1 Evidence that the DNA Endonuclease ARTEMIS also has Intrinsic 5' Exonuclease Activity Sicong

21 Pages·2014·3.09 MB·English

by  Michael Lieber

JBC Papers in Press. Published on February 5, 2014 as Manuscript M113.544874
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M113.544874

Evidence that the DNA Endonuclease ARTEMIS also has Intrinsic
5' Exonuclease Activity

Sicong Li1, Howard H. Chang1, Doris Niewolik2, Michael P. Hedrick3, Anthony B. Pinkerton3, Chris
Hassig3, Klaus Schwarz2, and Michael R. Lieber1*

1Departments of Pathology, Biochemistry & Molecular Biology,
Biological Sciences, and Molecular Microbiology & Immunology,
University of Southern California Keck School of Medicine
Norris Comprehensive Cancer Center, Los Angeles, CA, USA

2Institute for Transfusion Medicine, University Hospital, Ulm, Germany

3Sanford Burnham Medical Research Institute
La Jolla, CA

Running Title: Intrinsic 5' Exonuclease of ARTEMIS.
*Corresponding author. [email protected] (ph. 323 865 0568).
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Background: DNA-PKcs stimulates the endonucleolytic activities of ARTEMIS, but not the 5’ exonuclease ed
activity. fro
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Results: Point mutagenesis, small molecule inhibitors, and modulation of divalent cation concentrations all h
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affect both the endonuclease and 5’ exonuclease activities in parallel. ://w
Conclusion: ARTEMIS has intrinsic 5’ exonuclease activity, in addition to its known endonuclease activity. w
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Significance: ARTEMIS may use both its endo- and its 5’ exonuclease activity in NHEJ. .jb
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ARTEMIS is a member of the small molecule inhibitors, which do not b/
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metallo-β-lactamase protein family. block unrelated nucleases. We gu
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ARTEMIS has endonuclease activity at conclude that the 5' exonuclease is t o
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DNA hairpins and at 5’ and 3’ DNA intrinsic to ARTEMIS, making it relevant A
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overhangs of duplex DNA, and this to the role of ARTEMIS in ril 7
endonucleolytic activity is dependent nonhomologous DNA end joining. , 20
1
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upon DNA-PKcs. There has been ARTEMIS is a member of the metallo-β-
uncertainty about whether ARTEMIS lactamase superfamily of nucleases. The
also has 5’ exonuclease activity on metallo-β-lactamase family is characterized by
single-stranded DNA and 5' overhangs, a conserved metallo-β-lactamase and β-CASP
because this 5’ exonuclease is not domain, and their ability to hydrolyze DNA or
dependent upon DNA-PKcs. Here, we RNA. Two other mammalian members of the
show that the 5’ exonuclease and the same family, SNM1A and SNM1B (Apollo),
endonuclease activities co-purify. function in DNA interstrand cross-link repair (1)
Second, we show that a point mutant of and protection of telomeres (2). ARTEMIS,
ARTEMIS at a putative active site also known as SNM1C, functions in the
residue (H115A) markedly reduces both nonhomologous end joining (NHEJ) repair
the endonuclease activity and the 5’ pathway. On duplex DNA, ARTEMIS has
exonuclease activity. Third, divalent endonuclease activity on 5' and 3' DNA
cation effects on the 5’ exonuclease overhangs and at DNA hairpins, and these are
and the endonuclease parallel one all dependent on DNA-PKcs (3). In the context
another. Fourth, both the endonuclease of V(D)J recombination, the ARTEMIS:DNA-
activity and 5’ exonuclease activity of PKcs complex is required for the opening of
ARTEMIS can be blocked in parallel by hairpins generated by the RAG complex (3).
1
Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Patients lacking ARTEMIS suffer from severe Purified ARTEMIS protein also appears
combined immunodeficiency (SCID)(4). NHEJ to have 5’ exonuclease activity, which is DNA-
requires nucleases for resection of broken PKcs independent (3). The 5' exonuclease
DNA ends, polymerase to fill in the gaps, and activity co-fractionates with the known
the XLF:XRCC4:DNA ligase IV complex for endonucleolytic activities of ARTEMIS (12).
ligation (5). ARTEMIS is at least one of the Exonucleases are abundant in cells and are
nucleases participating in this process and is common contaminants. As other 5'
responsible for a subset of end processing exonucleases are purified away, the apparent
during NHEJ in response to ionizing radiation level of 5' exonuclease activity falls, but not to
(6). Patients or cells lacking ARTEMIS are zero, and the question arises whether the
deficient for repair caused by ionizing radiation residual level of 5' exonuclease activity is
(IR) (4,7) and for repair of breaks caused by intrinsic to ARTEMIS or a contaminating
topoisomerase II inhibitors (8). enzyme that co-fractionates with ARTEMIS.
In a biochemical system consisting of Like other members of the metallo-β-
purified proteins of the NHEJ pathway, lactamase family, it is thought that ARTEMIS
ARTEMIS, in complex with the serine/threonine requires the binding of divalent cations to two
kinase DNA-PKcs, removes 5' and 3' co-catalytic portions of the active site for
overhangs in order to join duplex DNA ends catalysis (13). Each catalytic site may hold
(9). ARTEMIS:DNA-PKcs is also able to open one Mg2+, Mn2+, or Zn2+ ion. In the metallo-β- D
hairpins both in vivo and in vitro. ARTEMIS lactamase family, the ligands for the divalent ow
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opens hairpins preferentially two nucleotides cations are generally conserved, and consist of loa
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past the tip of a perfect hairpin (3). At a 5’ a combination of histidine and aspartic acid ed
overhang, ARTEMIS:DNA-PKcs preferentially residues (14). Our previous studies have from
cuts directly at the double-strand/single-strand demonstrated that upon mutation of these http
junction (3,10). At a 3’ overhang, ARTEMIS conserved residues, the endonuclease activity ://w
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preferentially cuts 4 nucleotides into the single- of ARTEMIS:DNA-PKcs can be markedly w
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stranded overhang from the double reduced. However, the 5’ exonuclease activity c.o
strand/single-stranded DNA junction (11). All appeared to remain (13). This raised the brg/
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of the endonuclease activities of ARTEMIS on possibility that the 5’ exonuclease activity and g
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duplex DNA ends can be unified by a model the endonuclease activity of ARTEMIS use two est o
invoking binding to the single- to double-strand independent catalytic sites. However, an n A
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junction and occupying 4 nt along the single- alternative explanation is that previous ril 7
stranded portion, followed by nicking attempts at testing point mutants were carried , 2
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preferentially 3' of those 4 nt (3). out using 293T cells, a system known for high 19
In addition to its endonucleolytic action levels of background nucleases, and complete
on the single-strand to double-strand removal of contaminating 5' exonucleases may
transitions (overhangs and hairpins of duplex not have been successful.
DNA), ARTEMIS alone has a weaker single- Here, we present a point mutant of
stranded DNA endonuclease activity, such as ARTEMIS created using a baculovirus
poly dT (12). Like the endonuclease activity of expression system -- a system that more
ARTEMIS on duplex DNA substrates, the readily permits purification away from host cell
action of ARTEMIS on entirely single-stranded 5’ exonucleases. This mutation markedly
DNA is stimulated by the addition of DNA-PKcs reduces both the endonuclease as well as the
(12), which forms a complex with ARTEMIS 5' exonuclease activities of ARTEMIS. We
that is stable to well over 0.5 M monovalent have also identified small molecules that inhibit
salt (3). Our model for ARTEMIS both the ARTEMIS endonuclease and 5'
endonucleolytic action on single-stranded DNA exonuclease activities in parallel. These and
(ssDNA) proposes binding by ARTEMIS at other findings shed light on ARTEMIS nuclease
random locations along the ssDNA, followed by activity and expand the range of substrates
nicking, and these activities of ARTEMIS are that ARTEMIS can act upon. We discuss
increased by DNA-PKcs. these findings in the context of NHEJ.

2
EXPERIMENTAL PROCEDURES protein was eluted off using 250mM imidazole.
Oligonucleotides & DNA Substrates - Eluted protein was directly applied to DEAE-
Oligonucleotides used in this study were sepharose resin (GE Healthcare). Unbound
synthesized by Operon Biotechnologies,Inc. soluble fraction was collected and dialyzed
(Huntsville, AL) and Integrated DNA against Superose 12 buffer (500mM KCl,
Technologies, Inc. (San Diego, CA). We 25mM Tris-HCl, 1mM DTT). ARM14 fractions
purified the oligonucleotides using 12% or 15% eluted from Superose 12 were frozen down
denaturing PAGE and determined the and stored at -80°C. Expression and
concentration spectrophotometrically. DNA purification of DNA-PKcs from HeLa cells has
substrate 5’ end labeling was done with [γ- been previously described (15).
32P]ATP (3000 Ci/mmol) (PerkinElmer Life
Sciences, Boston, MA) and T4 polynucleotide Circular Dichroism - All CD studies were
kinase (New England Biolabs) according to the conducted on Jasco-810 Spectro Polarimeter.
manufacturer’s instructions. Substrates were A wavelength of 260nm was used at the start
incubated with [γ-32P]-ATP and T4 PNK for 30 and 190nm for the end reading. A data pitch of
min at 37°C. T4 PNK was subsequently 0.5nm was used with continuous reading and
inactivated by incubating samples at 72 °C for 20 accumulations per scan.
20 min. Unincorporated radioisotope was
removed by using G-25 Sephadex (Amersham Biochemical Nuclease Assay - The in
D
Biosciences, Inc.) spin-column vitro DNA nuclease assay was performed in a ow
n
chromatography. For the hairpin substrate, volume of 10ul with a buffer composition of loa
d
YM164-labeled oligonucleotide was diluted in a 25mM Tris-HCl (pH 7.5), 10mM KCl, 10mM ed
buffer containing 10 mM Tris-hydrochloride (pH MgCl2, and 1mM DTT. In the reaction, 50nM of from
8.0), 1 mM EDTA (pH 8.0), and 100 mM NaCl, single-stranded DNA or 20nM of hairpin DNA http
heated at 100°C for 5 min, allowed to cool to YM164 or SL23 was mixed with 50nM of ://w
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room temperature for 3 h, and then incubated ARM14 or 50nM of wild-type ARTEMIS. In w
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at 4 °C overnight. The sequences of the specified cases, 50nM of DNA-PKcs was c.o
oligonucleotides used in this study are as added with 0.5mM ATP. DNase I and brg/
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follows: JG 169 5’-TTT TTT TTT TTT TTT TTT micrococcal nuclease were obtained from New g
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TTT TTT TTT TTT-3’. JG 167 5’– AAA AAA England Biolabs and Sigma Chemical Co., est o
AAA AAA AAA AAA AAA AAA AAA AAA -3’. respectively. Reactions were then incubated n A
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YM164, 5’ -TTT TTG ATT ACT ACG GTA GTA at 37°C for 1h. After incubation, reactions ril 7
GCT ACG TAG CTA CTA CCG TAG TAA T-3’. were stopped, and analyzed on a 12% , 2
0
SL 23 5’- T*T*T*T*TT TTG CCA GCT GAC denaturing PAGE. 19
GCG CGT CAG CTG GC-3’.
Cells and Transfection - HEK293T cells
Protein Expression and Purification - were transfected by nucleofection using
ARM14 recombinant baculovirus with a C- AMAXA nucleofector kit V (Lonza). Equal
terminal 8x histidine tag was cloned into transfection efficiencies were confirmed by co-
baculodirect vector and transfected into SF21 transfection of an enhanced green fluorescent
according to protocol (Invitrogen). Virus was protein expression plasmid followed by FACS
harvested after 48hrs and used to infect Sf9 analysis.
cells in log phase growth. Wild type ARTEMIS
was purified as previously described (12). Immunoprecipitation and Immuno-
Harvested ARM14 infected Sf9 cells were blotting - The immunoprecipitation was
resuspended in Ni-NTA binding buffer (50mM performed as described (3). Myc-tagged
NaH PO (pH 7.8), 0.5M KCl, 2mM beta- ARTEMIS was immunoprecipitated and
2 4
mercaptoethanol, 10% glycerol, 0.1% Triton X- detected with anti-myc antibody (Invitrogen).
100, and 20mM imidazole (pH 7.8)) SV40 large T antigen was used as loading
supplemented with protease inhibitors. Cells control and was detected with anti-SV40 T-Ag
were lysed by sonication and applied to Ni- antibody (Santa Cruz Biotechnology, Santa
NTA agarose resin (Qiagen). Bound ARM14 Cruz, CA). Endogenous DNA-PKcs was
3
detected using anti-DNA-PKcs antibody Additionally, we performed a pull down
(Abcam). experiment to assess the ability of ARM14
protein to bind to endogenous DNA-PKcs.
ARTEMIS or ARM14 expression plasmids
RESULTS
were transfected into HEK293T cells. Myc-
ARTEMIS Endonuclease and
ARTEMIS and Myc-ARM14 were
5’Exonuclease Activities Co-Purify with
immunoprecipitated using anti-myc antibody.
WT ARTEMIS Protein - ARTEMIS has a
We assayed to determine if DNA-PKcs was
well-documented set of endonuclease
also co-immunoprecipitated in both cases. The
activities, all of which are stimulated by DNA-
presence of DNA-PKcs was assayed using
PKcs (3). The 5' exonuclease activity of
anti-DNA-PKcs antibody. We find that the
ARTEMIS co-fractionates with the ARTEMIS
ARM14 point mutant is able to bind to DNA-
endonucleolytic activities (Fig. 1). The 5'
PKcs with indistinguishable affinity as WT
exonuclease activity is seen as the 1 nt product
ARTEMIS (Fig. 2F).
at the bottom of Figure 1A. The 5'

exonuclease activity is not stimulated by DNA-
The ARM14 Mutant of ARTEMIS does
PKcs (3), and for this reason, we wondered
not Possess Hairpin Opening,
whether the 5' exonuclease is intrinsic to
Endonuclease, or 5’ Exonuclease
ARTEMIS.
Activity - We tested the activity of ARM14
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fractions collected from the Superose 12 ow
Expression and Purification of ARM14 n
column for nuclease action on a DNA hairpin lo
Point Mutant. - We created a point mutant ad
and ssDNA substrates, specifically a single- ed
hoifs AtidRiTnEe MatI Sa mbyin mo uatcaidtin 1g1 t5h eto c aonn saelarvneinde (Fig. stranded poly dT substrate. The WT ARTEMIS from
2A). The histidine at amino acid 115 is a endonuclease activity elutes as a symmetrical http
putative binding site for divalent cations and defined peak. Unlike WT ARTEMIS ://w
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protein, an equal amount of ARM14 mutant w
involved in the catalysis of DNA (13). This was .jb
protein had no endonuclease activity, on c
subsequently cloned into a baculodirect vector .o
(Invitrogen) to generate what we call the ssDNA (Fig. 2E, lane 2-5). Importantly, no 5’ brg/
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ARM14 baculovirus and mutant form of exonuclease activity was detected, as gu
demonstrated by the lack of a band at the 1 nt es
ARTEMIS protein. The ARM14 mutant and WT t o
human ARTEMIS-His proteins were both position at the bottom of the gel (Fig. 3A, lane n A
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expressed in Sf9 insect cells using a 2 versus lane 3). Therefore, the ARM14 ril 7
baculovirus expression system. Both the WT mutant does not exhibit any endonuclease , 2
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and ARM14 mutant proteins were purified activity or 5’ exonuclease activity. 19
In some DNA DSBs, the 5’ termini that
using Ni-NTA, an anion exchange step,
are generated can be 5’ hydroxyl rather than 5’
followed by a Superose 12 gel filtration column.
phosphate. We tested for the ability of the
Purified ARM14 mutant elutes as a relatively
intrinsic 5’ exonuclease activity of ARTEMIS to
sharp peak off of the Superose 12 column and
process a 3’ labeled substrate with a 5’
runs at 100 kDa on a 8% SDS-PAGE (Fig. 2B
hydroxyl group. We labeled the substrate at
and 2D). The ARM14 mutant runs at the same
the 3’ end by using TdT and dATP that has a
position (~100kDa) as the WT ARTEMIS on
radioactive phosphate at the alpha position.
8% SDS-PAGE (Fig. 2C).
To minimize addition of more than one
To ensure that the ARM14 mutant was
radiolabeled mononucleotide, we include a 5-
not denatured, we used circular dichroism (CD)
fold excess of ddTTP, which caps the 3’
on the WT ARTEMIS, on ARM14, and on
terminus. However, the final substrate is a
completely heat-denatured ARTEMIS. The WT
mixture of lengths due to incorporation of 1, 2,
ARTEMIS and ARM14 had similar CD profiles
or 3 (and smaller amounts of more)
(data not shown). Hence, purified ARM14
radiolabels, followed by one dideoxy
mutant has a conformation that is
mononucleotide. We tested the processitivity
indistinguishable from WT ARTEMIS by CD.
of the 5’ exonuclease activity of ARTEMIS
using this 3’ labeled substrate at three time
4
points. Unlike processive nucleases, activity. To distinguish this from its 5'
ARTEMIS is able to generate intermediate exonuclease activity, we utilized a ssDNA poly
products even at the 30 min time point (Fig. dA substrate. Single-stranded polypurine
3B, lane 4). Therefore, ARTEMIS acts substrates are not endonucleolytically cut as
nonprocessively and can process 5’ hydroxyl efficiently by ARTEMIS, which prefers
termini as shown here (Fig. 3B) and substrates polypyrimidine single-stranded DNA substrates
with 5’ phosphate termini, as we have shown (9). From our previous work, we knew that the
previously [Fig. 3B in ref. (12)]. 5' exonuclease activity of ARTEMIS is DNA-
We compared the activity of ARM14 PKcs independent (3). We tested the
with WT ARTEMIS on a hairpin substrate. To exonuclease activity of ARTEMIS in the
do this, we designed an oligonucleotide DNA presence and absence of DNA-PKcs (Fig. 4B,
substrate that is able to fold back on to itself lane 2 vs lane 3). Consistent with our previous
upon annealing to form a hairpin substrate findings, we saw no difference in the
(YM164. WT ARTEMIS protein shows weak exonuclease activity of purified WT ARTEMIS
hairpin opening, weak endonuclease activity, with or without DNA-PKcs. The addition of 20
as well as weak 5’ exonuclease activity on this uM of the inhibitor SBI-713 was able to inhibit
substrate in Mg2+-containing reactions that lack the 5' exonuclease activity of ARTEMIS, as
DNA-PKcs (Fig. 3C, lane 2). In contrast, the demonstrated by the lack of the 1 nt
ARM14 protein generated no hairpin nicking, exonuclease product (Fig. 4B, lane 4). This
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endonuclease products or 5’ exonuclease further demonstrates the 5’ exonuclease ow
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products even when we added DNA-PKcs to activity of ARTEMIS. Furthermore, we tested lo
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the reaction (Fig. 3C, lane 5). the ability of the ARM14 point mutant to ed
process the poly dA ssDNA. The ARM14 from
The Endonuclease and 5’ Exonuclease mutant does not have any apparent http
activities of ARTEMIS can be Inhibited endonuclease or 5’ exonuclease activity on this ://w
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in Parallel - We further tested the ability of substrate when compared to WT ARTEMIS w
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selected compounds to inhibit the hairpin (Fig. 4C, lane 3 vs. lane 2), indicating that the c
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opening, endonuclease, and 5’ exonuclease point mutation markedly reduces both brg/
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activities of ARTEMIS. Prior to the HTS, a endonuclease and 5’ exonuclease activities. g
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small screen showed that ampicillin, a β– We also tested the inhibitors in titrations est o
lactamase antibiotic that inhibits to determine if the 5’ exonuclease and the n A
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transpeptidase, is also inhibitory, probably endonuclease of ARTEMIS are inhibited over ril 7
because ARTEMIS is a member of the β- the same concentration ranges (Fig. 5). We , 2
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lactamase family. SBI-713 and SBI-116 are found that as the concentration of ampicillin or 19
two structurally distinct <500 mw small SBI-713 increases, there is a concomitant
molecules (not shown) that were identified inhibition of both the endonuclease and the 5’
using a high through-put screen (HTS) of a exonuclease activity of ARTEMIS. The level of
31,000 compound subset of the NIH’s MLSMR inhibition of the 5’ exo- and endonuclease
collection (16). activities is similar for both inhibitors. If the 5'
In the absence of any inhibitor, exonuclease were a contaminant, we would
ARTEMIS has endonuclease activity as well as not expect both the ARTEMIS endonuclease
5’ exonuclease activity on a hairpin substrate, and the contaminating 5’ exonuclease to be
YM164 (Fig. 4A, lane 2). However, at 20 uM, inhibited over the very same concentration
the SBI-713 dramatically inhibited both the ranges for each drug (Fig. 5).
endonuclease and the 5’ exonuclease activities We have also tested SBI-713 and
of ARTEMIS (Fig. 4A, lane 4). On the other ampicillin against other nucleases to assess
hand, 20 uM of SBI-116 did not inhibit either specificity for inhibition of Artemis. These
activity (Fig. 4A, lane 3). included DNase I and micrococcal nuclease
Given that ARTEMIS is an (MCN). SBI-713 does not inhibit either of
endonuclease, we wondered if ARTEMIS these counter-screen nucleases (Fig. 4D).
cutting at the 5’ most nt could merely reflect a In related studies, in 10 mM MgCl2
preferential cutting by ARTEMIS endonuclease reaction buffers, addition of ZnCl2 above 10 uM
5
inhibits ARTEMIS endonuclease activity (Fig. alter the activity of enzymes in the metallo-β-
6). It has been shown previously in certain lactamase family of nucleases (18). We found
cases that excess zinc can inhibit the that the addition of excess zinc (> 10 uM) to
enzymatic activity of zinc dependent enzymes. the reaction buffer decreases the
This is thought to be caused by excess zinc- endonuclease as well as the 5’ exonuclease
monohydroxide binding to the active form of activity of ARTEMIS. It seems unlikely that a
the zinc dependent enzyme, thereby contaminating 5’ exonuclease would also show
preventing the catalytic site from binding active this behavior, particularly at the same low
zinc (17). In the case of ARTEMIS, the (micromolar) concentrations of Zn2+.
inhibition by excess zinc is observed for the Fifth, we explored the possibility that the
endonuclease activity as well as the 5’ 5’ exonuclease activity seen in our gels is
exonuclease activity over the same Zn2+ actually the result of the ARTEMIS
concentration range (10 to 100 uM) (Fig. 6). endonuclease activity acting on the 5’ terminal
This is consistent with the 5’ exonuclease nucleotide. We created a poly dA ssDNA
activity of ARTEMIS sharing the same catalytic substrate that is not as readily
site as the endonuclease activity of ARTEMIS. endonucleolytically processed by the
ARTEMIS endonuclease activity, but
DISCUSSION conceivably might be processed by the 5’
Though a 5' DNA exonuclease activity exonuclease activity of ARTEMIS. Indeed, we
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co-purifies with ARTEMIS (Fig. 1), the 5' do observe 5’ exonuclease activity when using w
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exonuclease is not dependent on DNA-PKcs, this poly dA ssDNA substrate, indicating that loa
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wachteivrietiaess t(h5e' aAnRdT 3E' MovIeSr hDaNnAg eclnedaovnaugcel eaansde tphoely apcytrivimityid sineee nsu abts tthraet e5 ’ ise nadtt roibnu ata ble to 5’ d from
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hairpin opening) do depend on DNA-PKcs. For exonuclease activity and not merely due to the ttp
this reason, we did the further studies endonuclease activity of ARTEMIS. ://w
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described here to evaluate whether the 5’ Importantly, this 5’ exonuclease activity is w
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exonuclease is intrinsic to ARTEMIS. completely inhibited by the SBI-713 compound c.o
Here, we have provided several (Fig. 4). brg/
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independent lines of evidence that the 5’ From the evidence presented here we g
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exonuclease is intrinsic to ARTEMIS. First, the conclude that the 5’ exonuclease activity seen st o
5’ exonuclease and the endonuclease activities in purified ARTEMIS is intrinsic to the n A
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co-fractionate. ARTEMIS protein and not due to a co-purifying ril 7
Second, a point mutant of ARTEMIS is contaminating enzyme. , 2
0
1
markedly reduced in 5’ exonuclease activity in The metallo-β-lactamase family of 9
the presence and absence of DNA-PKcs. A nucleases uses several conserved catalytic
point mutant of ARTEMIS is very unlikely to residues to coordinate divalent cations. The
affect any contaminating 5’ exonuclease. This two most conserved residues within the
alone is strong evidence of an intrinsic 5’ catalytic site are histidine and aspartic acid
exonuclease. (14). We generated the ARM14 point mutant,
Third, both the endonuclease and the 5’ which changed a conserved histidine residue
exonuclease activity of ARTEMIS can be to an alanine residue. If both the
inhibited by small molecules that are not endonuclease activity of ARTEMIS and 5’
chelators. It is unlikely that any one inhibitor is exonuclease activity of ARTEMIS shared a
able to inhibit both ARTEMIS endonuclease common catalytic site, by disrupting the
activity and an unidentified contaminating 5’ divalent ligand in the site there should be a
exonuclease. It is even more unlikely that corresponding decrease in both endonuclease
these specific inhibitors would affect a as well as the 5’ exonuclease activity of
contaminating 5’ exonuclease at the same ARTEMIS, and this is what we observe here.
concentrations as they affect ARTEMIS We have previously purified ARM14
endonuclease activity. mutant from 293T cells using a two step
Fourth, disruption of the catalytic sites purification. Though this point mutant is
by divalent cation replacement is known to inactive for the hairpin opening and
6
endonuclease activity on a hairpin substrate, nuclease activities (both endo- and 5'
this version of ARM14 appeared to still retain exonuclease) are abolished by addition of
most of the 5’ exonuclease activity (13). We these same zinc specific chelators (data not
attribute the discrepancies in our findings with shown), suggesting that members of this family
insect cell-purified ARM14 in the current study use the same divalent cations in the catalytic
to our earlier study to the high levels of site (21). Biochemical studies have also
contaminating nucleases in mammalian cells demonstrated that human SMN1A and SMN1B
that are difficult to remove during purification. proteins, also members of the metallo-β-
The baculovirus system for ARTEMIS lactamase family and involved in inter-strand
production here contains much lower levels of crosslink DNA repair, are capable of acting as
background contaminating nucleases, which a 5’ exonuclease on single-stranded and
can be subsequently removed in our three step double-stranded DNA substrates (22), and this
purification. is its only biochemically defined enzymatic
Using a purification process consisting activity. Mutations of key catalytic residues
of phosphocellulose, nickel-NTA agarose, and within the metallo-β-lactamase domain also
hydroxyapatite, one group separated the usual abolish the 5’-3’ DNA processing ability of
bulk of other cellular 5’ exonuclease activity hSMN1a and hSMN1b. Therefore, 5’
away from the endonuclease activity of exonuclease activity is intrinsic to many
ARTEMIS (19). They concluded that the 5' members of the metallo-β-lactamase nuclease
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exonuclease activity was not intrinsic to family, and the catalytic site involved during ow
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ARTEMIS based on an assay that was much DNA processing resides in the metallo-β- loa
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less sensitive than our assay. Even at their lactamase domain. Our current study clearly ed
reduced sensitivity, we note that some 5' shows that ARTEMIS also has a similar 5' from
exonuclease activity is still apparent in their exonuclease activity. http
assay gels, which they did not point out (19). The 5’ exonuclease activity described ://w
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For example, they tested their purified here would permit Artemis to act on 1 and 2 nt w
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ARTEMIS on a single-stranded 5' radiolabeled 5’ overhangs more efficiently because such c.o
DNA substrate to assay for 5' exonuclease short 5’ overhangs are not optimal substrates brg/
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activity (Fig. 5B of ref. (19)). Though they for action by Artemis in an endonucleolytic g
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could detect no 5' exonuclease activity, their manner (3). This would be of particular utility es
t o
assay was insensitive to the extent that they for repair of ionizing radiation damage, which n A
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also could not detect any of the single-stranded might have short overhangs with either a 5’ ril 7
endonuclease activity of ARTEMIS (19). This phosphate or 5’ hydroxyl. , 2
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was due in part to exposure intensities of their 19
gels that were 10 to 20-fold lower than what we Acknowledgements: This work was
use to detect both single-stranded endo- and 5' supported by an NIH grant to M.R.L and the
exonuclease activities (12). Despite the fact BMBF grant 01GM1111F to K.S. We thank Dr.
that their gels show apparent residual 5' Ray Mosteller (USC) and members of the
exonuclease activity at their darker radiolabel Lieber lab for critically reading the manuscript.
exposures (Fig. 5C, lanes 10 & 11 in ref. (19)), We thank Dr. Ralf Langen (USC) for help
they nevertheless concluded that ARTEMIS obtaining the CD spectra. We thank Dr. David
has no intrinsic 5' exonuclease activity (19). Auld (Harvard) and Dr. Andrew Mason (Cal
Other members of the metallo-β- State Long Beach) for advice about zinc
lactamase family possess nuclease activities metalloenzymes. We thank Fusayo Yamamoto
similar to those of ARTEMIS. CPSF-73, an and Drs. T.C. Chung (Sanford-Burnham
enzyme involved in the 3' end processing of Research Institute) for conducting the high
histone pre-mRNA, has been shown to throughput screen of ARTEMIS inhibitors.
possess endonuclease activity as well as 5’ Abbreviations: NHEJ (nonhomologous DNA
exonuclease activity on single stranded RNA end joining); DNA-PKcs (DNA-dependent
substrates (18,20). The nuclease activities of protein kinase, catalytic subunit).
CPSF-73 can be abolished by the addition of
zinc specific chelators. We find that ARTEMIS
7
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sensitivity to the DNA interstrand cross-linking agent mitomycin C. Mol. Cell. Biol. 20, 4553-
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2. van Overbeek, M., and de Lange, T. (2006) Apollo, an Artemis-related nuclease, interacts with
TRF2 and protects human telomeres in S phase. Curr Biol 16, 1295-1302
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4. Moshous, D., Callebaut, I., Chasseval, R. d., Corneo, B., Cavazzana-Calvo, M., Diest, F. L.,
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Artemis, a novel DNA double-strand break repair/V(D)J recombination protein, is mutated in
human severe combined immune deficiency. Cell 105, 177-186
5. Lieber, M. R. (2010) The Mechanism of Double-Strand DNA Break Repair by the
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Fricke, A., Kempler, A., Parker, A. R., Jackson, S. P., Gennery, A., Jeggo, P. A., and Lobrich, w
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M. (2004) A pathway of double-strand break rejoining dependent upon ATM, Artemis, and loa
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proteins locating to gamma-H2AX foci. Mol. Cell 16, 715-724 d fro
7. Rooney, S., Alt, F. W., Lombard, D., Whitlow, S., Eckersdorff, M., Fleming, J., Fugmann, S., m
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Ferguson, D. O., Schatz, D. G., and Sekiguchi, J. (2003) Defective DNA repair and increased ttp
genomic instability in Artemis-deficient murine cells. J. Exp. Med. 197, 553-565 ://w
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8. Kurosawa, A., Koyama, H., Takayama, S., Miki, K., Ayusawa, D., Fujii, M., Iiizumi, S., and w
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M. R. (2008) A biochemically defined system for coding joint formation in human V(D)J st o
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recombination. Molecular Cell 31, 485-497 A
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10. Yannone, S. M., Khan, I. S., Zhou, R. Z., Zhou, T., Valerie, K., and Povirk, L. F. (2008) ril 7
Coordinate 5' and 3' endonucleolytic trimming of terminally blocked blunt DNA double-strand , 2
0
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break ends by Artemis nuclease and DNA-dependent protein kinase. Nucleic Acids Res 36, 9
3354-3365
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Artemis. DNA Repair (Amst) 9, 429-437
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20. Mandel, C. R., Kaneko, S., Zhang, H., Gebauer, D., Vethantham, V., Manley, J. L., and Tong,
L. (2006) Polyadenylation factor CPSF-73 is the pre-mRNA 3'-end-processing endonuclease.
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the mRNA 3' processing endonuclease. Rna 10, 565-573
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and SNM1B/Apollo DNA repair exonucleases. J Biol Chem 287, 26254-26267

FIGURE LEGENDS
Figure 1. Mono Q purified ARTEMIS exonuclease activity correlates with its
endonuclease activity.
A, 20 nM of 5' radiolabeled ssDNA substrate (JG169) was incubated with 1ul of Mono Q fractions in a
D
o
10ul reaction. The reaction was performed in 25 mM Tris-Cl (pH 8.0), 10 mM MnCl , 50 ug/mL BSA, w
2 n
and 1 mM DTT for 60 min at 37°C. Lane 2 contains pre-Mono Q ARTEMIS, and lanes 3-18 contain loa
d
e
Mono Q fractions 8-23. The 5' exonuclease activity is seen as the 1 nt product at the bottom of the d fro
gel. m
h
B, The signals of remaining substrate (30nt position), endonucleolytic products (2-29nt position), and ttp
exonucleoytic product (1nt position) were quantified by boxing the corresponding bands and ://w
w
measuring the summed band intensities using Bio-Rad Quantify One software. The signal % was w
.jb
plotted as a function of fraction number. The top graph plots the substrate and endonucleolytic c.o
rg
product signals. The bottom graph plots the 5' exonucleolytic product signal. b/
y
C, An SDS PAGE gel is stained with Coomassie Blue. The gel shows that purified Artemis from the g
u
e
Mono Q column runs at the 100 kDa position. Fractions 10 – 18 correlate with peak activity as well st o
n
as ARTEMIS protein abundance. A
p
ril 7
Figure 2. Size exclusion chromatography and characterization of a single amino acid , 2
0
1
point mutant of ARTEMIS, the ARM14 mutant. 9
A, The ARM14 mutant was constructed by creating a point mutation in the putative active site of
ARTEMIS, specifically histidine 115 to alanine, followed by cloning into a baculodirect vector.

B, An SDS-PAGE gel was stained with Coomassie Blue. The gel shows that the ARM14 mutant
protein has a gel mobility of ~100kDa.

C, ARM14 and wild type ARTEMIS were both run on an 8% SDS-PAGE gel and stained with
Coomassie Blue. Purified ARM14 mutant (lane 2) and wild type ARTEMIS (lane 3) have gel mobility
positions at ~100 kDa, 2 ug of each was loaded onto the SDS-PAGE. The molecular weight marker
is in lane 1.

D, The UV profile of ARM14 during Superose 12 size exclusion chromatography is shown. Purified
ARM 14 mutants elutes as a ~450 kDa multimer at fraction 15 on the Superose 12 size exclusion
column.

E, Fractions across the Superose 12 UV peak were collected and assayed for nuclease activity. In
each reaction, 50 nM of ARM14 mutant was incubated with 50 nM of ssDNA poly dT substrate
(JG169) in a 10 ul reaction. Reactions were incubated for 60 min at 37°C, stopped, and run on 12%
9
denaturing PAGE. The ARM14 mutant fractions 15-18 do not show any endonuclease or 5’
exonuclease activity.

F, ARM14 binds endogenous DNA-PKcs. Empty vector, wildtype (ART-WT) or mutant ARTEMIS
(ARM14) expressing plasmids were transfected into HEK293T cells, and the myc-tagged ART-
WT/ARM14 was immunoprecipitated (IP) in the presence (+) or absence (-) of anti-myc antibody from
cell lysates. N.a. not applicable. ART-WT/ARM14, co-precipitated endogenous DNA-PKcs, and the
loading control SV40 large T-antigen were detected with the corresponding antibodies by Western
blot analysis. The position of protein standards is given at the left in kDa. Transfection efficiencies
were between 83 and 86%.

Figure 3. Endonuclease and 5’ exonuclease activity of WT ARTEMIS and the
ARTEMIS point mutant, ARM14.
A, The activity of ARM14 mutant was assayed for activity and compared to that of WT ARTEMIS. 50
nM of ARM14 mutant or WT ARTEMIS was incubated with 50 nM of ssDNA poly dT substrate in a 10
ul reaction. Reactions were incubated for 60 min at 37°C, stopped, and run on 12% denaturing
PAGE. The 5' exonuclease activity is seen as the 1 nt product at the bottom of the gel.

B, The activity of WT ARTEMIS was assayed for activity on a 3’ labeled substrate to determine the
D
processitivity of the 5’ exonuclease activity. 50 nM of WT ARTEMIS was incubated with 50 nM of ow
n
ssDNA poly dT substrate (3’ labeled by Tdt) in a 10 ul reaction. Reactions were incubated for either lo
a
d
2, 5, or 30 min as indicated. Where compound SBI-713 is used, 20 uM was added to the reaction. ed
from
C, 50 nM of hairpin DNA substrate (YM164) was incubated with 50 nM WT ARTEMIS or 50 nM of http
ARM14 in a 10 ul reaction. Reactions were incubated at 37°C for 60 min. In lanes where DNA-PKcs ://w
w
was added, 50 nM of DNA-PKcs, 0.5 mM of ATP, and 0.25 uM of cold DNA YM8/9 were also added. w
.jb
Lane 1 contains only the hairpin substrate. Lane 2 contains ARTEMIS, which does have low dsDNA c
.o
endonucleolytic activity without DNA-PKcs being present, but it has high 5’ exonucleolytic activity (1 brg/
y
nt product at bottom of gel). Lane 4 and 5 are the ARM14 point mutant of ARTEMIS. Lane 3 WT g
u
ARTEMIS plus DNA-PKcs, which generates a hairpin opened product, and 5’ overhang cleavage es
t o
products. Lane 6 is the control lane containing DNA-PKcs, but no ARTEMIS. n A
p
ril 7
Figure 4. Small molecule inhibitors block both the 5' exonuclease and the , 2
0
endonucleolytic activities of ARTEMIS on hairpin DNA. 19
A, Addition of compound SBI-713 inhibits the endonuclease and 5’ exonuclease activities of
ARTEMIS. 50 nM of hairpin DNA substrate (YM164) was incubated with 60 nM ARTEMIS in a 10ul
reaction. Reactions were incubated at 37°C for 60 min. Compound SBI-116 or SBI-713 was added
at 20 uM in the indicated reactions. The 5' exonuclease activity is seen as the 1 nt product at the
bottom of the gel.
B, ARTEMIS has weak endonuclease activity on a single-stranded poly dA substrate, but has 5'
exonuclease activity. 50 nM of ssDNA substrate (JG167) was incubated with 60 nM of ARTEMIS in
addition to 75 nM of DNA-PKcs and 0.5 mM ATP where applicable. 20 uM of the inhibitor SBI-713
was added to the indicated reactions. Reactions were incubated for 1hr at 37°C. The 5' exonuclease
activity is seen as the 1 nt product at the bottom of the gel. The Klenow ladder was generated by
adding 2 units of Klenow (NEB) to 50 nM of substrate JG 167. Reactions were incubated for the
indicated time periods at 37°C.
C, Point mutant ARM14 does not have endonuclease or 5’ exonuclease activity on poly dA substrate.
50 nM of ssDNA substrate (JG167) was incubated with 60 nM of ARTEMIS or 60 nM of ARM14 point
mutant. Reactions were incubated for 1hr at 37°C. The 5' exonuclease activity is seen as the 1 nt
product at the bottom of the gel.
D, The specificity of compound SBI-713 was tested against DNase I and micrococcal nuclease. 100
nM of DNase I or 2.5 uM of micrococcal nuclease (Sigma) was incubated with 20 nM of the hairpin
10