Characterization of Lhr-Core DNA helicase and manganese-dependent DNA nuclease components of a bacterial gene cluster encoding nucleic acid repair enzymes

Article abstract of DOI:10.1074/jbc.RA118.005296

Lhr is a large superfamily 2 helicase present in mycobacteria and a moderate range of other bacterial taxa. A shorter version of Lhr, here referred to as Lhr-Core, is distributed widely in bacteria, where it is often encoded in a gene cluster along with predicted binuclear metallo-phosphoesterase (MPE), ATP-dependent DNA ligase, and metallo-β-lactamase exonuclease enzymes. Here we characterized the Lhr-Core and MPE proteins from Pseudomonas putida. We report that P. putida Lhr-Core is an ssDNA-dependent ATPase/dATPase (K_m, 0.37 mM ATP; k_cat, 3.3 s^–1), an ATP-dependent 3'-to-5' single-stranded DNA translocase, and an ATP-dependent 3'-to-5' helicase. Lhr-Core unwinds 3'-tailed duplexes in which the loading/tracking strand is DNA and the displaced strand is either DNA or RNA. We found that P. putida MPE is a manganese-dependent phosphodiesterase that releases p-nitrophenol from bis-p-nitrophenyl phosphate (k_cat, 212 s^–1) and p-nitrophenyl-5'-thymidylate (k_cat, 34 s^-1) but displays no detectable phosphomonoesterase activity against p-nitrophenyl phosphate. MPE is also a manganese-dependent DNA endonuclease that sequentially converts a closed-circle plasmid DNA to nicked circle and linear forms prior to degrading the linear DNA to produce progressively smaller fragments. The biochemical activities of MPE and a structure predicted in Phyre2 point to MPE as a new bacterial homolog of Mre11. Genetic linkage of a helicase and DNA nuclease with a ligase and a putative exonuclease (a predicted homolog of the SNM1/Apollo family of nucleases) suggests that these enzymes comprise or participate in a bacterial DNA repair pathway.

Full text of DOI:10.1074/jbc.RA118.005296

cro
ARTICLE
Characterization of Lhr-Core DNA helicase and manganese-
dependent DNA nuclease components of a bacterial gene
cluster encoding nucleic acid repair enzymes
Received for publication, August 9, 2018, and in revised form, September 11, 2018 Published, Papers in Press, September 17, 2018, DOI 10.1074/jbc.RA118.005296
Anam Ejaz and X Stewart Shuman1
From the Molecular Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York 10065
Edited by Patrick Sung
Lhr is a large superfamily 2 helicase present in mycobacteria
and a moderate range of other bacterial taxa. A shorter version
of Lhr, here referred to as Lhr-Core, is distributed widely in
bacteria, where it is often encoded in a gene cluster along with
predicted binuclear metallo-phosphoesterase (MPE), ATP-de-
pendent DNA ligase, and metallo-␤-lactamase exonuclease
enzymes. Here we characterized the Lhr-Core and MPE pro-
teins from Pseudomonas putida. We report that P. putida Lhr-
Core is an ssDNA-dependent ATPase/dATPase (Km, 0.37 mM
ATP; kcat, 3.3 s؊1), an ATP-dependent 3؅-to-5؅ single-stranded
DNA translocase, and an ATP-dependent 3؅-to-5؅ helicase. Lhr-
Core unwinds 3؅-tailed duplexes in which the loading/tracking
strand is DNA and the displaced strand is either DNA or RNA.
We found that P. putida MPE is a manganese-dependent phos-
phodiesterase that releases p-nitrophenol from bis-p-nitrophe-
nyl phosphate (kcat, 212 s؊1) and p-nitrophenyl-5؅-thymidylate
(kcat, 34 s؊1) but displays no detectable phosphomonoesterase
activity against p-nitrophenyl phosphate. MPE is also a man-
ganese-dependent DNA endonuclease that sequentially con-
verts a closed-circle plasmid DNA to nicked circle and linear
forms prior to degrading the linear DNA to produce progres-
sively smaller fragments. The biochemical activities of MPE and
a structure predicted in Phyre2 point to MPE as a new bacterial
homolog of Mre11. Genetic linkage of a helicase and DNA
nuclease with a ligase and a putative exonuclease (a predicted
homolog of the SNM1/Apollo family of nucleases) suggests that
these enzymes comprise or participate in a bacterial DNA repair
pathway.
tional 3Ј-to-5Ј translocation along single-strand DNA (ssDNA)
and to unwind duplexes en route. Lhr is up-regulated in myco-
bacteria in response to DNA damage (3, 4), and deletion of the
lhr gene sensitizes M. smegmatis to killing by mitomycin C.3
Full-length Lhr homologs are found in bacteria from eight
phyla, being especially prevalent in Actinobacteria and Proteo-
bacteria (1).
The ATPase, DNA translocase, and helicase activities of
mycobacterial Lhr are encompassed within the N-terminal
856-aa segment of the 1507-aa polypeptide (1). The crystal
structure of Lhr-(1–856) in complex with AMPPNP⅐Mg2ϩ and
a “loading/tracking” 3Ј DNA single strand revealed that the
enzyme consists of two N-terminal RecA-like domains that
bind the AMPPNP⅐Mg2ϩ, a central winged helix domain, and a
C-terminal domain of novel tertiary structure (2). All four
domains make contributions to Lhr’s unique DNA interface.
Homologs of Lhr-(1–856), all of similar size (ϳ800–900 aa),
are present in the proteomes of scores of diverse bacterial and
archaeal taxa. We will henceforth refer to this shorter Lhr-like
protein as Lhr-Core. The striking feature of the bacterial Lhr-
Core clade is its genetic organization. Most commonly, Lhr-
Core is encoded in a co-oriented four-gene cluster comprising a
putative exonuclease (Exo) of the metallo-␤-lactamase enzyme
family, an ATP-dependent DNA ligase, Lhr-Core, and a mem-
ber of the binuclear metallo-phosphoesterase (MPE) enzyme
family. We refer to this operon-like arrangement of the four
genes as a “class I” cluster (Fig. 1). Variations on this theme
include cases where one or more additional co-oriented ORFs,
encoding proteins unrelated to nucleic acid enzymology, are
inserted between the DNA ligase and Lhr-Core genes (class Ia
cluster) (Fig. 1). For example, in the class Ia cluster of Pseu-
domonas putida, the intervening ORF encodes a member of the
succinyl glutamate desuccinylase/aspartoacylase enzyme fam-
ily. In another variation, the nuclease and ligase proteins are
fused and encoded by a single continuous ORF upstream of
Lhr-Core (class Ib cluster) (Fig. 1). A distinctive arrangement of
the four genes is found in other bacteria in which the Exo-ligase
cassette has been inverted en bloc so that they are transcribed
divergently from the Lhr-Core and MPE genes; these are desig-
nated class II and IIa clusters (Fig. 1). Finally, other bacteria and
archaea have a two-gene Lhr-Core⅐MPE cassette (dubbed a
class III cluster) that is not linked to Exo or ligase genes (Fig. 1).
Mycobacterium smegmatis Lhr is the founding member of a
new clade of superfamily 2 DNA helicases by virtue of its signa-
ture domain structure and distinctive DNA interface (1, 2). Lhr
is a 1507-amino acid (aa)2 monomeric nucleic acid-dependent
ATPase/dATPase that uses ATP hydrolysis to drive unidirec-
This work was supported by National Institutes of Health Grants AI64693 and
P30CA008748. The authors declare that they have no conflicts of interest
with the contents of this article. The content is solely the responsibility of
the authors and does not necessarily represent the official views of the
National Institutes of Health.
1 To whom correspondence should be addressed. E-mail: s-shuman@
ski.mskcc.org.
2 The abbreviations used are: aa, amino acids; ssDNA, single-strand DNA; Exo,
exonuclease; MPE, metallo-phosphoesterase; SA, streptavidin; NHEJ, non-
homologous end-joining; MBL, metallo-␤-lactamase; pNPP, p-nitrophenyl
phosphate; AMPPNP, adenosine 5Ј-(␤,␥-imino)triphosphate.
3 A. Ejaz, A. Fay, M. Glickman, and S. Shuman, unpublished data.
J. Biol. Chem. (2018) 293(45) 17491–17504 17491
© 2018 Ejaz and Shuman. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.

Bacterial nucleic acid repair enzyme gene cluster
Figure 1. Genetic clustering of bacterial Lhr-Core and three putative nucleic acid repair enzymes. The bacterial ORF encoding Lhr-Core (green arrows) is
typically located within a gene cluster that includes ORFs encoding a binuclear MPE (magenta arrows), an ATP-dependent DNA ligase (gold arrows), and an
exonucleaseofthemetallo-␤-lactamaseenzymefamily(cyanarrows). Theorderandorientationsoftheclusteredgenesmayvaryamongtaxaandareclassified
accordingly in the figure. A bacterial species exemplifying each class is indicated on the left. Where additional, functionally unrelated ORFs are inserted into the
gene cluster, these are denoted by gray arrows. The cases in which only Lhr-Core and MPE comprise a two-gene cluster are designated as class III in the
schematic.
The wide prevalence of a heretofore uncharacterized in P. putida Lhr-Core. A gap in the sequence alignment at res-
gene cluster specifying putative helicase, nuclease, and ligase idue 280 of P. putida Lhr-Core corresponds to a disordered
enzymes suggests to us the existence of a new bacterial nucleic surface loop in the second RecA domain of the M. smegmatis
acid repair pathway centered around Lhr-Core. To understand Lhr structure (in red font in Fig. 2) that is not conserved in the
whether and how these enzymes might contribute to nucleic P. putida protein.
acid repair, we undertook here to purify and characterize bio-
Lhr-Core is a DNA-dependent ATPase
chemically the Lhr-Core and MPE proteins specified by the
class Ia cluster of P. putida. We report that the P. putida Lhr-
Core is a bona fide ATP-driven 3Ј-to-5Ј translocase and DNA
helicase and that the MPE is a manganese-dependent phospho-
diesterase/DNA endonuclease with structural and functional
similarity to Mre11.
To evaluate the enzymatic and physical properties of
P. putida Lhr-Core, we produced the protein in Escherichia coli
as a His10Smt3 fusion and purified it from a soluble extract by
serial nickel affinity, tag removal, and gel filtration steps. Lhr-
Core eluted as a monomer during gel filtration. SDS-PAGE
affirmed the purity of the Lhr-Core preparation (Fig. 3A). Reac-
tion of Lhr-Core with 1 mM [␣-32P]ATP in the presence of 5 mM
magnesium and 5 ␮M 24-mer ssDNA oligonucleotide cofactor
Results
P. putida Lhr-Core
The816-aaP. putidaKT2440Lhr-Corepolypeptideishomo- resulted in the hydrolysis of [␣-32P]ATP to [␣-32P]ADP, as
logous to the 856-aa ATPase/helicase domain of the 1507-a- monitored by polyethyleneimine-cellulose TLC. The extent of
mino acid M. smegmatis Lhr protein (Fig. 2). A primary struc- ATP hydrolysis increased in proportion to the amount of input
ture alignment of P. putida Lhr-Core and M. smegmatis Lhr Lhr-Core, and 60% of the available ATP was hydrolyzed at 1 ␮M
highlights 325 positions of amino acid side chain identity/sim- enzyme (Fig. 3A). The ATPase specific activity, calculated from
ilarity (Fig. 2). The conserved positions include eight side the slope of the titration curve in the linear range of enzyme
chains that make atomic contacts to AMPPNP⅐Mg2ϩ in the dependence, was 2.56 nmol ATP hydrolyzed per 1 pmol Lhr-
M. smegmatis Lhr crystal structure (shaded gold in Fig. 2) and Core in 15 min, which translates into an estimated turnover
seven side chains that contact the ssDNA loading strand number of 2.8 sϪ1. ATPase activity depended on addition of
(shaded green in Fig. 2) (2). In particular, the four amino acids a divalent cation. Divalent cation cofactor specificity was
found to be essential in M. smegmatis Lhr for the coupling of assessed at 0.5, 1, 2.5, 5, and 10 mM magnesium, manganese,
ATP hydrolysis to duplex unwinding (Thr-145, Arg-279, Ile- calcium, cobalt, copper, nickel, and zinc (Fig. 3B). Magnesium
528, and Trp-597; denoted by triangles in Fig. 2) are conserved and calcium were the superior cofactors, supporting vigorous
17492 J. Biol. Chem. (2018) 293(45) 17491–17504

Bacterial nucleic acid repair enzyme gene cluster
Figure 2. Homology of Lhr-Core and Lhr. The top panel depicts, in cartoon form, the domain organization of the 1507-aa M. smegmatis Lhr protein, as
revealed by the crystal structure of its autonomous helicase domain (aa 1–856). Lhr-Core proteins, exemplified by the 816-aa P. putida enzyme, lack the large,
structurally uncharacterized C-terminal domain present in full-length Lhr. The bottom panel shows an alignment of the primary structures of P. putida Lhr-Core
(Ppu) and M. smegmatis Lhr (Msm). Positions of side chain identity/similarity are denoted by ●. Gaps in the alignment are indicated by Ϫ. A disordered segment
oftheMsmLhrtertiarystructurethathasnocounterpartinPpuLhr-Coreisshowninredfont. ConservedLhraminoacidsthatcontactATP⅐Mg2ϩ arehighlighted
in gold shading; conserved side chains that contact the ssDNA loading strand are shaded in green. Four conserved Lhr amino acids essential for coupling of ATP
hydrolysis to duplex unwinding are denoted by triangles.
ATP hydrolysis across the full range of concentrations tested. and became progressively less active at 2.5, 5, and 10 mM. Zinc
Manganese was effective at 0.5 to 2.5 mM and slightly less so at supported activity narrowly at 0.5 to 1 mM but was ineffective at
5 and 10 mM. Cobalt and nickel functioned best at 0.5 and 1 mM a concentration of 2.5 mM or higher. By contrast, copper was
J. Biol. Chem. (2018) 293(45) 17491–17504 17493

Bacterial nucleic acid repair enzyme gene cluster
Figure 3. Characterization of the Lhr-Core ATPase. A, left panel, an aliquot (5 ␮g) of the Superdex-200 fraction of P. putida Lhr-Core was analyzed by
SDS-PAGE. The Coomassie Blue–stained gel is shown. The positions and sizes (kilodaltons) of marker polypeptides are indicated on the left. Right panel, ATPase
reaction mixtures (10 ␮l) containing 20 mM Tris-HCl (pH 7.5), 30 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 mM [␣-32P]ATP (10 nmol ATP), 5 ␮M 24-mer ssDNA
oligonucleotide (5Ј-GGGTCGCAATTGATTCCGATAGTG), and Lhr-Core protein as specified (0.125, 0.25, 0.5, 1, 2, 4, 6, 8, or 10 pmol, corresponding to 0.0125,
0.025, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, or 1 ␮M Lhr-Core) were incubated at 37 °C for 15 min. The extent of [32P]ADP formation is plotted as a function of input enzyme.
Each datum is the average of three independent enzyme titration experiments Ϯ S.E. B, divalent cation specificity. Reaction mixtures (10 ␮l) containing 20 mM
Tris-HCl (pH 7.5), 30 mM NaCl, 1 mM DTT, 1 mM [␣-32P]ATP (10 nmol ATP), 5 ␮M 24-mer ssDNA, 0.5 ␮M (5 pmol) Lhr-Core, and increasing concentrations of the
indicated divalent cations (chloride salt) were incubated at 37 °C for 15 min. The extent of ATP hydrolysis is plotted as a function of metal concentration. Each
datum is the average of three separate metal titration experiments Ϯ S.E. C, NTP specificity. Reaction mixtures (10 ␮l) containing 20 mM Tris-HCl (pH 7.5), 30 mM
NaCl, 5mM MgCl2, 1mM DTT, 5␮M 24-merssDNA, 0.5␮M Lhr-Core, and1mM oftheindicatedNTPordNTPwereincubatedat37 °Cfor30min. Thereactionswere
quenched with 990 ␮l of malachite green reagent (Biomol Research Laboratories). Phosphate release was quantified by measuring A620 and interpolating
the value to a phosphate standard curve. The values were corrected for the low levels of phosphate measured in control reaction mixtures containing
1 mM of the indicated NTP/dNTP but no added enzyme. Data are the average of three separate experiments Ϯ S.E. D, steady-state kinetics. Reaction
mixtures (40 ␮l) containing 20 mM Tris-HCl (pH 7.5), 30 mM NaCl, 5 mM MgCl2, 1 mM DTT, 5 ␮M 24-mer ssDNA, 1 ␮M Lhr-Core, and either 0.0625, 0.125, 0.25,
0.5, or 1.0 mM [␣-32P]ATP were incubated at 37 °C. Aliquots (10 ␮l) were withdrawn at 10, 20, and 30 s (for 0.03125, 0.0625, 0.125, 0.25, and 0.5 mM ATP)
or 15, 30, and 45 s (for 1.0 and 2.0 mM ATP) and quenched immediately with formic acid. The extents of ATP hydrolysis were plotted as a function of time
for each ATP concentration, and the initial rates were derived by linear regression analysis in Prism. The initial rates (picomoles per second) were divided
by the molar amount of input enzyme to obtain a turnover number, V (sϪ1), which is plotted as a function of ATP concentration. Each datum is the
average of three separate experiments Ϯ S.E. A nonlinear regression curve fit of the data to the Michaelis–Menten equation (in Prism) is shown. The Km
and kcat values are indicated.
ineffective at 0.5 and 1 mM but supported activity at 5 and 10 hydrolyzed ATP and dATP; the other rNTPs and dNTPs were
mM. All subsequent assays of Lhr-Core activity employed mag- hydrolyzed poorly or not at all (Fig. 3C).
nesium as the metal cofactor.
Steady-state kinetics of ATP hydrolysis
NTP substrate specificity
We determined steady-state kinetic parameters by measur-
Nucleotide specificity was examined by colorimetric assay of ing the velocity of ATP hydrolysis as a function of ATP concen-
the release of Pi from unlabeled ribonucleotides ATP, GTP, tration in the presence of 5 mM magnesium and 5 ␮M 24-mer
CTP, or UTP and the deoxynucleotides dATP, dGTP, dCTP, ssDNA cofactor (Fig. 3D). From a nonlinear regression curve fit
and dTTP, each at 1 mM concentration. Lhr-Core specifically of the data to the Michaelis–Menten equation, we calculated
17494 J. Biol. Chem. (2018) 293(45) 17491–17504

Bacterial nucleic acid repair enzyme gene cluster
biotinylated 34-mer DNA during native PAGE (Fig. 5). The
translocation assay scores the motor-dependent displacement
of SA from DNA in the presence of ATP and excess free biotin,
which instantly binds to free SA and precludes SA rebinding to
the labeled DNA. The rationale of the assay is that directional
tracking of the motor along the DNA single strand will displace
SA from one DNA end but not the other. As depicted in Fig. 5A,
the enzyme acts as a “cow catcher” on a locomotive engine.
When moving 3Ј to 5Ј, it can displace SA as it collides with the
5Ј biotin–SA. In contrast, a 3Ј biotin–SA complex is not
expected to be displaced by a 3Ј-to-5Ј translocase because the
motor moves away from the SA and simply falls off the free 5Ј
end. The converse outcomes apply to a 5Ј-to-3Ј translocase,
whereby it displaces a 3Ј biotin–SA complex but not a 5Ј
biotin–SA adduct. The instructive finding was that Lhr-Core
displaced the SA from a 5Ј biotin–SA complex on the 34-mer
ssDNA to yield the free 32P-labeled 34-mer strand but was
unable to displace SA from the 3Ј biotin–SA complex tested in
parallel. Stripping of the 5Ј biotin–SA complex by Lhr-Core to
liberate free DNA depended on ATP and magnesium (Fig. 5A).
Enzyme titration experiments with 0.1 ␮M 5Ј biotinylated
34-mer substrate in the presence of 1 mM ATP and 5 mM mag-
nesium revealed that the extent of SA displacement was pro-
portional to the amount of input enzyme and that 90% of the
biotin–SA complex was dissociated at saturating enzyme. In
the linear range of enzyme dependence, Lhr-Core displaced
0.99 Ϯ 0.05 pmol of 5Ј biotin-SA per 1 pmol of Lhr-Core (Fig.
5B). The kinetic profile of SA displacement by 0.5 ␮M Lhr-Core
during a 10-min reaction with 0.1 ␮M of 5Ј biotinylated sub-
strate is shown in Fig. 5C. The data fit well by nonlinear regres-
sion to a two-phase association with apparent rates constants of
Figure 4. Dependence of ATP hydrolysis by Lhr-Core on ssDNA cofactor.
Reaction mixtures (10 ␮l) containing 20 mM Tris-HCl (pH 7.5), 1 mM DTT, 5 mM
MgCl2, 1 mM [␣-32P]ATP, 0.4 ␮M Lhr-Core, and increasing amounts of 24-mer,
18-mer, 12-mer, or 6-mer ssDNA oligodeoxynucleotides as specified (0.06,
0.13, 0.25, 0.5, 1, 2, 3, 4, 5, or 10 pmol, corresponding to 0.006, 0.013, 0.025,
0.05, 0.1, 0.2, 0.3, 0.4, 0.5, or 1 ␮M ssDNA) were incubated at 37 °C for 15 min.
The extent of ATP hydrolysis is plotted as a function of input oligonucleotide.
Each datum is the average of three separate DNA titration experiments Ϯ S.E.
A nonlinear regression curve fit of the data to a one-site specific binding
model (in Prism) is shown. The nucleobase sequences of the ssDNA oligonu-
cleotides and the calculated Kd values are shown below the graph.
Ϫ1
3.35 Ϯ 0.87 minϪ1 (percent fast 53 Ϯ 11) and 0.49 Ϯ 0.17 min
that Lhr-Core has a Km of 0.37 Ϯ 0.038 mM ATP and a kcat of
Ϫ1
(Fig. 5C).
3.33 Ϯ 0.12 s
.
Lhr-Core is a 3؅-to-5؅ helicase
Single-strand DNA length requirement for ATP hydrolysis
In light of the translocase activity demonstrated above, we
tested Lhr-Core for helicase activity with a series of 3Ј-tailed
duplex substrates consisting of a 24-bp duplex with a 15-mer 3Ј
single-strand tail to serve as a loading strand (Fig. 6). The
39-mer loading/tracking strand was a synthetic DNA or RNA
oligonucleotide of identical nucleobase sequence (excepting U
for T in RNA). The 5Ј 32P-labeled 24-mer displaced strand
was a DNA or RNA oligonucleotide of identical nucleobase
sequence complementary to that of the 39-mer strand. The
helicase assay format entailed preincubation of Lhr-Core and
labeled nucleic acid substrate, followed by initiation of unwind-
ing by addition of ATP, with simultaneous addition of a “trap”
We tested the ability of ssDNAs of varying length to activate
ATP hydrolysis by Lhr-Core. Titration of the oligonucleotides
revealed a hyperbolic dependence of ATP hydrolysis on the
amount of 24-mer, 18-mer, or 12-mer strands with similar
extents of hydrolysis at saturating DNA concentrations (Fig. 4).
Nonlinear regression fitting of the data to a one-site binding
model in Prism yielded apparent Kd values as follows: 44 nM
24-mer, 65 nM 18-mer, and 113 nM 12-mer. Shortening the
DNA cofactor to a 6-mer caused a marked shift to the right in
the titration curve, with an apparent Kd value of 725 nM 6-mer.
Lhr-Core is a 3؅-to-5؅ DNA translocase
NTP hydrolysis by nucleic acid–dependent NTP phospho- of excess unlabeled 24-mer displaced strand. The trap strand
hydrolases is often coupled to mechanical work, either duplex minimizes reannealing of any radiolabeled 24-mer that was
unwinding or displacement of protein–nucleic acid complexes, unwound by Lhr-Core, and it competes with the loading strand
as a consequence of translocation of the phosphohydrolase for binding to any free Lhr-Core or Lhr-Core that dissociated
enzyme along the nucleic acid. To address whether Lhr-Core from the labeled 3Ј-tailed duplex without unwinding it. Conse-
has translocase activity, we employed a streptavidin (SA) dis- quently, the assay predominantly gauges a single round of
placement assay (1, 2, 6) as follows. 5Ј 32P-labeled 34-mer DNA strand displacement by Lhr-Core bound to the labeled 3Ј-tailed
oligonucleotides containing a single biotin moiety at the fourth duplex prior to the onset of ATP hydrolysis. The salient find-
inter-nucleotide from the 5Ј end or the second inter-nucleotide ings from the assay were as follows. Lhr-Core unwound
from the 3Ј end were preincubated with excess SA to form a 3Ј-tailed RNA–DNA and DNA–DNA substrates to yield a
stable SA–DNA complex that was easily resolved from the free radiolabeled free single-strand RNA and DNA, respectively,
J. Biol. Chem. (2018) 293(45) 17491–17504 17495

Bacterial nucleic acid repair enzyme gene cluster
Figure 5. Lhr-Core DNA translocase activity. A, streptavidin displacement assays were performed as described under “Experimental procedures.” The
nucleobase sequences of the 5Ј or 3Ј biotinylated 34-mer ssDNAs are shown at bottom; B denotes the position of the biotin spacer. The complete translocase
reaction mixtures contained 20 mM Tris-HCl (pH 7.5), 30 mM NaCl, 5 mM MgCl2 (where denoted by ϩ over the lane), 1 mM DTT, 1 mM ATP (where indicated by
ϩ), 0.1 ␮M 32P-labeled 5Ј biotinylated DNA attached to SA (on the left side) or 3Ј biotinylated DNA attached to SA (on the right side), and 2 ␮M Lhr-Core (where
indicated by ϩ). Control mixtures lacking Lhr-Core and SA were included in the leftmost lanes of each series. Other components were omitted where indicated
by Ϫ above the lanes. Reactions lacking magnesium were supplemented with 10 mM EDTA where indicated by ϩ. The reaction products were analyzed by
nativePAGE, andthe32P-labeledDNAwasvisualizedbyautoradiographyofthedriedgel. ThespeciescorrespondingtotheSA–DNAcomplexandfreeDNAare
indicated on the left. B, translocase reaction mixtures (10 ␮l) containing 20 mM Tris-HCl (pH 7.5), 30 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 mM ATP, 0.1 ␮M
32P-labeled5ЈbiotinylatedSA–DNAcomplex, andLhr-Coreasspecified(0.08, 0.16, 0.31, 0.63, 1.25, 2.5, 5, or10pmol, correspondingto0.008, 0.016, 0.031, 0.063,
0.125, 0.25, 0.5, or 1 ␮M Lhr-Core) were incubated at 37 °C for 15 min. The products were analyzed by native PAGE. The extent of translocation ((free
DNA)/(SA-DNA ϩ free DNA)) is plotted as a function of input enzyme. Each datum represents the average of three separate enzyme titrations Ϯ S.E. C, reaction
mixtures (100 ␮l) containing 20 mM Tris-HCl (pH 7.5), 30 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 mM ATP, 0.1 ␮M 32P-labeled 5Ј-biotinylated SA-DNA complex, and
0.5 ␮M Lhr were incubated at 37 °C. Aliquots (10 ␮l) were withdrawn at the times specified and quenched by adding 3 ␮l of a solution containing 200 mM EDTA,
0.6% SDS and 25% glycerol, and 20 ␮M unlabeled single-stranded 59-mer DNA oligonucleotide. The products were analyzed by native PAGE. The extent of
translocation is plotted as a function of reaction time. Each datum represents the average of three separate time course experiments Ϯ S.E.
that migrated faster than the input tailed duplex during native sition metal ion center to catalyze phosphomonoester or phos-
PAGE and comigrated with free 24-mer generated by thermal phodiester hydrolysis (7). Structurally and biochemically well-
denaturation of the substrates (Fig. 6, lanes ⌬). Lhr-Core failed
to unwind an RNA–RNA duplex, and Lhr-Core did not unwind
a DNA–RNA hybrid with a 39-mer RNA loading strand (Fig. 6).
These results establish that Lhr-Core is a unidirectional motor,
powered by ATP hydrolysis, that tracks 3Ј to 5Ј along a DNA
loading strand and unwinds duplexes en route.
studied binuclear MPE enzymes play diverse physiological
roles, e.g. as agents of signal transduction (phosphoprotein
phosphatase calcineurin), RNA processing (RNA debranching
enzyme Dbr1), DNA repair (DNA nuclease Mre11), and RNA
repair (bacterial Pnkp phosphoesterase) (8–12). Submission of
the P. putida MPE amino acid sequence to the Phyre2 structure
prediction server (13) generated a “top hit” structure model
(99.7% confidence) of MPE based on homology to the crystal
structure of Methanocaldococcus jannaschii Mre11 bound to
P. putida MPE
P. putida gene PP_1102 encodes a 216-aa protein (Fig. 7, top
panel) that is a member of the binuclear MPE enzyme super- manganese (PDB code 3AZU). The modeled active site of
family. MPE enzymes characteristically utilize a binuclear tran- P. putida MPE is shown in Fig. 7, bottom panel, with two
17496 J. Biol. Chem. (2018) 293(45) 17491–17504

Bacterial nucleic acid repair enzyme gene cluster
manganese ions (superimposed from the M. jannaschii Mre11
structure) coordinated by conserved MPE side chains Asp-33,
His-35, Asp-78, Asn-112, His-113, His-142, His-156, and His-
158. MPE His-113 is predicted to interact with the scissile phos-
phate of the substrate. These eight active site residues are high-
lighted in green shading in the amino acid sequence in Fig. 7.
MPE is a manganese-dependent p-nitrophenyl
phosphodiesterase
To gain insight into the catalytic activities of P. putida MPE,
we produced the protein in E. coli as a His10Smt3 fusion and
purified it from a soluble extract by serial nickel affinity, tag
removal, and gel filtration steps. MPE eluted as a monomer
during gel filtration. The protein was tested for its ability to
hydrolyze the generic non-nucleotide phosphoester substrates
p-nitrophenyl phosphate (a phosphomonoester) and bis-p-ni-
trophenyl phosphate (a phosphodiester). Hydrolysis of these
compounds liberates a chromogenic product, p-nitrophenol,
that is easily quantified by its absorbance at 410 nm. MPE read-
ily converted 10 mM bis-p-nitrophenyl phosphate into p-nitro-
phenol in the presence of a divalent cation. When various met-
als were tested at 2 mM concentration, the divalent cation
requirement for bis-p-nitrophenyl phosphate hydrolysis was
satisfied best by manganese (Fig. 8A). Cobalt and cadmium
were 69% and 50% as effective as manganese; nickel was 6% as
effective. Other divalent cations added at the same concentra-
tion (calcium, magnesium, copper, and zinc) were ineffective.
No p-nitrophenol release was detected when 1 ␮g of MPE was
reacted with 10 mM p-nitrophenyl phosphate and any of the
divalent metals tested. We conclude that MPE is a manganese-
dependent phosphodiesterase.
Figure 6. Lhr-Core helicase activity. Reaction mixtures (10 ␮l) contained 20
mM Tris-HCl (pH 7.5), 30 mM NaCl, 5 mM MgCl2 1 mM DTT, 1 mM ATP, 0.1 ␮M of
the indicated 3Ј-tailed duplex substrate (depicted at the bottom, with the 5Ј
32P label denoted by ●), and 0.5 ␮M Lhr-Core (indicated by ϩ). Lhr-Core was
omitted from the reaction mixtures in lanes marked Ϫ. Reaction mixtures
lackingLhr-Corethatwereheat-denaturedpriortoPAGEareincludedinlanes
marked ⌬. The reaction products were analyzed by native PAGE and visual-
ized by autoradiography.
The extent of hydrolysis of 10 mM bis-p-nitrophenyl phos-
phate during a 30-min reaction with MPE in the presence of 2
mM manganese was proportional to the amount of input
enzyme (Fig. 8B). The amount of product formed by 1 ␮g of
MPE in 30 min represented hydrolysis of 87% of the input bis-
p-nitrophenyl phosphate. From the slope of the titration curve
in the linear range of enzyme dependence, we calculated that
46.2 Ϯ 1.6 nmol of bis-p-nitrophenyl phosphate were hydro-
lyzed per 1 pmol of MPE during a 30-min reaction. The kinetic
profiles of p-nitrophenol release by 3.125, 6.25, and 25 pmol
MPE during a 30-min reaction with 10 mM bis-p-nitrophenyl
phosphate revealed that the rates and extents of product for-
mation were proportional to the input enzyme (Fig. 8C). For-
mation of p-nitrophenol by MPE displayed a hyperbolic depen-
dence on the concentration of bis-p-nitrophenyl phosphate
(Fig. 8D). From a nonlinear regression fit of the data to the
Michaelis–Menten equation, we calculated a Km of 4.85 mM
Ϫ1
and a kcat of 212 s
.
To further query the substrate specificity of MPE, we assayed
the enzyme for activity with p-nitrophenyl-5Ј-thymidylate
(pNP-TMP), a phosphodiesterase-nuclease substrate (Fig. 9).
MPE catalyzed the hydrolysis of 10 mM pNP-TMP to yield p-ni-
trophenol. pNP-TMP hydrolysis was even more strictly depen-
dent on manganese compared with bis-p-nitrophenyl phos-
phate hydrolysis under the same experimental conditions.
Other divalent metals added at the same concentration were
largely ineffective as cofactors for pNP-TMP hydrolysis, with
Figure7. P. putidaMPEandahomologymodelofitsactivesite. Toppanel,
the amino acid sequence of P. putida MPE polypeptide, with the predicted
metal and phosphate ligands highlighted in green shading. Bottom panel,
stereo view of the model of the P. putida MPE active site generated in Phyre2
based on homology to the crystal structure of M. jannaschii Mre11 in complex
with manganese (PDB code 3AUZ). The P. putida MPE active site side chains
are depicted as stick models. The two manganese ions are shown as magenta
spheres.
J. Biol. Chem. (2018) 293(45) 17491–17504 17497

Bacterial nucleic acid repair enzyme gene cluster
Figure 8. MPE is a manganese-dependent p-nitrophenyl phosphodiesterase. A, reaction mixtures (50 ␮l) containing 100 mM Tris-HCl (pH 8.0), 30 mM NaCl,
10 mM pNPP or bis-pNPP, 1 ␮g MPE (41.2 pmol, corresponding to 0.82 ␮M MPE), and either no divalent cation (lane Ϫ) or 2 mM calcium, cadmium, cobalt,
copper, magnesium, manganese, nickel, or zinc (as chloride salts) were incubated at 37 °C for 30 min. The extents of p-nitrophenol formation are plotted in bar
graph format. Each datum represents the average of three separate experiments Ϯ S.E. B, reaction mixtures (50 ␮l) containing 100 mM Tris-HCl (pH 8.0), 30 mM
NaCl, 2 mM MnCl2, 10 mM bis-pNPP, and MPE as specified (0.78, 1.56, 3.13, 6.25, 12.5, 25, or 50 pmol, corresponding to 0.016, 0.031, 0.063, 0.125, 0.25, 0.5, or 1
␮M MPE) were incubated 37 °C for 30 min. The extent of p-nitrophenol formation is plotted as a function of input enzyme. Each datum represents the average
of three independent enzyme titration experiments Ϯ S.E. C, reaction mixtures (500 ␮l) containing 100 mM Tris-HCl (pH 8.0), 30 mM NaCl, 2 mM MnCl2, 10 mM
bis-pNPP, and either 3.125 pmol (filled circles), 6.25 pmol (open squares), or 25 pmol (open circles) MPE protein were incubated at 37 °C. Aliquots (50 ␮l) were
withdrawnatspecifiedtimesandquenchedwithEDTAandNa2CO3. Theextentofp-nitrophenolformationisplottedasafunctionofreactiontime. Eachdatum
represents the average of three independent time course experiments Ϯ S.E. D, steady-state kinetic parameters for hydrolysis of bis-pNPP. Reaction mixtures
(50 ␮l) containing 20 mM Tris-HCl (pH 7.5), 30 mM NaCl, 2 mM MnCl2, 0.125 ␮M (6.25 pmol) MPE, and 0.625, 1.25, 2.5, 5, or 10 mM bis-pNPP were incubated at 37 °C
for 1 min. p-nitrophenol formation (nanomoles per minute) is plotted as a function of bis-pNPP concentration. Each datum is the average of three separate
experiments Ϯ S.E. A nonlinear regression curve fit of the data to the Michaelis–Menten equation (in Prism) is shown. The Km and kcat values are indicated.
cadmium, cobalt, and nickel supporting 4–10% the level of circle DNA into nicked-circle DNA within 1 to 2 min, and the
activity seen with manganese (Fig. 8A). We determined steady- nicked-circle DNA was in turn converted to 2.7-kb linear DNA
state kinetic parameters for Mn2ϩ-dependent hydrolysis of
pNP-TMP as a function of pNP-TMP concentration. From a
between 2 and 10 min (Fig. 10A). Between 5 and 45 min, the
linear DNAs were progressively shortened (Fig. 10A).
nonlinear regression fit of the data to the Michaelis–Menten
The order of events in the time course of the MPE reaction
with pUC19 was echoed when we analyzed the products of a
30-min reaction at varying levels of input MPE (Fig. 10B). At
limiting MPE (0.5 ␮g), the closed-circle DNA was converted to
nicked-circle DNA. Increasing MPE to 1 and 2 ␮g prompted the
appearance of linear DNA, and a further increase to 4 ␮g MPE
resulted in digestion to DNA fragments of less than 1 kb (Fig.
10B). No digestion of the pUC19 DNA substrate was observed
when 2 mM magnesium was added as the divalent cation (Fig.
10B). We conclude that P. putida MPE is a manganese-depen-
dent DNA endonuclease.
Ϫ1
equation, we calculated a Km of 11.3 mM and a kcat of 34 s
.
These results indicate that MPE has vigorous phosphodiester-
ase activity and does not hydrolyze phosphomonoesters.
MPE is a Mn2؉-dependent DNase
In light of the predicted homology of P. putida MPE to
Mre11 and its ability of hydrolyze p-nitrophenyl-5Ј-thymidy-
late to p-nitrophenol and TMP (Fig. 9), we tested MPE for
deoxynuclease activity by reacting the protein with pUC19
plasmid DNA in the presence of 2 mM manganese. Aliquots of
the reaction mixture were withdrawn at 1, 2, 5, 10, 20, 30, and 45
min and analyzed by native agarose gel electrophoresis in the
presence of ethidium bromide to stain the DNA (Fig. 10A). The
To check that the observed phosphodiesterase and nuclease
activities are inherent to P. putida MPE, we produced and puri-
fied a mutated version of MPE in which the putative active site
input pUC19 DNA substrate comprised a mixture of closed residue Asp-78 (Fig. 7) was changed to alanine. SDS-PAGE
circle (the major species), nicked circle, and dimer circle forms showed comparable purity of the WT and D78A proteins (Fig.
(Fig. 10A, time 0). Reaction with MPE converted the closed- 11A). The D78A mutation reduced manganese-dependent bis-
17498 J. Biol. Chem. (2018) 293(45) 17491–17504

Bacterial nucleic acid repair enzyme gene cluster
Figure 9. Hydrolysis of p-nitrophenyl-5؅-thymidylate. A, divalent cation specificity. Reaction mixtures (50 ␮l) containing 100 mM Tris-HCl (pH 8.0), 30 mM
NaCl, 10 mM pNP-TMP, 1 ␮g MPE (41.2 pmol, corresponding to 0.82 ␮M MPE), and either no divalent cation (lane Ϫ) or 2 mM calcium, cadmium, cobalt, copper,
magnesium, manganese, nickel, or zinc (as chloride salts) were incubated at 37 °C for 30 min. The extent of p-nitrophenol formation is plotted in bar graph
format. Each datum represents the average of three separate experiments Ϯ S.E. B, kinetic parameters. Reaction mixtures (50 ␮l) containing 100 mM Tris-HCl
(pH 8.0), 30 mM NaCl, 2 mM MnCl2, 0.5 ␮M (25 pmol) MPE, and 0.625, 1.25, 2.5, 5, or 10 mM pNP-TMP were incubated at 37 °C for 1 min. p-nitrophenol formation
(nanomoles per minute) is plotted as a function of pNP-TMP concentration. Each datum is the average of three separate experiments Ϯ S.E. A nonlinear
regression curve fit of the data to the Michaelis–Menten equation (in Prism) is shown. The Km and kcat values are indicated.
Figure 10. MPE is a manganese-dependent DNase. A, reaction mixtures containing (per 20 ␮l) 20 mM Tris-HCl (pH 8.0), 30 mM NaCl, 2 mM MnCl2, 800 ng of
circular pUC19 DNA, and 4 ␮g MPE (165 pmol, 8.24 ␮M MPE) were incubated at 37 °C. Aliquots (20 ␮l) were withdrawn at the specified times and quenched
immediately by adding 10 ␮l of a solution containing 50% glycerol, 50 mM EDTA, and 0.3% SDS. The reaction products were analyzed by electrophoresis
through a 0.7% native agarose gel and visualized by staining with ethidium bromide. The closed circle, nicked circle, and linear forms of pUC19 DNA are
indicated on the left. The positions and sizes (kilobase pairs) of linear DNA markers are shown on the right. B, reaction mixtures (20 ␮l) containing 20 mM Tris-HCl
(pH 8.0), 30 mM NaCl, 800 ng of circular pUC19 DNA, 2 mM MnCl2 or MgCl2 (where indicated by ϩ), and MPE as indicated (0.5, 1, 2, or 4 ␮g, corresponding to 1.03,
2.06, 4.12, or 8.24 ␮M MPE) were incubated at 37 °C for 30 min. The reaction products were analyzed and visualized as specified in A.
p-nitrophenylphosphatase activity to 6% of the WT (Fig. 11B)
We show here that P. putida Lhr-Core is an ssDNA-depen-
and similarly attenuated the DNA endonuclease activity dent ATPase/dATPase, an ATP-dependent 3Ј-to-5Ј ssDNA
translocase, and an ATP-dependent 3Ј-to-5Ј helicase. Lhr-Core
unwinds 3Ј-tailed duplexes in which the loading/tracking
strand is DNA and the displaced strand is either DNA or RNA.
These properties of P. putida Lhr-Core echo those of M. smeg-
matis Lhr, with a few differences: P. putida Lhr-Core ATPase is
most active with magnesium as the metal cofactor, whereas
M. smegmatis Lhr-Core ATPase is most active with calcium (1),
and M. smegmatis Lhr prefers to unwind a 3Ј-tailed duplex with
a DNA loading strand in which the displaced strand is RNA (1).
We report that P. putida MPE is a manganese-dependent
(Fig. 11C).
Discussion
Interest in Lhr helicase is driven by its distinctive domain
organization and mode of ssDNA binding and the strong induc-
tion of its expression after DNA damage in mycobacteria (1–4).
Having solved the crystal structure of the M. smegmatis Lhr-
(1–856) (2) and noting the widespread distribution of Lhr-Core
homologs in diverse bacteria as part of a gene cluster of putative
nucleic acid repair enzymes (Fig. 1), we were eager to biochem-
ically characterize Lhr-Core and its cluster neighbor MPE from phosphodiesterase that releases p-nitrophenol from bis-p-ni-
an exemplary bacterium; in this case, P. putida.
trophenyl phosphate and p-nitrophenyl-5Ј-thymidylate but
J. Biol. Chem. (2018) 293(45) 17491–17504 17499

Bacterial nucleic acid repair enzyme gene cluster
Figure 11. Effect of mutating MPE active site residue Asp-78. A, MPE purity. Aliquots (5 ␮g) of the Superdex preparations of WT MPE and the D78A mutant
were analyzed by SDS-PAGE. The Coomassie Blue–stained gel is shown. The position and sizes (kilodaltons) of marker polypeptides are indicated on the left. B,
phosphodiesterase reaction mixtures (50 ␮l) containing 100 mM Tris-HCl (pH 8.0), 30 mM NaCl, 10 mM bis-pNPP, 2 mM MnCl2, and 1 ␮g (41.2 pmol, 0.82 ␮M) WT
or D78A MPE were incubated at 37 °C for 30 min. The extent of formation of p-nitrophenol is plotted. Each datum represents the average of three separate
experiments Ϯ S.E. C, DNase reaction mixtures (20 ␮l) containing 20 mM Tris-HCl (pH 8.0), 30 mM NaCl, 800 ng of circular pUC19 DNA, 2 mM MnCl2, and either
no enzyme (lane Ϫ) or 4 ␮g WT or D78A MPE (165 pmol, 8.24 ␮M MPE) were incubated at 37 °C for 30 min. The reaction products were analyzed by
electrophoresis through a 0.7% native agarose gel and visualized by staining with ethidium bromide. The closed circle, nicked circle, and linear forms of pUC19
DNA are indicated on the right. The positions and sizes (kilobase pairs) of linear DNA markers are shown on the left.
displays no detectable phosphomonoesterase activity against
The fourth gene in the Lhr-Core cluster encodes a putative
p-nitrophenyl phosphate. The key finding regarding a putative nuclease of the metallo-␤-lactamase (MBL) family. MBL
nucleic acid repair function is that MPE is a manganese-depen- nucleases that play key roles in RNA and DNA transactions
dent DNA endonuclease that sequentially converts a closed- include the endoribonucleases tRNAase Z (3Ј processing of
circle plasmid DNA to nicked-circle and linear forms prior to tRNA) and CPSF-73 (3Ј end formation of mRNA), the DNA 5Ј
degrading the linear DNA to produce progressively smaller exonucleases SNM1A and SNM1B/Apollo (repair of inter-
fragments. The biochemical activities of MPE and structure strand DNA cross-links), and the DNA endonuclease Artemis
prediction in Phyre2 point to MPE as a new bacterial homolog (DNA break repair via NHEJ) (16, 17). The catalytic unit of MBL
of Mre11. Mre11 is the manganese-dependent nuclease subunit nucleases consists of an MBL domain and a ␤-CASP domain.
of a DNA end–processing complex (which also includes an The phosphodiesterase active site is formed by two catalytic
SMC-like ATPase subunit, Rad50) that functions in cellular zinc ions coordinated by an ensemble of histidine and aspartate
pathways of double-strand break repair, e.g. homologous side chains in the MBL domain (16). Phyre2 analysis of the
recombination and nonhomologous end-joining (NHEJ) (14). putative 338-aa P. putida Exo protein returned a structural
The purified recombinant P. putida MPE enzyme is active as a model (100% confidence) based on homology to the crystal
DNA nuclease in the absence of other P. putida proteins. structure of human SNM1B/Apollo (18). The fold of the Phyre2
Although Lhr-Core is an ATPase, it is structurally and func- model of P. putida Exo (Fig. 12A) consists of MBL and ␤-CASP
tionally distant from Rad50-like SMC proteins, particularly domains. The Phyre2-generated primary structure alignment
because it lacks their characteristic coiled-coil structures. Pre- of P. putida Exo and human Apollo highlights 79 positions of
liminary experiments using the recombinant P. putida Lhr- side chain identity/similarity spanning the segment of P. putida
Core and MPE proteins showed that they did not form a het- Exo from amino acids 8 to 304 (Fig. 12C). A stereo view of the
eromeric complex when mixed in vitro and that addition of structural model of the active site of P. putida Exo is shown in
Lhr-Core and ATP did not stimulate the DNase activity of Fig. 12B, superimposed on the active site ligands of the Apollo
MPE.
crystal structure (PDB code 5AHO), these being two zinc ions,
Clustering on the bacterial chromosome of genes encoding a zinc-bound water, and tartrate anion posited to mimic the
enzymes with related biochemical activities is suggestive of scissile phosphodiester (18). The histidine and aspartate side
their participation in a common physiological pathway. In this chains that coordinate the zinc ions and tartrate in Apollo are
case, we find that bona fide helicase (Lhr-Core) and DNA conserved in P. putida Exo (Fig. 12C). Although this in silico
endonuclease (MPE) enzymes are genetically linked to a puta- exercise affirms that P. putida Exo is likely to be a nuclease, our
tive DNA ligase and a putative exonuclease. The 552-aa aim to biochemically characterize P. putida Exo has been sty-
P. putida DNA ligase belongs unambiguously to the ATP-de- mied by the insolubility of the protein when produced in E. coli
pendent DNA ligase clade; per BLAST search with its amino via the same protocols used to make Lhr-Core and MPE.
acid sequence and analysis in Phyre2 (13), the ligase consists of
Nonetheless, it is tempting at this juncture to speculate that
an N-terminal DNA-binding domain, a central adenylyltrans- Exo is a candidate ortholog of SNM1A, SNM1B/Apollo, or
ferase domain, and a C-terminal OB domain, which are homo- Artemis and thereby might play a role, along with the helicase
logous to the equivalent domains of human DNA ligase I (15). Lhr-Core, endonuclease MPE, and DNA ligase, in a dedicated
17500 J. Biol. Chem. (2018) 293(45) 17491–17504

Bacterial nucleic acid repair enzyme gene cluster
Figure 12. Structural model of P. putida Exo. A, Phyre2-modeled tertiary structure of Exo based the on the crystal structure of the human 5Ј exonuclease
Apollo (18) and consisting of metallo-␤-lactamase and ␤-CASP domains. The N and C termini are indicated. B, stereo view of the active site of the Phyre2-
modeled Exo superimposed on the active site ligands of the Apollo crystal structure (PDB code 5AHO), these being two zinc ions (green spheres), a zinc-bound
water (red sphere), and a tartrate anion (stick model with gray carbons) posited to mimic the scissile phosphodiester (18). The Exo histidine and aspartate side
chains that coordinate the zinc ions and tartrate are shown as stick models with beige carbons. Atomic contacts are denoted by dashed lines. C, Phyre2-based
alignmentoftheaminoacidsequencesofPpuExoandhumanApollo. Positionsofsidechainidentity/similarityareindicatedbydotsabovethesequence. Gaps
in the alignment are denoted by dashes. The conserved active site residues depicted in B are indicated by arrowheads below the Apollo sequence.
DNA repair pathway, potentially involving inter-strand DNA have a class III Lhr-Core⅐MPE cluster, Caulobacter crescentus
cross-links (an appealing idea in light of the apparent role of and Dinoroseobacter shibae (neither of which has a full-length
mycobacterial Lhr and eukaryal SNM1s in mitomycin C sensi- Lhr), are sensitized to cisplatin when the Lhr-Core or MPE
tivity)3 or NHEJ.
genes are disrupted by transposons (20).
It is worth noting that the P. putida KT2440 proteome (5)
A potential role for the MPE nuclease in NHEJ is attractive
has, in addition to the Lhr-Core helicase that is part of the given MPE’s relatedness to Mre11. Bacterial NHEJ, as under-
class Ia cluster, a full-length Lhr protein (1434 aa; NCBI stood in M. smegmatis, is genetically dependent on the end-
NP_743222.1) encoded elsewhere on the bacterial chromo- binding protein Ku and the multifunctional end processing and
some (locus tag PP_1061). To our knowledge, there has been no sealing enzyme LigD (21). Mycobacterial NHEJ is mutagenic,
report of the effects of genetically ablating these enzymes in by virtue of the addition and excision of nucleotides at the DSB
P. putida. However, a recent study via genome-wide trans- ends prior to end sealing by DNA ligase (21). Nucleotide addi-
poson mutagenesis of P. fluorescens indicated that the full- tion at DSB ends is performed by the polymerase module of
length Lhr and each of the four enzymes of the Exo⅐ligase⅐Lhr- LigD (21), but the nuclease(s) responsible for end resection dur-
Core⅐MPE cluster were inessential for viability and that strains ing bacterial NHEJ are unknown. Although M. smegmatis and
with transposon inserts in these genes were not sensitized to Mycobacterium tuberculosis lack the Exo⅐ligase⅐Lhr-Core⅐MPE
the DNA cross-linking agent cisplatin (20). It is conceivable gene cluster, the cluster is present in many Pseudomonas spe-
that Pseudomonas fluorescens Lhr and Lhr-Core are genetically cies (albeit not in Pseudomonas aeruginosa). P. putida, like
redundant in this regard. On the other hand, two bacteria that P. aeruginosa (22), has both Ku and LigD. We speculate that
J. Biol. Chem. (2018) 293(45) 17491–17504 17501

Bacterial nucleic acid repair enzyme gene cluster
one or more of the enzymes in the Exo⅐ligase⅐Lhr-Core⅐MPE The polypeptide compositions of the eluate fractions were
gene cluster can contribute to NHEJ, either in a pathway paral- monitored by SDS-PAGE. The recombinant proteins eluted in
the 200 and 500 mM imidazole fractions, which were pooled,
supplemented with the Smt3-specific protease Ulp1 (at a
His10Smt3-Lhr-Core:Ulp1 or His10Smt3-MPE:Ulp1 ratio of
1000:1), and then dialyzed overnight against 4 liters of buffer C
(20 mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM DTT, 20 mM
imidazole, 10% glycerol). The tag-free proteins were separated
from His10Smt3 by applying the dialysates to a 2-ml nickel-
nitrilotriacetic acid–agarose column that had been equilibrated
with buffer C; the Lhr-Core and MPE proteins were recovered
in the flow-through fractions. The tag-free Lhr-Core and MPE
preparations were then subjected to gel filtration through a
120-ml Superdex-200 column equilibrated with buffer D (20
mM Tris-HCl (pH 8.0), 300 mM NaCl, 1 mM DTT, 10% glycerol).
The gel filtration column had been calibrated with a mixture of
size standards: thyroglobulin, ␥-globulin, ovalbumin, myoglo-
bin, and vitamin B12. Lhr-Core chromatographed as a single
symmetrical peak at an elution volume of 75 ml; interpolation
to the standard curve (of the log of the molecular weights of
standards versus their elution volume) indicated a native size of
88 kDa, consistent with the calculated size of 90 kDa from the
Lhr-Core amino acid sequence. MPE eluted as a single peak at
86 ml, corresponding to a native size of 28 kDa, versus the cal-
culated size of 24 kDa. We surmise that Lhr-Core and MPE are
monomers in solution. Peak Superdex fractions were pooled
and concentrated by centrifugal ultrafiltration. Protein concen-
trations were determined with Bio-Rad dye reagent using BSA
as the standard. The final yield of Lhr-Core was 7 mg per liter of
bacterial culture. The yields of WT MPE and mutant MPE-
D78A were 25 mg per liter and 3 mg per liter, respectively.
lel to Ku-LigD or in the Ku-LigD pathway as backup activities,
e.g. for the DNA ligase (21).
Finally, we refer the interested reader to an Lhr-Core–based
subsystem in the SEED viewer (19) that tabulates the distribu-
tion of Lhr-Core in bacterial and archaeal genomes and its clus-
tering with MPE, DNA ligase, and/or metallo-␤-lactamase Exo
enzymes. In brief, Lhr-Core is clustered genetically with the
other three enzymes in various bacteria belonging to the
phyla Proteobacteria, Bacteroidetes, Chlamydiae, Cyano-
bacteria, Planctomycetes, Verrucomicrobia, and Gemmati-
monadetes. The class III cluster arrangement Lhr-Core⅐MPE
is found in several archaeal taxa from the phyla Euryar-
chaeota and Korarchaeota.
Experimental procedures
Purification of recombinant Lhr-Core and MPE proteins
The ORFs encoding full-length P. putida Lhr-Core (PP_
1103; NCBI accession number NP_743264) and MPE
(PP_1102; NCBI accession NP_743263.1) were PCR-amplified
from P. putida strain KT2440 genomic DNA (5) with primers
that introduced a BamHI site immediately flanking the start
codon and a HindIII site downstream of the stop codon. The
PCR products were digested with BamHI and HindIII and
inserted between the BamHI and HindIII sites of pET28b-
His10Smt3 to generate expression plasmids encoding the Lhr-
Core or MPE polypeptides fused to an N-terminal His10Smt3
tag. MPE mutation D78A was introduced into the expression
plasmid by PCR with mutagenic primers. All of the plasmid
inserts were sequenced to verify that no unintended coding
changes were acquired during amplification and cloning.
pET28b-His10Smt3-Lhr-Core and pET28b-His10Smt3-MPE
plasmids were transformed into E. coli BL21(DE3) cells. Cul-
tures (2 liters) amplified from single kanamycin-resistant trans-
formants were grown at 37 °C in Terrific Broth containing 60
␮g/ml kanamycin until the A600 reached 0.8–1.0. The cultures
were chilled on ice for 1 h, adjusted to 2% (v/v) ethanol and 0.3
mM isopropyl-␤-D-thiogalactopyranoside, and incubated for
16 h at 17 °C with constant shaking. All subsequent steps were
performed at 4 °C. Cells were harvested by centrifugation and
resuspended in 25 ml of buffer A (50 mM Tris-HCl (pH 8.0), 500
mM NaCl, 1 mM DTT, 20 mM imidazole, 10% sucrose) contain-
ing one protease inhibitor mixture tablet (Roche). Lysozyme
was added to a concentration of 1 mg/ml. After incubation for
1 h, the lysate was sonicated to reduce viscosity, and the insol-
uble material was removed by centrifugation at 38,000 ϫ g for
1 h. The supernatant was mixed for 1 h with 3 ml of nickel-
nitrilotriacetic acid–agarose resin (Qiagen) that had been
equilibrated with buffer A. The resin was recovered by centrif-
ugation and resuspended in 30 ml of buffer A. The resin was
again recovered by centrifugation and then resuspended in 20
ml of buffer B (50 mM Tris-HCl (pH 8.0), 500 mM NaCl, 1 mM
DTT, 10% glycerol) containing 50 mM imidazole and poured
into a column. After washing the column with 10 ml of 3 M KCl
Lhr-Core ATPase assay
Reaction mixtures (10 ␮l) containing 20 mM Tris-HCl (pH
7.5), 1 mM DTT, 5 mM MgCl2, 1 mM [␣-32P]ATP, 5 ␮M 24-mer
ssDNA oligonucleotide, and Lhr-Core were incubated for 15
min at 37 °C. Reactions were quenched by adding 2 ␮l of 5 M
formic acid. An aliquot (2 ␮l) of the mixture was spotted on a
polyethyleneimine–cellulose TLC plate. Ascending TLC was
performed with 0.45 M ammonium sulfate as the mobile phase.
The extent of conversion of [␣-32P]ATP to [␣-32P]ADP was
quantified by scanning the TLC plate with a Fujix BAS2500
imager.
Streptavidin displacement assay of Lhr-Core translocation on
DNA
Synthetic 34-mer oligodeoxynucleotides containing a Bio-
tin-ON inter-nucleotide spacer at the fourth position from the
5Ј end or the second position from the 3Ј end were 5Ј 32P-
labeled by reactions with T4 Pnk and [␥-32P]ATP. The labeled
DNAs were purified by electrophoresis through a native 18%
polyacrylamide gel and eluted from an excised gel slice by over-
night incubation at 4 °C in 400 ␮l of 10 mM Tris-HCl (pH 7.5), 1
mM EDTA, 50 mM NaCl. Translocation reaction mixtures (10
␮l) containing 20 mM Tris-HCl (pH 7.5), 1 mM DTT, 5 mM
MgCl2, 1 mM ATP, 0.1 ␮M (1 pmol) biotinylated 32P-labeled
solution, the bound material was eluted stepwise with 5-ml ali- DNA, and 4 ␮M streptavidin (Sigma) were preincubated at
quots of buffer B containing 100, 200, and 500 mM imidazole. room temperature for 10 min to form SA–DNA complexes.
17502 J. Biol. Chem. (2018) 293(45) 17491–17504

Bacterial nucleic acid repair enzyme gene cluster
The mixtures were supplemented with 40 ␮M free biotin at the specified times by adding 10 ␮l of a solution containing
(Fisher), and the translocation reactions were initiated by add- 50% glycerol, 50 mM EDTA, and 0.3% SDS. The mixtures were
ing Lhr-Core. After incubation at 37 °C for the specified times, extracted serially with equal volumes of phenol-CHCl3 and
the reactions were quenched by adding 3 ␮l of a solution con- CHCl3. The aqueous phase samples were then analyzed by elec-
taining 200 mM EDTA, 0.6% SDS, 25% glycerol, and 20 ␮M unla- trophoresis through a horizontal 0.7% agarose gel containing 45
beled single-stranded 59-mer DNA oligonucleotide. The reac- mM Tris borate, 1.2 mM EDTA, and 0.5 ␮g/ml ethidium bro-
tion products were analyzed by electrophoresis through a mide. Ethidium bromide–stained DNA was visualized via UV
15-cm native 18% polyacrylamide gel containing 89 mM Tris transillumination.
borate and 2.5 mM EDTA. The free 32P-labeled 34-mer DNA
and the SA–DNA complexes were visualized and quantified by
scanning the gel with a Fujix BAS-2500 imaging apparatus.
Author contributions—A. E. and S. S. conceptualization; A. E. and
S. S. formal analysis; A. E. and S. S. investigation; A. E. and S. S. writ-
ing-original draft; A. E. and S. S. writing-review and editing; S. S.
funding acquisition; S. S. project administration.
Lhr-Core helicase assay
5Ј 32P-labeled synthetic 24-mer DNA or RNA oligonucleo-
tides were prepared by reaction with T4 polynucleotide kinase Acknowledgment—We thank Valérie de Crécy-Lagard for advice and
(Pnk) and [␥-32P]ATP. The kinase reaction mixture was heated
assistance with preparing the SEED subsystem.
to 95 °C to inactivate T4 Pnk. The labeled DNA or RNA strand
was then annealed to a 3-fold excess of complementary 39-mer
References
DNA or RNA strand to form 3Ј-tailed duplex helicase sub-
1. Ordonez, H., and Shuman, S. (2013) Mycobacterium smegmatis Lhr is a
strates. The tailed duplexes were purified by electrophoresis
DNA-dependent ATPase and a 3Ј-to-5Ј DNA translocase and helicase
through a native 12% polyacrylamide gel and eluted from an
that prefers to unwind 3Ј-tailed RNA:DNA hybrids. J. Biol. Chem. 288,
excised gel slice by overnight incubation at 4 °C in 400 ␮l of
either 10 mM Tris-HCl (pH 6.8), 1 mM EDTA, 50 mM NaCl for
RNA-containing substrates or 10 mM Tris-HCl (pH 7.5), 1 mM
EDTA, 50 mM NaCl for the all-DNA substrate. Helicase reac-
tion mixtures (10 ␮l) containing 20 mM Tris-HCl (pH 7.5), 1
mM DTT, 5 mM MgCl2, 50 nM (0.5 pmol) 32P-labeled tailed
duplex substrate, and Lhr-Core as specified were preincubated
for 10 min at room temperature. The unwinding reaction was
initiated by addition of 1 mM ATP and a 10-fold excess of an
unlabeled oligonucleotide identical to the labeled strand of the
helicase substrate. Addition of an excess of unlabeled strand
was necessary to prevent the spontaneous reannealing of the
unwound 32P-labeled strand. The reaction mixtures were incu-
bated for 30 min at 37 °C and then quenched by adding 1.5 ␮l of
a solution containing 3.5% SDS, 80 mM EDTA, and 10 ␮g pro-
teinase K. After protease digestion for 5 min at 37 °C, the mix-
tures were supplemented with 4 ␮l of 50% glycerol, 0.3% bro-
mphenol blue. The reaction products were analyzed by
electrophoresis through a 15-cm 10% polyacrylamide gel in 89
mM Tris borate, 2.5 mM EDTA. The products were visualized by
autoradiography of the dried gel.
14125–14134 CrossRef Medline
2. Ejaz, A., Ordonez, H., Jacewicz, A., Ferrao, R., and Shuman, S. (2018)
Structure of mycobacterial 3Ј-to-5Ј RNA:DNA helicase Lhr bound to a
ssDNA tracking strand highlights distinctive features of a novel family of
bacterial helicases. Nucleic Acids Res. 46, 442–455 CrossRef Medline
3. Rand, L., Hinds, J., Springer, B., Sander, P., Buxton, R. S., and Davis, E. O.
(2003) The majority of inducible DNA repair genes in Mycobacterium
tuberculosis are induced independently of RecA. Mol. Microbiol. 50,
1031–1042 CrossRef Medline
4. Boshoff, H. I., Reed, M. B., Barry, C. E., 3rd, and Mizrahi, V. (2003) DnaE2
polymerase contributes to in vivo survival and the emergence of drug
resistance in Mycobacterium tuberculosis. Cell 113, 183–193 CrossRef
Medline
5. Belda, E., van Heck, R. G., José Lopez-Sanchez, M., Cruveiller, S., Barbe, V.,
Fraser, C., Klenk, H. P., Petersen, J., Morgat, A., Nikel, P. I., Vallenet, D.,
Rouy, Z., Sekowska, A., Martins Dos Santos, V. A., de Lorenzo, V., et al.
(2016) The revisited genome of Pseudomonas putida KT2440 enlightens
its value as a robust metabolic chassis. Environ. Microbiol. 18, 3403–3424
CrossRef Medline
6. Morris, P. D., and Raney, K. D. (1999) DNA helicases displace strepta-
vidin from biotin-labeled oligonucleotides. Biochemistry 38,
5164–5171 CrossRef Medline
7. Matange, N., Podobnik, M., and Visweswariah, S. S. (2015) Metallophos-
phoesterases: structural fidelity with functional promiscuity. Biochem. J.
467: 201–216 CrossRef Medline
8. Rusnak, F., and Mertz, P. (2000) Calcineurin: form and function. Physiol.
Rev. 80, 1483–1521 CrossRef Medline
MPE hydrolysis of p-nitrophenyl phosphoesters
p-nitrophenyl phosphate (pNPP), bis-p-nitrophenyl phos-
phate (bis-pNPP) and p-nitrophenyl-5Ј-thymidylate (pNP-
TMP) were purchased from Sigma. Reaction mixtures (25 ␮l)
containing 100 mM Tris-HCl (pH 8.0), 10 mM p-nitrophenyl
phosphoester, divalent cation, and MPE as specified were incu-
bated for 30 min at 37 °C. The reactions were quenched by
adding 50 ␮l of 50 mM EDTA and then 0.9 ml of 1 M Na2CO3.
Release of p-nitrophenol was determined by measuring A410
and interpolating the value to a p-nitrophenol standard curve.
9. Hopfner, K. P., Karcher, A., Craig, L., Woo, T. T., Carney, J. P., and Tainer,
J. A. (2001) Structural biochemistry and interaction architecture of the
DNA double-strand break repair Mre11 nuclease and Rad50-ATPase. Cell
105, 473–485 CrossRef Medline
10. Schwer, B., Khalid, F., and Shuman, S. (2016) Mechanistic insights into the
manganese-dependent phosphodiesterase activity of yeast Dbr1 with bis-
p-nitrophenylphosphate and branched RNA substrates. RNA 22,
1819–1827 CrossRef Medline
11. Clark, N. E., Katolik, A., Roberts, K. M., Taylor, A. B., Holloway, S. P.,
Schuermann, J. P., Montemayor, E. J., Stevens, S. W., Fitzpatrick, P. F.,
Damha, M. J., and Hart, P. J. (2106) Metal dependence and branched RNA
cocrystal structures of the RNA lariat debranching enzyme Dbr1. Proc.
Natl. Acad. Sci. U.S.A. 113, 14727–14732
MPE DNA nuclease assay
Reaction mixtures (20 ␮l) containing 20 mM Tris-HCl (pH
8.0), 2 mM MnCl2, 800 ng of pUC19 plasmid DNA, and MPE as
specified were incubated at 37 °C. The reactions were quenched
12. Wang, L. K., Smith, P., and Shuman, S. (2013) Structure and mechanism of
the 2Ј,3Ј phosphatase component of the bacterial Pnkp-Hen1 RNA repair
system. Nucleic Acids Res. 41, 5864–5873 CrossRef Medline
J. Biol. Chem. (2018) 293(45) 17491–17504 17503

Bacterial nucleic acid repair enzyme gene cluster
13. Kelley, L. A., Mezulis, S., Yates, C. M., Wass, M. N., and Sternberg, M. J.
(2015) The Phyre2 web portal for protein modeling, prediction and anal-
ysis. Nat. Protoc. 10, 845–858 CrossRef Medline
19. Overbeek, R., Begley, T., Butler, R. M., Choudhuri, J. V., Chuang, H. Y.,
Cohoon, M., de Crécy-Lagard, V., Diaz, N., Disz, T., Edwards, R., Fonstein,
M., Frank, E. D., Gerdes, S., Glass, E. M., Goesmann, A., et al. (2005) The
subsystems approach to genome annotation and its use in the project to
annotate 1000 genomes. Nucleic Acids Res. 33, 5691–5702 CrossRef
Medline
14. Paull, T. T. (2018) 20 years of Mre11 biology: no end in sight. Mol. Cell 71,
419–427 CrossRef Medline
15. Pascal, J. M., O’Brien, P. J., Tomkinson, A. E., and Ellenberger, T. (2004)
Human DNA ligase I completely encircles and partially unwinds nicked 20. Price, M. N., Wetmore, K. M., Waters, R. J., Callaghan, M., Ray, J., Liu, H.,
DNA. Nature 432, 473–478 CrossRef Medline
Kuehl, J. V., Melnyk, R. A., Lamson, J. S., Suh, Y., Carlson, H. K., Esquivel,
Z., Sadeeshkumar, H., Chakraborty, R., Zane, G. M., et al. (2018) Mutant
phenotypes for thousands of bacterial genes of unknown function. Nature
557, 503–509 CrossRef Medline
16. Dominski, Z. (2007) Nucleases of the metallo-␤-lactamase family and
their role in DNA and RNA metabolism. Crit. Rev. Biochem. Mol. Biol. 42,
67–93 CrossRef Medline
17. Chang, H. H., and Lieber, M. R. (2016) Structure-specific nuclease activi- 21. Aniukwu, J., Glickman, M. S., and Shuman, S. (2008) The pathways and
ties of Artemis and the Artemis:DNA-PKcs complex. Nucleic Acids Res.
44, 4991–4997 CrossRef Medline
outcomes of mycobacterial NHEJ depend on the structure of the broken
DNA ends. Genes Dev. 22, 512–527 CrossRef Medline
18. Allerston, C. K., Lee, S. Y., Newman, J. A., Schofield, C. J., McHugh, P. J., 22. Zhu, H., and Shuman, S. (2010) Gap filling activities of Pseudomonas
and Gileadi, O. (2015) The structure of the SNM1A and SNM1B/Apollo
nuclease domains reveal a potential basis for their distinct DNA pro-
cessing activities. Nucleic Acids Res. 43, 11047–11060 CrossRef Medline
LigD polymerase and functional interactions of LigD with the DNA
end-binding Ku protein. J. Biol. Chem. 285, 4815–4825 CrossRef
Medline
17504 J. Biol. Chem. (2018) 293(45) 17491–17504

Characterization of Lhr-Core DNA helicase and manganese- dependent DNA
nuclease components of a bacterial gene cluster encoding nucleic acid repair
enzymes
Anam Ejaz and Stewart Shuman
J. Biol. Chem. 2018, 293:17491-17504.
doi: 10.1074/jbc.RA118.005296 originally published online September 17, 2018
Access the most updated version of this article at doi: 10.1074/jbc.RA118.005296
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
This article cites 22 references, 6 of which can be accessed free at
http://www.jbc.org/content/293/45/17491.full.html#ref-list-1