\documentclass[10pt]{article}

\usepackage{fullpage}
\usepackage{setspace}
\usepackage{parskip}
\usepackage{titlesec}
\usepackage[section]{placeins}
\usepackage{xcolor}
\usepackage{breakcites}
\usepackage{lineno}
\usepackage{hyphenat}





\PassOptionsToPackage{hyphens}{url}
\usepackage[colorlinks = true,
            linkcolor = blue,
            urlcolor  = blue,
            citecolor = blue,
            anchorcolor = blue]{hyperref}
\usepackage{etoolbox}
\makeatletter
\patchcmd\@combinedblfloats{\box\@outputbox}{\unvbox\@outputbox}{}{%
  \errmessage{\noexpand\@combinedblfloats could not be patched}%
}%
\makeatother


\usepackage{natbib}




\renewenvironment{abstract}
  {{\bfseries\noindent{\abstractname}\par\nobreak}\footnotesize}
  {\bigskip}

\titlespacing{\section}{0pt}{*3}{*1}
\titlespacing{\subsection}{0pt}{*2}{*0.5}
\titlespacing{\subsubsection}{0pt}{*1.5}{0pt}


\usepackage{authblk}


\usepackage{graphicx}
\usepackage[space]{grffile}
\usepackage{latexsym}
\usepackage{textcomp}
\usepackage{longtable}
\usepackage{tabulary}
\usepackage{booktabs,array,multirow}
\usepackage{amsfonts,amsmath,amssymb}
\providecommand\citet{\cite}
\providecommand\citep{\cite}
\providecommand\citealt{\cite}
% You can conditionalize code for latexml or normal latex using this.
\newif\iflatexml\latexmlfalse
\providecommand{\tightlist}{\setlength{\itemsep}{0pt}\setlength{\parskip}{0pt}}%

\AtBeginDocument{\DeclareGraphicsExtensions{.pdf,.PDF,.eps,.EPS,.png,.PNG,.tif,.TIF,.jpg,.JPG,.jpeg,.JPEG}}

\usepackage[utf8]{inputenc}
\usepackage[greek,ngerman,english]{babel}



\usepackage{float}








\begin{document}

\title{E. coli DNA Repair Helicase Lhr is also a Uracil-DNA Glycosylase}



\author[1]{Ed Bolt}%
\author[1]{Ryan J. Buckley}%
\author[2]{Karl M. Hanson}%
\author[2]{Nadia R. Ahmed}%
\author[2]{Christopher Cooper}%
\affil[1]{University of Nottingham School of Life Sciences}%
\affil[2]{University of Huddersfield School of Applied Sciences}%


\vspace{-1em}



  
  \date{June 08, 2024}


\begingroup
\let\center\flushleft
\let\endcenter\endflushleft
\maketitle
\endgroup





\selectlanguage{english}
\begin{abstract}
DNA glycosylases protect genetic fidelity during DNA replication by
removing potentially mutagenic chemically damaged DNA bases. Bacterial
Lhr proteins are well-characterized DNA repair helicases that are fused
to additional 600-700 amino acids of unknown function, but with
structural homology to SecB chaperones and AlkZ DNA glycosylases. Here
we identify that \emph{E. coli} Lhr is a uracil-DNA glycosylase that
depends on an active site aspartic acid residue. We show that the Lhr
DNA helicase activity is functionally independent of the uracil-DNA
glycosylase activity, but that the helicase domains are required for
fully active uracil DNA glycosylase activity. Consistent with uracil DNA
glycosylase activity, deletion of \emph{lhr} from the \emph{E. coli}
chromosome sensitized cells to oxidative stress that triggers cytosine
deamination to uracil. The ability of Lhr to translocate single-stranded
DNA and remove uracil bases suggests a surveillance role to seek and
remove potentially mutagenic base changes during replication stress.%
\end{abstract}%



\sloppy


\emph{E. coli} \textbf{DNA Repair Helicase Lhr is also a Uracil-DNA
Glycosylase}

Ryan J. Buckley\textsuperscript{1}, Karl M. Hanson\textsuperscript{2},
Nadia R. Ahmed\textsuperscript{2}, Christopher D. O.
Cooper\textsuperscript{2 +} and Edward L. Bolt\textsuperscript{1*}

\textsuperscript{1}University of Nottingham, School of Life Sciences, UK

\textsuperscript{2}School of Biological and Geographical Sciences,
School of Applied Sciences, University of Huddersfield, UK.

*Correspondence to ed.bolt@nottingham.ac.uk

\textbf{Summary}

DNA glycosylases protect genetic fidelity during DNA replication by
removing potentially mutagenic chemically damaged DNA bases. Bacterial
Lhr proteins are well-characterized DNA repair helicases that are fused
to additional 600-700 amino acids of unknown function, but with
structural homology to SecB chaperones and AlkZ DNA glycosylases. Here
we identify that \emph{E. coli} Lhr is a uracil-DNA glycosylase that
depends on an active site aspartic acid residue. We show that the Lhr
DNA helicase activity is functionally independent of the uracil-DNA
glycosylase activity, but that the helicase domains are required for
fully active uracil DNA glycosylase activity. Consistent with uracil DNA
glycosylase activity, deletion of \emph{lhr} from the \emph{E.
coli}chromosome sensitized cells to oxidative stress that triggers
cytosine deamination to uracil. The ability of Lhr to translocate
single-stranded DNA and remove uracil bases suggests a surveillance role
to seek and remove potentially mutagenic base changes during replication
stress.

\textbf{Keywords:} Helicase; glycosylase; DNA repair; uracil; DNA
replication

\textbf{Introduction}

Lhr (\emph{L} arge \emph{h} elicase\emph{-r} elated) proteins are
ATP-dependent 3' to 5' DNA translocases within the Superfamily 2
helicases {[}1{]}. The founder member of Lhr proteins was identified in
bacteria {[}2{]}, and subsequently Lhr was found to be widely
distributed across all clades of archaea {[}3{]}. High amino acid
sequence identity (typically about 30\%) between archaeal and bacterial
Lhr proteins is limited to 800-900 amino acids that form helicase
domains from the Lhr N-terminus ? called the `Lhr-Core' {[}4{]}.
Biochemical analyses of the Lhr-Core from the
bacteria\emph{Mycobacterium smegmatis} and \emph{Pseudomonas putida} and
from the archaeon \emph{Methanothermobacter thermautotrophicus} have
characterized Lhr translocation and helicase mechanism {[}5-7{]}, and
crystal structures of Lhr-Cores highlight similarities with
translocation by the archaeal DNA repair helicase Hel308 {[}6, 8{]},
especially in interactions between their winged helix and RecA-like
domains {[}9, 10{]}.

In addition to the Lhr-Core, bacterial Lhr proteins extend to 1400-1600
amino acids, in a C-terminal protein region of unknown function, called
Lhr-CTD (Lhr-C-terminal domains). Structural modelling of the bacterial
Lhr-CTD {[}11{]} and a subsequent cryo-EM structure {[}12{]}, provided
intriguing clues to Lhr-CTD function, including the presence of an array
of tandem winged helix domains characteristic of the HTH\_42 superfamily
of proteins that have structural homology to the DNA glycosylase AlkZ
{[}11{]}. Genetic analyses of the effects on bacterial and archaeal
cells of deleting the \emph{lhr} gene revealed mild sensitivities to
agents that cause replication stress?UV irradiation {[}13{]} and
azidothymidine (AZT) {[}14{]} ? and transcriptional up-regulation
of\emph{lhr} in response to mitomycin C {[}15{]}. In this work we report
new insights about how Lhr contributes to DNA repair in bacteria. We
demonstrate that the \emph{E. coli} Lhr protein has uracil-DNA
glycosylase activity, in addition to its well-characterized
ATP-dependent DNA translocase functions, and that cells lacking Lhr are
sensitive to oxidative stress.

\textbf{Results}

\emph{E. coli} \textbf{Lhr is an uracil DNA glycosylase requiring an
active site aspartate}

We investigated whether \emph{E. coli} Lhr is capable of DNA glycosylase
activity, as suggested from structural similarities between glycosylases
and the uncharacterized C-terminal region of \emph{E. coli} Lhr
(Lhr-CTD, LHR amino acids 876-1538) {[}11, 12{]} (\emph{Figure 1A}
).\emph{E. coli} Lhr-CTD protein fragment and full-length Lhr protein
(1538 amino acid) were purified (\emph{Figure 1B} ) ? when Lhr-CTD (50
-- 800 nM) mixed with a Cy5-end labelled 37-nt ssDNA molecule modified
to contain a single uracil nucleotide located 18 nucleotides from the 5'
ssDNA end (uracil-ssDNA) a single product was observed on alkaline
treatment of reactions (\emph{Figure 1C} , compare lanes 1-6 with 7-12),
indicating DNA strand breakage at a DNA abasic site consistent with
glycosylase activity. No product was observed from Lhr-CTD mixed with
the same ssDNA lacking chemical modification (\emph{Figure 1C} lanes
13-24).

To validate the uracil-DNA glycosylase activity of \emph{E. coli}Lhr-CTD
we sought to identify single amino acid substitutions that would
inactivate it. Sequence alignment of \emph{E. coli} Lhr-CTD and AlkZ,
with which it has structural similarity {[}11, 12{]}, were unproductive
at identifying highly conserved residues because Lhr-CTD lacks the `QxQ'
motif characteristic of AlkZ protein active sites {[}16{]}, therefore
suggesting an alternate catalytic mechanism in LHR. Alternatively,
potential active site amino acids were identified through visual
scrutiny of the Phyre2 {[}17{]} predicted model of the \emph{E.
coli}Lhr-CTD, and in particular the positioning of side chains proximal
to a proposed glycosylase active site (Figure 1D). Purified
Lhr-CTD\textsuperscript{D1536A} (50 -- 800 nM), gave no product when
titrated into the uracil modified ssDNA after alkaline treatment,
compared with Lhr-CTD (\emph{Figure 1E} ). We then tested whether
substitution of the Lhr Asp-1536 residue inactivated uracil-DNA
glycosylase active site chemistry or had some other effect on the
protein that perturbed DNA binding. Unmutated Lhr-CTD was unable to form
stable complex with DNA in EMSAs, when compared with full length Lhr
(\emph{Figure 2A} ), therefore we purified and tested full
Lhr\textsuperscript{D1536A}. Lhr was also active as an uracil-DNA
glycosylase compared with Lhr-CTD (\emph{Figure 2B} ), but the
Lhr\textsuperscript{D1536A} mutation inactivated glycosylase activity in
agreement with inactive Lhr-CTD\textsuperscript{D1536A} fragment
(\emph{Figure 2C} ). In EMSAs Lhr\textsuperscript{D1536A} formed stable
complex with DNA similarly to Lhr (\emph{Figure 2D} ), therefore we
conclude that Lhr is a uracil DNA glycosylase that requires an active
site aspartic acid residue.

\textbf{DNA glycosylase activity of \emph{E. coli} Lhr is independent
from its DNA helicase activity}

Full length Lhr was substantially more active than Lhr-CTD as a
uracil-DNA glycosylase when measured in assays as a function of time
(Figure 3A) ? this may be explained by much more stable DNA binding by
full length Lhr compared with Lhr-CTD that was observed in EMSAs (Figure
2A). We therefore continued to use full-length Lhr to further
investigate uracil-DNA glycosylase function against flayed duplex DNA
molecules that are substrates for unwinding by the Lhr 3' to 5' DNA
helicase activity {[}11{]}. For this work the duplex substrate was
formed from annealing uracil-containing ssDNA with its unmodified
complementary DNA strand, with uracil positioned 8nt from the fork
branchpoint, 18 nt from the Cy5-DNA 5' end. Measured as a function of
time, Lhr generated glycosylase product from the uracil duplex at least
5-fold more effectively than when incubated with uracil-ssDNA
(\emph{Figure 3B} ), and Lhr was more active than Lhr-CTD on the
uracil-fork DNA (\emph{Figure 3C} ). Neither Lhr or Lhr-CTD gave any
glycosylase product when uracil was substituted for a single
8-oxoguanine residue at the same position in DNA (\emph{Figure 3D} ).
The product of Lhr from uracil-DNA single strands or duplex migrated
close to the 16 nt marker, indicating that the same glycosylase product
was formed from both substrates. Glycosylase assay conditions in Figures
1-3 included Mg\textsuperscript{2+} in the reaction buffer, but Lhr was
also active in buffers containing EDTA, Mn\textsuperscript{2+} and
Ca\textsuperscript{2+} instead of Mg\textsuperscript{2+} (\emph{Figure
3E} lanes 1-4) but was inactive as a glycosylase on DNA lacking a uracil
residue (\emph{Figure 3E} lanes 5-8). Lhr\textsuperscript{D1536} that is
inactive as a uracil-DNA glycosylase was proficient at fork DNA
unwinding (\emph{Figure 3F} ), and we therefore conclude that the uracil
glycosylase activity of Lhr is functionally distinct from helicase
activity, but we observe that glycosylase activity is enhanced when the
helicase domains are present, by contributing to DNA binding
(\emph{Figure 2A} ).

\emph{E. coli} \textbf{cells lacking LHR are sensitive to oxidative
stress}

Oxidative damage to DNA in \emph{E. coli} cells includes deamination of
cytosine to uracil and further oxidized uracil derivatives {[}18-21{]},
triggering cytosine to adenine transversion mutations. We therefore
assessed for a contribution from Lhr to DNA repair in \emph{E.
coli}cells that is consistent with \emph{in vitro} uracil-DNA
glycosylase activity. The \emph{lhr} gene was deleted in \emph{E. coli}
MG1655 (\selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} ) by recombineering, and we removed the inactivating
antibiotic resistance marker, verified by sequencing across the deletion
site. We first tested \selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} cells for sensitivity to
azidothymidine (AZT), a previously reported phenotype for Lhr in\emph{E.
coli} cells {[}14{]}. In a viability plate assay after growing cells in
broth (LB) containing a fixed 7.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL AZT we observed 10-fold reduced
viability of \selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} cells compared with wild type cells
(\emph{Figure 4A} ), and similar moderate sensitivity of
\selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr}cells across AZT concentrations (\emph{Figure 4B} ), agreeing
with the previous study {[}14{]}. We next measured survival of cells
when grown in media containing hydrogen peroxide as a potent oxidizing
agent. Hydrogen peroxide (12.5 mM) added to growth media after cells had
reached OD\textsubscript{600} of 0.3 resulted in significantly reduced
growth of \selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} cells in exponential phase compared with wild type
cells (\emph{Figure 4C} ). This agreed with 10-100-fold reduced cell
viability compared with wild type cells when \selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} cells grown in
the same way, but without hydrogen peroxide, were then spotted onto LB
agar containing increasing concentrations of hydrogen peroxide to count
their viability (\emph{Figure 4D} ).

\textbf{Discussion}

We provide biochemical evidence that \emph{E. coli} Lhr is a uracil-DNA
glycosylase (Lhr-UDG), a new function for bacterial Lhr proteins
alongside their well-characterized 3' to 5' single DNA translocation
activity that is stimulated by forked or flayed DNA substrates {[}5, 7,
11{]}. We show that the Lhr-UDG activity requires an active site
aspartate residue (Asp-1536), similarly to the active site aspartate
general base (Asp-62) that is essential for major groups of UNG/UDG
proteins {[}22{]}. The Lhr UDG function is positioned in the previously
uncharacterized Lhr-CTD ? though this fragment of Lhr was proficient as
a `stand-alone' uracil-DNA glycosylase, its activity was significantly
increased by the presence of the Lhr helicase domains, probably by the
helicase domains providing more stable DNA binding compared with
Lhr-CTD, observed in EMSAs. Inactivating the Lhr-UDG activity did not
inactivate DNA unwinding by Lhr, providing further support for the DNA
binding functions of Lhr being concentrated in the helicase domains.

Loss of Lhr from bacterial cells (\selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} ) causes mild sensitivity
to AZT {[}14{]}, a phenotype we also observed after generating
\selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} cells and removing the inserted antibiotic resistance
marker. We identified that \selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} cells were also significantly
more sensitive than \emph{lhr\textsuperscript{+}} cells to oxidative
stress induced by hydrogen peroxide, which is one of several routes
causing genetic damage by cytosine deamination in bacterial cells. This
therefore supports our \emph{in vitro} observations of Lhr-UDG function.
UDGs are ubiquitous in nature, although this is the first report of a
UDG fused to a DNA helicase. \emph{E. coli} has a canonical UDG enzyme
that functions in global DNA repair coupled with stable DNA replication
? upregulation of Mycobacterial Lhr in response to mitomycin C treatment
{[}15{]}, and the sensitivity of \emph{E. coli} cells to the polymerase
inhibitor AZT when they lack \emph{lhr} , may indicate that Lhr is
activated as part of bacterial responses to specific forms of
replication-stress. In this context removal of uracil from DNA by Lhr
may protect genetic fidelity at sites that are overcoming blocked DNA
replication. We cannot exclude that Lhr may be able to remove other
lesions or chemical modifications from DNA, although we observed that it
was inactive as a glycosylase against 8-oxoguanine, suggesting that is
has at least specificity for recognizing pyrimidine damage over purines.

\textbf{Experimental Procedures}

\textbf{Proteins}

DNA sequences of primers and substrates, plasmids and \emph{E.
coli}strains are detailed in Supplementary data. \emph{E. coli} MG1655
gene b1653, encoding Lhr, was PCR-amplified from genomic DNA, and cloned
into pET14b using \emph{Nde} I and \emph{Hin} DIII restriction sites
generating pRJB28 for expression of hexa-histidine tagged Lhr. Lhr-CTD
(amino acids 876-1538) was amplified and cloned by ligation independent
cloning (LIC) The resulting plasmid based on vector pNH-TrxT (GU269914.1
{[}23{]}). These plasmids were used to generate
Lhr\textsuperscript{D1536A} using mutagenic primers in PCR by Q5 hot
start polymerase, and resulting reactions were treated with \emph{Dpn}
I, T4 polynucleotide kinase, and DNA ligase. Plasmid DNA was extracted
and sequenced from colonies after transforming reaction mixtures into
\emph{E. coli} .

Lhr and C-Lhr proteins were over-expressed in \emph{E. coli} Rosetta 2
cells grown in MU broth with ampicillin and chloramphenicol selection.
Cells were grown with shaking at 37\selectlanguage{ngerman}°C to OD\textsubscript{600} of 1.2
and transferred to an ice slurry for cooling before addition of IPTG
(0.8 mM). Growth was continued for 10 hours at 18°C, cells were
harvested and resuspended in 20 mM HEPES pH 8.0, 1.5 M ammonium sulfate,
20 mM imidazole, 10\% (w/v) glycerol (Ni-NTA buffer A) containing 0.1 mM
phenylmethylsulphonyl fluoride (PMSF). This process and purification was
also followed for obtaining Lhr\textsuperscript{D1536A} protein. Cells
were thawed and sonicated on ice, clarified by centrifugation, and
soluble proteins were loaded into a 5 ml butyl sepharose column
equilibrated with 20 mM HEPES pH 8.0, 1.5 M ammonium sulfate, 10\% (w/v)
glycerol (hydrophobic salt buffer A). The column was washed with 20 mM
HEPES pH 8.0, 900 mM ammonium sulfate. Then a 5 ml Ni-NTA column
pre-equilibrated with Ni-NTA buffer A was attached in tandem with the
butyl sepharose column and columns washed with 20 mM HEPES pH 8.0 and
10\% glycerol until no proteins were detectable by UV monitoring as
eluting from the columns. The butyl sepharose column was removed and Lhr
was eluted from the Ni-NTA column by increasing imidazole to 500 mM in
20 mM HEPEs and 10\% glycerol. Lhr-containing fractions were pooled and
dialyzed overnight into 20 mM HEPES pH 8.0, 150 mM NaCl, 10\% (w/v)
glycerol (low salt buffer A) and loaded into a 1 ml Q-sepharose column.
Lhr eluted in an increasing gradient of NaCl to 1.5 M. Lhr fractions
were pooled and dialyzed overnight for storing in 20 mM HEPES pH 8.0,
150 mM NaCl and 35\% (w/v) glycerol for aliquoting, flash freezing, and
storage at -80°C. \emph{E. coli} uracil-DNA glycosylase and
formamidopyrimidine DNA glycosylase (Fpg) control proteins (Figure 3)
were purchased from New England Biolabs.

\emph{In vitro} \textbf{DNA binding, unwinding and glycosylase assays}

DNA strands for substrate formation (supplemental data) were synthesized
with Cy5 end-label. DNA binding was assessed using electrophoretic
mobility shift assays (EMSAs). Reactions were incubated at 37°C for 20
minutes in helicase buffer (HB); 20 mM Tris pH 7.5, 10\% (v/v) glycerol,
100 \selectlanguage{greek}µ\selectlanguage{english}g/ml BSA, using 12.5 nM Cy5-fluorescently labelled DNA substrate,
25 mM DTT and 5 mM EDTA, and then placed on to ice for 10 minutes.
Orange G and 80\% (v/v) glycerol (OG) was added to load reactions onto a
5\% acrylamide TBE gel that was electrophoresed for 1 hour 30 minutes at
140 V. Gels were imaged using a Typhoon phosphor-imager (Amersham) at
633 nm using a R765 filter for Cy5 detection.

DNA unwinding assays were at 37\selectlanguage{ngerman}°C in reactions containing buffer HB,
12.5 nM Cy5-fluorescently labelled DNA substrate, 25 mM DTT, 1.25 \selectlanguage{greek}µ\selectlanguage{english}M
unlabeled `trap' DNA, 5 mM ATP and 5 mM CaCl\textsubscript{2}. Reactions
were pre-incubated at 37\selectlanguage{ngerman}°C for 5 minutes without the `trap' or ATP
before they were added together to start the reactions for 30 minutes at
37°C, stopped by addition of stock stop solution (4 \selectlanguage{greek}µ\selectlanguage{english}l per 20 \selectlanguage{greek}µ\selectlanguage{english}l
reaction); 2 mg/mL proteinase K in 200 mM EDTA and 2.5\% (w/v) SDS. OG
dye was added for electrophoresis through a 10\% acrylamide TBE gel for
45 minutes at 150 V. Gels were imaged using a Typhoon phosphor-imager
(Amersham) at 633 nm using a R765 filter for Cy5 detection.

DNA glycosylase reactions were at 37\selectlanguage{ngerman}°C in reaction mixtures containing
buffer HB, 12.5 nM Cy5-fluorescently labelled DNA substrate, 25 mM DTT,
5 mM ATP, 4 mM MnCl\textsubscript{2} and 4 mM CaCl\textsubscript{2}.
Reactions were pre-incubated at 37°C before being initiated by addition
of Lhr protein and (unless in a time course assay) allowed to continue
for 30 minutes before addition of stock stop solution and 4 \selectlanguage{greek}µ\selectlanguage{english}l of 1 M
NaOH. Reaction samples were boiled for 5 minutes and formamide added
before loading into a 15\% denaturing (8 M urea) acrylamide TBE gel for
4 hours at 5 watts per gel. Gels were imaged using a Typhoon
phosphor-imager (Amersham) at 633 nm using a R765 filter for Cy5
detection, generating TIFFs that were measured using Gel Analyzer 19.1
(Lazar) software. Graphs of glycosylase activity were generated using
Prism (GraphPad).

\subsubsection*{\texorpdfstring{Generation of a chromosomal deletion
of\emph{E. coli
lhr}}{Generation of a chromosomal deletion ofE. coli lhr}}

{\label{generation-of-a-chromosomal-deletion-of-e.-coli-lhr}}

DNA constructs and strain genotypes are presented in the Supplementary
material. \emph{Lhr} deletion was by recombineering {[}24{]} and P1
transduction of an FRT (FLP recognition target) flanked
Kan\textsuperscript{r} marker. To generate an effective P1 stock the
overnight culture was used to inoculate 8 ml of Mu broth containing 6 mM
CaCl\textsubscript{2}. A sample of the cells (0.1 mL) grown at 37\selectlanguage{ngerman}°C to
OD\textsubscript{600} 0.8-1.0 in a shaking water bath was added to four
overlay tubes each containing 3 mL of 0.4\% w/v Mu broth agar held at
42°C. P1 phage stock was diluted 10-100-fold in MC buffer (100 mM
MgSO\textsubscript{4}, 5 mM CaCl\textsubscript{2}) and 0.05 mL, 0.1 mL
or 0.2 mL of this diluted phage was added to the overlay tubes
containing cells and molten agar and gently mixed. The remaining tube
was left without phage as a control. The contents of each overlay tube
was poured onto P1 agar plates and left to set for overnight growth at
37°C for 18 hours. Soft agar from phage-lysed plates was added to 1 ml
of MC buffer (100 mM MgSO\textsubscript{4} and 5 mM
CaCl\textsubscript{2}) and 0.5 ml of chloroform for vigorous mixing
before centrifugation at 5752 rcf for 20 minutes at 4°C. The supernatant
was retrieved and mixed with chloroform (0.5 mL) for storage at 4°C as a
P1 phage stock. MG1655 recipient strain was grown in a Mu Broth to
OD\textsubscript{600} 0.8 using a shaking water bath. Cells were
pelleted, resuspended in 1 ml of MC buffer, and left at 25°C for 10
minutes. 0.2 ml of cells were added into 3 overlay tubes containing 0
ml, 0.05 ml and 0.2 ml of P1 lysate produced previously and incubated
for 30 minutes at 37°C. Cell/P1 lysate mix was added to 2.5 ml of 0.6\%
agar, mixed gently and poured onto Mu Broth agar plates containing 30
\selectlanguage{greek}µ\selectlanguage{english}g/ml kanamycin and left to set. Plates were grown for 1-2 days, lid-up,
at 37\selectlanguage{ngerman}°C to allow colonies to develop. Colonies were then picked and
purified by streaking onto Mu broth agar plates containing no
antibiotic. This was repeated 3 times before plating again onto agar
containing 30 \selectlanguage{greek}µ\selectlanguage{english}g/ml kanamycin for confirmation of gene knockout and
Kan\textsuperscript{r}-FTR insertion.

Successful P1 treated MG1655 cells were transformed with pCP20.
Transformants were picked and used to inoculate 8 ml of Mu broth
containing no antibiotic. Culture was grown overnight at 45\selectlanguage{ngerman}°C in a
shaking water bath FLP recombinase expression and plasmid curation.
Cells were then streaked onto Mu Broth agar plates to produce single
colonies and grown at 37°C overnight. Colonies were re-streaked 3 times
before replica plating onto Mu Broth agar plates containing 50 \selectlanguage{greek}µ\selectlanguage{english}g/ml
ampicillin, 30 \selectlanguage{greek}µ\selectlanguage{english}g/ml kanamycin and then no antibiotic to confirm loss of
the pCP20 plasmid. Isolates which only grew on the no antibiotic agar
plates were grown overnight for glycerol stock production and streaked a
further time for colony PCR diagnostic confirmation.

\subsubsection*{\texorpdfstring{\emph{E. coli} viability spot
assays}{E. coli viability spot assays}}

{\label{e.-coli-viability-spot-assays}}

Cell viabilities were measured from liquid cultures grown to
OD\textsubscript{600} 0.3-0.4 in a shaking water bath at 37\selectlanguage{ngerman}°C monitored
in the growth tubes by using a Spectronic 20+. Cells were then treated
by addition to the growth media of hydrogen peroxide
(H\textsubscript{2}O\textsubscript{2}) or AZT at concentrations stated
in the results. Cells were grown for a further 30 minutes and then
serially diluted into 1x M9 medium to arrest growth for spotting (10 ul)
on to agar plates grown overnight in a 37°C incubator. For comparing
growth curves cells were grown to OD\textsubscript{600} 0.3-0.4 and then
transferred into a 24-well flat-bottomed plate and
H\textsubscript{2}O\textsubscript{2} was added to appropriate wells to
the given concentration from a 0.98 M stock. Growth in the plates was
monitored with orbital shaking in a FLUOstar microplate reader (BMG
Labtech). OD\textsubscript{600} readings were taken every 30 minutes in
this time, and data was extracted and analyzed using Prism (GraphPad)
software.

\textbf{Author Contributions}

ELB and CDOC designed the project and with RJB wrote the paper. RJB,
KMH, NA and CDOC performed experiments, analyzed data, and generated
images.

\textbf{Acknowledgments}

The work was supported by BBSRC grant BB/T006625-1 (ELB) and the BBSRC
Doctoral Training Partnership (ELB/RJB).

\textbf{References}

1. Hajj, M., et al., \emph{Phylogenetic Diversity of Lhr Proteins and
Biochemical Activities of the Thermococcales aLhr2 DNA/RNA
Helicase.}Biomolecules, 2021. \textbf{11} (7).

2. Reuven, N.B., et al., \emph{The gene for the longest known
Escherichia coli protein is a member of helicase superfamily II.} J
Bacteriol, 1995. \textbf{177} (19): p. 5393-400.

3. Chamieh, H., H. Ibrahim, and J. Kozah, \emph{Genome-wide
identification of SF1 and SF2 helicases from archaea.} Gene,
2016.\textbf{576} (1 Pt 2): p. 214-28.

4. Ejaz, A., Y. Goldgur, and S. Shuman, \emph{Activity and structure of
Pseudomonas putida MPE, a manganese-dependent single-strand DNA
endonuclease encoded in a nucleic acid repair gene cluster.} J Biol
Chem, 2019. \textbf{294} (19): p. 7931-7941.

5. Ordonez, H. and S. Shuman, \emph{Mycobacterium smegmatis Lhr Is a
DNA-dependent ATPase and a 3'-to-5' DNA translocase and helicase that
prefers to unwind 3'-tailed RNA:DNA hybrids.} J Biol Chem,
2013.\textbf{288} (20): p. 14125-34.

6. Ejaz, A., et al., \emph{Structure of mycobacterial 3'-to-5' RNA:DNA
helicase Lhr bound to a ssDNA tracking strand highlights distinctive
features of a novel family of bacterial helicases.} Nucleic Acids Res,
2018. \textbf{46} (1): p. 442-455.

7. Ejaz, A. and S. Shuman, \emph{Characterization of Lhr-Core DNA
helicase and manganese- dependent DNA nuclease components of a bacterial
gene cluster encoding nucleic acid repair enzymes.} J Biol Chem,
2018.\textbf{293} (45): p. 17491-17504.

8. Buttner, K., S. Nehring, and K.P. Hopfner, \emph{Structural basis for
DNA duplex separation by a superfamily-2 helicase.} Nat Struct Mol Biol,
2007. \textbf{14} (7): p. 647-52.

9. Northall, S.J., et al., \emph{DNA binding and unwinding by Hel308
helicase requires dual functions of a winged helix domain.} DNA Repair
(Amst), 2017. \textbf{57} : p. 125-132.

10. Johnson, S.J. and R.N. Jackson, \emph{Ski2-like RNA helicase
structures: common themes and complex assemblies.} RNA Biol,
2013.\textbf{10} (1): p. 33-43.

11. Buckley, R.J., et al., \emph{Mechanistic insights into Lhr helicase
function in DNA repair.} Biochem J, 2020. \textbf{477} (16): p.
2935-2947.

12. Warren, G.M., et al., \emph{Oligomeric quaternary structure of
Escherichia coli and Mycobacterium smegmatis Lhr helicases is nucleated
by a novel C-terminal domain composed of five winged-helix
modules.}Nucleic Acids Res, 2021. \textbf{49} (7): p. 3876-3887.

13. van Wolferen, M., X. Ma, and S.V. Albers, \emph{DNA Processing
Proteins Involved in the UV-Induced Stress Response of Sulfolobales.} J
Bacteriol, 2015. \textbf{197} (18): p. 2941-51.

14. Cooper, D.L., D.C. Boyle, and S.T. Lovett, \emph{Genetic analysis of
Escherichia coli RadA: functional motifs and genetic interactions.} Mol
Microbiol, 2015. \textbf{95} (5): p. 769-79.

15. Rand, L., et al., \emph{The majority of inducible DNA repair genes
in Mycobacterium tuberculosis are induced independently of RecA.} Mol
Microbiol, 2003. \textbf{50} (3): p. 1031-42.

16. Mullins, E.A., et al., \emph{Structure of a DNA glycosylase that
unhooks interstrand cross-links.} Proc Natl Acad Sci U S A,
2017.\textbf{114} (17): p. 4400-4405.

17. Kelley, L.A., et al., \emph{The Phyre2 web portal for protein
modeling, prediction and analysis.} Nat Protoc, 2015. \textbf{10} (6):
p. 845-58.

18. Almatarneh, M.H., C.G. Flinn, and R.A. Poirier, \emph{Mechanisms for
the deamination reaction of cytosine with H2O/OH(-) and 2H2O/OH(-): a
computational study.} J Chem Inf Model, 2008. \textbf{48} (4): p.
831-43.

19. Cadet, J. and J.R. Wagner, \emph{DNA base damage by reactive oxygen
species, oxidizing agents, and UV radiation.} Cold Spring Harb Perspect
Biol, 2013. \textbf{5} (2).

20. Kreutzer, D.A. and J.M. Essigmann, \emph{Oxidized, deaminated
cytosines are a source of C --\textgreater{} T transitions in vivo.}Proc
Natl Acad Sci U S A, 1998. \textbf{95} (7): p. 3578-82.

21. Krokan, H.E., F. Drablos, and G. Slupphaug, \emph{Uracil in
DNA--occurrence, consequences and repair.} Oncogene, 2002.\textbf{21}
(58): p. 8935-48.

22. Aravind, L. and E.V. Koonin, \emph{The alpha/beta fold uracil DNA
glycosylases: a common origin with diverse fates.} Genome Biol,
2000.\textbf{1} (4): p. RESEARCH0007.

23. Savitsky, P., et al., \emph{High-throughput production of human
proteins for crystallization: the SGC experience.} J Struct Biol,
2010.\textbf{172} (1): p. 3-13.

24. Datsenko, K.A. and B.L. Wanner, \emph{One-step inactivation of
chromosomal genes in Escherichia coli K-12 using PCR products.} Proc
Natl Acad Sci U S A, 2000. \textbf{97} (12): p. 6640-6645.

Figure Legends

Figure 1: The \emph{E. coli} Lhr-CTD is a uracil DNA glycosylase
requiring a catalytic aspartic acid

(A) AlphaFold 2 structural model of \emph{E. coli} Lhr that is based on
strong homology with the cryo-EM structure of Lhr helicase core and
Lhr-CTD from \emph{M. smegmatis} , respectively PDB: 5V9X and PDB:7LHL.
The \emph{E. coli} Lhr-core helicase (amino acids 1-897) contains RecA
domains, a beta-sheet bundle (\selectlanguage{greek}β\selectlanguage{english}) 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 \selectlanguage{greek}β/δ
\selectlanguage{english}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 \emph{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-CTD\textsuperscript{D1536A}proteins 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-CTD\textsuperscript{D1536A}.

Figure 2: Lhr\textsuperscript{D1536A} is inactive as a glycosylase but
binds to DNA

(\textbf{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.

(\textbf{B} ) Products of Lhr glycosylase activity seen in an 18\%
acrylamide denaturing TBE gel were absent when reactions contained
Lhr\textsuperscript{D1536A}. Proteins were used at 25, 50, 100 and 200
nM, with 12.5 nM of d-uracil containing 5' Cy5-ssDNA substrate.

(\textbf{C} ) EMSA showing that Lhr\textsuperscript{D1536A} and Lhr
(12.5, 25, 50, 100 and 200 nM) form stable complex with DNA in a 5\%
acrylamide TBE gel

\textbf{Figure 3:} \textbf{Lhr is inactive against 8-oxoguanine, and its
uracil DNA glycosylase activity on duplex DNA functions independently
from Lhr helicase activity}

(\textbf{A} ) Time-dependent uracil DNA glycosylase activity of Lhr (50
nM) compared with Lhr\textsuperscript{D1536A}. The data shows means of
glycosylase activity (n=3, with bars for standard error) alongside a
representative gel used for quantification.

(\textbf{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.

(\textbf{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 \emph{E. coli} uracil DNA glycosylase.

(\textbf{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.

(\textbf{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).

(\textbf{F} ) DNA unwinding by Lhr and
Lhr\textsuperscript{D1536A}proteins (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.

\textbf{Figure 4: \emph{E. coli} cells lacking Lhr are sensitive to
oxidative stress}

(\textbf{A} ) Viability spot tests showing moderately increased
sensitivity of \selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} cells to AZT (7.5 \selectlanguage{greek}µ\selectlanguage{english}g/mL) compared with wild
type (wt) cells, and (\textbf{B} ) represented in viability curves when
\selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} and wild type cells were grown independently in media
containing AZT at 2.5, 5, 7.5 or 10 \selectlanguage{greek}µ\selectlanguage{english}g/mL. The plots show grow relative
to wild type cells grown in media lacking AZT.

(\textbf{B} ) Growth of \selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} and wild type cells monitored in 96
well plates in media containing 12.5 mM
H\textsubscript{2}O\textsubscript{2}, and (\textbf{C} ) viability spot
tests comparing \selectlanguage{greek}Δ\selectlanguage{english}\emph{lhr} and wild type cells grown in
H\textsubscript{2}O\textsubscript{2} added to media at 1.5625, 3.75,
6.25 and 12.5 nM.

\textbf{Hosted file}

\verb`Main Figures - Final.pptx`  available at \url{https://authorea.com/users/576754/articles/619423-e-coli-dna-repair-helicase-lhr-is-also-a-uracil-dna-glycosylase}

\selectlanguage{english}
\FloatBarrier
\end{document}

