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Figure Legends
Figure 1: The E. coli Lhr-CTD is a uracil DNA glycosylase
requiring a catalytic aspartic acid
(A) AlphaFold 2 structural model of E. coli Lhr that is based on
strong homology with the cryo-EM structure of Lhr helicase core and
Lhr-CTD from M. smegmatis , respectively PDB: 5V9X and PDB:7LHL.
The E. coli Lhr-core helicase (amino acids 1-897) contains RecA
domains, a beta-sheet bundle (β) and a winged helix domain (WH) as
indicated. Lhr-CTD (amino acids 898-1538) comprises folds with
structural homology to SecB chaperones and AlkZ glycosylases, as
indicted.
(B) Coomassie stained SDS-PAGE acrylamide gels showing purified Lhr and
Lhr-CTD, with molecular mass ladder (M) values in kDa.
(C) Products from mixing Lhr-CTD (50, 100, 200, 400 and 800 nM) with 5’
Cy5-ssDNA (12.5 nM) containing a d-Uracil base 18 located nucleotides
from the fluorescent moiety as indicated (lanes 1-12), seen in a 15%
denaturing acrylamide TBE gel. Addition of NaOH (lanes 8-12) causes β/δ
elimination at the site of the abasic DNA product, resulting in DNA
backbone cleavage. This confirms glycosylase protein activity. Marker
(M) is made from known lengths of 5’ Cy5 ssDNA.
(D) As for (C) in reactions containing unmodified 5’ Cy5-ssDNA (12.5
nM).
(E) Phyre2 structural model of E. coli Lhr-CTD with predicted
active site residues as labelled, including Lhr-CTD residue Asp-1536
that we mutated in this work.
(F) Products from mixing Lhr-CTD and Lhr-CTDD1536Aproteins with 12.5 mM d-uracil containing 5’ Cy5-ssDNA substrate, viewed
in a 18% acrylamide denaturing TBE gel. Product formation is shown
every 5 minutes for 30 minutes, observing no glycosylase activity from
Lhr-CTDD1536A.
Figure 2: LhrD1536A is inactive as a glycosylase but
binds to DNA
(A ) EMSA assays showing Lhr (12.5, 25, 50, 100 and 200nM)
complexes bound to DNA (12.5 nM) that are stable migrating through a 5%
acrylamide TBE gel, compared with Lhr-CTD at the same concentrations.
(B ) Products of Lhr glycosylase activity seen in an 18%
acrylamide denaturing TBE gel were absent when reactions contained
LhrD1536A. Proteins were used at 25, 50, 100 and 200
nM, with 12.5 nM of d-uracil containing 5’ Cy5-ssDNA substrate.
(C ) EMSA showing that LhrD1536A and Lhr
(12.5, 25, 50, 100 and 200 nM) form stable complex with DNA in a 5%
acrylamide TBE gel
Figure 3: Lhr is inactive against 8-oxoguanine, and its
uracil DNA glycosylase activity on duplex DNA functions independently
from Lhr helicase activity
(A ) Time-dependent uracil DNA glycosylase activity of Lhr (50
nM) compared with LhrD1536A. The data shows means of
glycosylase activity (n=3, with bars for standard error) alongside a
representative gel used for quantification.
(B ) Comparison of Lhr (50 nM) glycosylase activity on ss-, ds-
and forked d-uracil containing DNA substrates (12.5 nM) as a function of
time, with samples taken at time points indicated plots are means of
two independent experiments showing standard error bars.
(C ) Time-course assays (10, 20, 30 minutes) showing products
from Lhr and Lhr-CTD (each 80 nM) mixed with the preferred flayed duplex
uracil-DNA, seen in an 18% acrylamide denaturing TBE gel. Known length
DNA strands are shown (M) and the positive control reaction (+ve) is
product from 5 units of E. coli uracil DNA glycosylase.
(D ) As for (C) except d-uracil DNA was replaced with otherwise
identical 8-oxo-d-Guanine DNA, and the control reaction (+ve) shows
product formed by 5 units of formamidopyrimidine DNA glycosylase (Fpg)
protein.
(E ) Lhr (80 nM) uracil-DNA glycosylase activity seen as
products in 18% acrylamide denaturing TBE gels (lanes 1-4), after
30-minute reactions in either EDTA, manganese or calcium, each replacing
magnesium as indicated, compared with unmodified DNA (lanes 5-8).
(F ) DNA unwinding by Lhr and LhrD1536Aproteins (12.5, 25, 50, 100 and 200 nM) on 12.5 nM of 5’ Cy5 labelled
flayed duplex DNA, seen in 10% acrylamide TBE gel.