to xanthine
which represented a fraction of the sample much smaller than the bypass
efficiencies observed (ca. 5%). Microsoft Powerpoint file (64 KB).
Microsoft Word file Supplemental.doc describing strain and plasmid
construction, protein purification, and detailed oligonucleotide substrate
synthesis methods (36 KB).