Autoinhibition of the Nuclease ARTEMIS is Mediated by a Physical Interaction between Its Catalytic

33 Pages·2017·1.6 MB·English

JBC Papers in Press. Published on January 12, 2017 as Manuscript M116.770461
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M116.770461
Autoinhibition of the Nuclease ARTEMIS

Autoinhibition of the Nuclease ARTEMIS
is Mediated by a Physical Interaction between Its Catalytic
and C-terminal Domains

Doris Niewolik1, Ingrid Peter2, Carmen Butscher2 and Klaus Schwarz1,2
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1Institute for Transfusion Medicine, University of Ulm, Ulm, Germany ://w
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2Institute for Clinical Transfusion Medicine and Immunogenetics Ulm, German Red Cross Blood .jb
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Service Baden-Wuerttemberg-Hessen, Ulm, Germany .org
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Running title: Autoinhibition of the Nuclease ARTEMIS , 2
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Corresponding author: Doris Niewolik, Institute for Transfusion Medicine, University of Ulm,
Helmholtzstrasse 10, 89081 Ulm, Germany, Telefone: [phone], FAX: [phone],
[email protected]

Keywords: ARTEMIS, Autoinhibition, DNA-PK, DNA Repair, Immunodeficiency, NHEJ, Protein
Phosphorylation, Protein-Protein Interaction, V(D)J Recombination
1

Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Autoinhibition of the Nuclease ARTEMIS

ABSTRACT RAG1 and RAG2 cut sequence-specifically
between the coding and signal sequences of the
The nuclease ARTEMIS is essential for the
V-, D- and J-gene segments, generating
development of B and T lymphocytes. It is
physiological DNA double strand breaks (DSB).
required for opening DNA hairpins generated
At the coding joints RAG1/2 generate hairpin
during antigen receptor gene assembly from
variable (V), diversity (D) and joining (J) structures, which subsequently are specifically
subgenic elements (V(D)J recombination). As a opened by the nuclease ARTEMIS before
member of the non-homologous end joining religation can take place via the
pathway it is also involved in repairing a subset
XRCC4/XLF/DNA Ligase IV complex (3). Loss
of pathological DNA double-strand breaks. Loss
of ARTEMIS function results in the absence of
of ARTEMIS function therefore results in
B and T lymphocytes and in cellular
radiosensitive severe combined
hypersensitivity to ionizing radiation (IR).
immunodeficiency (RS-SCID). The hairpin-
opening activity is dependent on the DNA- Patients diagnosed with this severe combined
dependent protein kinase catalytic subunit immunodeficiency with sensitivity to IR (RS-
(DNA-PKcs), which can bind to and SCID) suffer from recurrent infections during
phosphorylate ARTEMIS. The ARTEMIS C-
infancy and treatment by stem cell
terminus is dispensable for cellular V(D)J
transplantation is necessary for survival. Thus
recombination and in vitro nuclease assays with
ARTEMIS is part of the Non-Homologous End D
C-terminally truncated ARTEMIS show ow
DNA-PKcs-independent hairpin-opening Joining (NHEJ) pathway, which is important for nlo
a
activity. Therefore it has been postulated that the repair of naturally occurring and exogenously de
d
ARTEMIS is regulated via autoinhibition by its induced DNA DSB. ARTEMIS is composed of fro
m
C-terminus. To obtain evidence for the 692 amino acids and it can be divided into three h
autoinhibition model, we performed co- distinct regions: The metallo-β-lactamase ttp://w
immunoprecipitation experiments with w
homology domain (aa 1-155) and the β-CASP w
combinations of ARTEMIS mutants. We show .jb
that an N-terminal fragment comprising the domain (aa 156-385, metallo-β-lactamase- c.o
rg
catalytic domain can interact both with itself and associated CPSF ARTEMIS SNM1 PSO2), b/
y
with a C-terminal fragment. Amino acid which together constitute the highly conserved g
u
e
exchanges N456A+S457A+E458Q in the C- s
catalytic domain, and the C-terminal region (aa t o
terminus of full-length ARTEMIS resulted in n
386-692) (4–6). The majority of ARTEMIS- A
AunRmTaEskMinISg of atchteiv Nity-t ermiinn us caenldlu lianr incrVe(aDse)dJ deficient RS-SCID patients have gross deletions, pril 6, 2
recombination assays. Mutations in ARTEMIS- missense or nonsense mutations in the catalytic 01
9
deficient patients impaired the interaction with domain (7). In addition to these null alleles,
the C-terminus and also affected protein stability. hypomorphic alleles have been described in
The interaction between the N- and C-terminal patients with combined immunodeficiency (CID)
domains was not DNA-PKcs dependent and
of varying severity and in radiosensitive Omenn
phosphomimetic mutations in the C-terminal
Syndrome (8–11). These mutations are
domain did not result in unmasking of the
associated with low numbers of polyclonal B and
catalytic domain. Our experiments provide
strong evidence that a physical interaction T lymphocytes and they may predispose patients
between the C-terminal and catalytic domains to lymphoma (8).
mediates ARTEMIS autoinhibition.
ARTEMIS associates with the DNA-
INTRODUCTION dependent protein kinase catalytic subunit
(DNA-PKcs) and acquires endonucleolytic
The nuclease ARTEMIS is an essential factor of
activity on hairpins and DNA overhangs, as
V(D)J recombination, which is an obligatory step shown by in vitro nuclease assays (2). In
in the development of lymphocytes of the addition, ARTEMIS has an intrinsic, DNA-PKcs
adaptive immune system (1, 2). During V(D)J independent exonucleolytic activity, which also
resides in the N-terminal part of the protein (2,
recombination the lymphocyte specific
12). In NHEJ mediated general DNA repair,
recombination-activating gene (RAG) products
ARTEMIS is necessary for processing the DNA
2
Autoinhibition of the Nuclease ARTEMIS

termini of a subset of DNA DSB induced by IR ARTEMIS-deficient patients affect the N:C, but
and other DNA damaging agents (13). not the N:N interaction and in addition influence
ARTEMIS is an in vitro substrate of the protein protein stability. The presented data provide
kinases DNA-PKcs, Ataxia telangiectasia evidence that the formerly postulated ARTEMIS
mutated (ATM) and Ataxia telangiectasia and autoinhibition is mediated by a physical
Rad3 related (ATR) and the majority of interaction between the catalytic and the C-
phosphorylation sites are localized in the C- terminal domains of the protein.
terminal tail of the protein (14–17). In response
to DNA damage ARTEMIS is RESULTS
hyperphosphorylated (13, 14, 16, 18–20). The
ARTEMIS Can Self-interact – To test,
DNA-PKcs binding region of ARTEMIS is
whether ARTEMIS is capable of self-interaction,
located between amino acid positions 398 and
we transiently co-expressed wild type ARTEMIS
403 (17, 21). The exact role of the ARTEMIS C-
and different deletion mutants (Figure 1A),
terminal tail still is unclear, in particular in light
containing at their C-termini either a myc or V5
of the fact that it is dispensable for V(D)J
tag. In order to avoid indirect co-
recombination and DNA repair (5, 21).
immunoprecipitation, we performed the
Strikingly, a C-terminally truncated ARTEMIS
experiments in the CHO derived cell line V-3,
mutant, which retains DNA-PKcs interaction,
which is devoid of endogenous DNA-PKcs.
has DNA-PKcs independent activity on hairpins
Upon immunoprecipitation with anti-myc
in the in vitro nuclease assay. This result is
suggestive of the C-terminal region having a antibody, eluates and input lysates were tested in Dow
Western blots. These were sequentially probed n
negative regulatory function, which may be lo
with anti-V5 antibody to test for co- ad
relieved by DNA-PKcs mediated e
d
phosphorylation (21). However, mutation of immunoprecipitation and anti-myc antibody to fro
detect the immunoprecipitate. Full length m
DNA-PKcs phosphorylation sites in the C- h
terminal tail of ARTEMIS has no effect on in cAoR-iTmEmMuInSo p(rAecRipTi-tWateTd 1b-6y 92i)ts ewlfa s (Fsipgeucriefi ca1lBly, ttp://w
vivo V(D)J recombination and DNA repair w
lanes 1-3). The analyses of N- and C-terminal w
properties, rather autophosphorylation of .jb
fragments showed, that the N-terminal (ARM 1- c
DNA-PKcs was shown to regulate ARTEMIS .o
382), but not the C-terminal part (ARM 414-692) rg
endonucleolytic activities (16). Recently, DNA- b/
of the ARTEMIS protein is capable of self- y
Ligase IV (22–24) and Pax transactivation g
interaction (Figure 1B, lanes 5 and 14, ue
domain interaction protein (PTIP) (25) have respectively). In addition, the non-overlapping st o
n
been shown to specifically interact with the C- N- and C-terminal fragments show reciprocal co- Ap
terminal part of ARTEMIS. immunoprecipitations (Figure 1B, lanes 7 and ril 6
NHEJ factors are in the focus as targets for 12). To further characterize these two , 201
independent interactions which we denote N:N 9
cancer therapy, because of the reliance of tumor
and N:C interactions, we tested additional
cell division on DNA repair mechanisms (26).
ARTEMIS deletion constructs. Deletion of
Elucidation of the exact activation mechanisms
amino acids 1 to 7 of the N-terminal fragment
of potential target proteins is a prerequisite for
(ARM 8-382) retained co-immunoprecipitation
the development of novel therapeutic options. In
of ARM 1-382, but resulted in loss of the
case of the nuclease ARTEMIS advance has
interaction with ARM 414-692 (Figure 1B, lanes
been impeded by the lack of any structural data
6 and 13, respectively). Deletion mutant ARM
of the full length protein. Because of these
474-692 shows reduced expression as compared
limitations we decided to employ classical
to ARM 414-692. Therefore in addition we
biochemical methods to challenge the hypothesis
performed a co-immunoprecipitation, in which
of ARTEMIS being regulated by autoinhibition.
cell lysate containing ARM 414-692 was diluted
Using co-immunoprecipitation experiments we
1:4 with vector cell lysate (Figure 1B, lane 10).
demonstrate, that ARTEMIS interacts with itself
Under these conditions, the amount of
in two ways: The N-terminus can bind both to
immunoprecipitated ARM 414-692 is
itself (N:N interaction) and to the C-terminus
comparable to that of mutant ARM 474-692,
(N:C interaction). In full length ARTEMIS
which appears as a doublet band. Whereas co-
specific amino acid exchanges within the C-
immunoprecipitation of the N-terminal fragment
terminus impair the N:C interaction and confer
(ARM 1-382) is detectable for ARM 414-692 it
an increase in V(D)J recombination activity. We
is not for ARM 474-692 (Figure 1B, compare
show, that missense mutations found in
3
Autoinhibition of the Nuclease ARTEMIS

lanes 8 and 10). Thus, amino acids between not dependent on the 7 N-terminal residues (N:N
positions 414 and 474 in the C-terminal fragment interaction, Figure 1B, lanes 4-6).
are critical for the N:C interaction.
Single Point Mutations in the ARTEMIS N-
The extended C-terminus of ARTEMIS Terminus also Affect V(D)J Recombination
contains several DNA-PKcs and ATM Activity – Previously, we have observed that
phosphorylation sites and it was speculated to deletion of the 7 N-terminal amino acids of full
have a regulatory function. Therefore we length ARTEMIS (ARM 8-692 in Figure 1A)
concentrated our further analyses on the abolishes V(D)J recombination activity (9). To
characterization of the interaction between the determine the functional effect of individual
N- and C-terminal ARTEMIS fragments. Since amino acid exchanges in the N-terminal
deletion of the first 7 amino acids at the N- fragment we performed V(D)J recombination
terminus is critical for the N:C interaction assays in ARTEMIS-deficient human primary
(Figure 1B, compare lanes 12 and 13) we tested skin fibroblasts. As controls, full length
the effect of single amino acid exchanges in ARTEMIS (ART-WT 1-692) as well as ARM
conserved positions within this region (see 8-692 and ARM 8-382 were included in the
Figure 1C). Expression levels of these mutant analyses and the expression levels of these
proteins are reduced compared to ARM 1-382, proteins, in the absence of endogenous
this has to be taken into account in the ARTEMIS, were analyzed in Western blots
interpretation of the results of the co- (Figure 2). The results of three independent
D
immunoprecipitation experiments. With the cellular V(D)J recombination experiments are o
w
exception of amino acid exchange P12A all shown in Table 1. The recombination activity of nlo
a
mutations abolished co-immunoprecipitation by C-terminally truncated ARTEMIS (ARM 1-382) de
d
the C-terminus (Figure 1D). Thus, amino acids is reduced 10-14 fold compared to ART-WT. fro
m
S2, F4, G6, E10 and Y11 in the N-terminal However, since expression of ARM 1-382 is h
fragment are critical for the N:C interaction. decreased about 100-fold as compared to ART- ttp
Next, we wanted to determine amino acids in the WT (Figure 2, compare lanes1-4 with lane 7), its ://w
w
C-terminal fragment, which are crucial for V(D)J recombination activity must exceed that w.jb
binding to the N-terminus. Analyses of of ART-WT. Deletion of the first 7 amino acids c.o
additional deletion mutants revealed, that both in the full length (ARM 8-692) and C- brg/
binding is lost between amino acid positions 439 terminally truncated (ARM 8-382) proteins, y g
u
to 454 (data not shown). We therefore prepared results in reduction of V(D)J recombination es
C-terminal ARTEMIS mutants (aa 383 – 692) in activity to basal levels (- ART and - RAG1). In t on
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which two or three adjacent amino acids were the case of ARM 8-692, this may in part be p
exchanged to alanine or in the case of acidic accounted for by the low expression levels as ril 6
, 2
amino acids (E and D) to their corresponding compared to ART-WT (Figure 2, lanes 1 - 5). 0
1
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amides (Q and N). The expression levels of the Yet, expression levels of ARM 1-382 and ARM
different C-terminal proteins vary considerably 8-382 are comparable (Figure 2, lanes 7 and 8,
(Figure 1E, compare anti-V5 and anti-HPRT in respectively), indicating that in the context of the
input lanes). However, the capability to bind the C-terminally truncated proteins, the first 7 amino
N-terminal fragment (ARM 1-382) clearly is lost acids are important for V(D)J recombination
upon mutation of amino acids from positions 448 activity. Each of the single point mutations tested
to 462 (Figure 1E). abolished the V(D)J recombination activity of
the C-terminally truncated ARM 1-382. This
The experiments depicted in Figure 1 define holds true also for mutation P12A, which
two non-overlapping regions of the ARTEMIS showed binding activity towards the ARTEMIS
protein that bind to each other in co- C-terminus in co-immunoprecipitation
immunoprecipitation experiments. Conserved experiments (Figure 1D, lane 7).
amino acids at the very N-terminus of ARTEMIS
and in the C-terminal fragment are important for Full Length Wild Type ARTEMIS Cannot
the N:C interaction. For simplicity we refer to Interact with the Isolated C-terminal Fragment –
these separable protein binding regions hereafter The experiments shown in Figure 1 were
as N- and C-terminal interaction domains. The performed in the absence of endogenous
N-terminal domain was also shown to have the DNA-PKcs, in order to avoid indirect co-
capability to self-interact and this interaction is immunoprecipitation. Next, we wanted to
establish whether full length ARTEMIS also can
4
Autoinhibition of the Nuclease ARTEMIS

interact with the N- and C-terminal fragments within the core of the above defined C-terminal
and if so, whether binding of endogenous interaction domain (aa 448 – 462, see Figure 1C
DNA-PKcs has an influence on these and E). Deletion of the 7 N-terminal amino acids
interactions. Hence we carried out co- in the context of the latter mutant results in loss
immunoprecipitation experiments in HEK293T of the interaction (ARM
cells. As expected mutations in (ARM 8-692+N456A+S457A+E458Q, compare Figure
1-692+L401G+R402N) and deletion of the 4, lanes 12 and 13). This indicates that the
DNA-PKcs binding site (ARM 1-382) abolish observed co-immunoprecipitation indeed reflects
co-immunoprecipitation of endogenous binding to the N-terminus of full length
DNA-PKcs (Figure 3, compare lanes 1 – 5 to 10 ARTEMIS. The results illustrated in Figure 4 are
– 14 and 19 – 22, respectively). ARTEMIS self- in support of the hypothesis, that in ART-WT,
interaction is seen for both full length proteins, the N-terminal domain is masked by the C-
as well as for the two N-terminal fragments terminus due to self-interaction. Disabling the
ARM 1-413 and ARM 1-382, the latter of which N:C interaction by mutation of amino acids 456
does not contain the DNA-PKcs binding site – 458, and to a lesser extend also of amino acids
(Figure 3, lanes 1 – 4 and 10 – 13). Surprisingly, 454 + 455 (Figure 4, lanes 11 and 12,
the C-terminal fragment (ARM 414-692) is not respectively) liberates the N-terminus of full
co-immunoprecipitated by full length length ARTEMIS, which then is available for
ARTEMIS, irrespective of whether or not the interaction with the C-terminal fragment (ARM
precipitated ARTEMIS protein binds DNA-PKcs 414-692), as demonstrated by co-
D
(Figure 3, lanes 5 and 14). In HEK293T cells immunoprecipitation. ow
n
specific interactions are observed both between lo
a
the N-terminal (Figure 3, lanes 19 - 20) and the Binding of the C-terminal Fragment to Full de
d
non-overlapping N- and C-terminal fragments Length ARTEMIS and to the N-terminal fro
m
(Figure 3, lanes 21-22). Fragment is independent of the DNA Ligase IV h
Interaction Site – The experiment described in ttp
While interaction with DNA-PKcs apparently the previous section was performed in CHO V-3 ://w
w
is not the reason why the C-terminal fragment cells, in the absence of endogenous DNA-PKcs. w.jb
cannot bind to the N-terminus in ART-WT, other Subsequent co-immunoprecipitation experiments c.o
protein:protein interactions could be responsible were conducted with lysates prepared from brg/
for masking the N-terminus. Both intra- or HEK293T cells, transiently transfected with y g
u
intermolecular interactions between the N- and combinations of ARTEMIS expression plasmids. es
C-termini of full length ARTEMIS or These experiments were optimized in two ways: t on
A
dimerization via the N:N interaction could Firstly, they were performed in a reverse p
theoretically be an explanation. To test this experimental set up, in which the V5-tagged full ril 6
, 2
hypothesis we analyzed the complete cassette of length ARTEMIS protein is co- 0
1
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mutations that define the C-terminal interaction immunoprecipitated by the myc-tagged C-
domain (see Figure 1D) in full length context (aa terminal fragment. This increases the sensitivity
1 – 692, wild type or mutant), asking whether of the assay, since full length ARTEMIS is
any of the mutant proteins can interact with the expressed much more strongly than the C-
isolated C-terminal fragment (ARM 414-692). terminal fragment. Secondly, the C-terminal
This experiment was again performed in CHO fragment was enlarged so that it contains the
V-3 cells, in order to avoid competition for DNA-PKcs binding site (ARM 348-692, see
binding with endogenous DNA-PKcs. In Figure 1A) and in addition an N-terminal tag,
accordance with the results shown in Figure 1, which improves expression levels. With these
binding between the C- and N-terminal modifications co-immunoprecipitation of
fragments is observed and this interaction is lost, endogenous DNA-PKcs can be monitored as an
if the first 7 amino acids at the N-terminus are internal control. In accordance with the results
deleted (Figure 4, lanes 9 and 10). Strikingly, obtained in CHO V-3 cells (Figure 4) ART-WT
two full length mutant proteins show co- cannot bind the C-terminal fragment, while the
immunoprecipitation of the isolated C-terminal mutant (ARM 1-692+N456A+S457A+E458Q)
fragment (Figure 4, lanes 11 and 12) while all and the N-terminal fragment can (Figure 5, lanes
others, like ART-WT do not. Both of these 5, 9 and 1, respectively). Amino acid exchanges
mutant proteins (ARM 1-692+E454Q+S455A N456A+S457A+E458Q in the
and ARM 1-692+N456A+S457A+E458Q) have immunoprecipitated C-terminal fragment,
exchanges at invariant amino acid positions abolish co-immunoprecipitation both of the N-
5
Autoinhibition of the Nuclease ARTEMIS

terminal fragment and of the full length mutant DNA-dependence (Figure 7, lanes 7 – 9). In the
(Figure 5, lanes 2 and 10), but have no effect on case of immunoprecipitation of the isolated C-
the DNA-PKcs interaction. Therefore, the terminal fragment (Max/ARM 348-692/myc),
interactions between the C-terminal fragment co-immunoprecipitation of both endogenous
and the N-terminus either as part of full length DNA-PKcs and full length mutant ARTEMIS
ARTEMIS or as isolated fragment (ARM 1-382) (ARM 1-692+N456A+S457A+E458Q) show no
have the same specificities. A DNA Ligase IV difference with or without ethidium bromide
interaction site in ARTEMIS (22) lies in (Figure 7, lanes 1 – 3). Therefore, the newly
proximity to the C-terminal domain described in described N:C interaction of ARTEMIS is DNA-
this work. Therefore we tested whether amino independent.
acid exchange W489A, which is crucial for
Ligase IV interaction also affected ARTEMIS Mutations in ARTEMIS-deficient Patients
self-interaction. W489A does not have an effect Impair the N:C Interaction of Full Length
on the N:C interaction, neither in the context of ARTEMIS – Mutations at the very N-terminus of
the isolated fragments, nor with respect to ARTEMIS abolish N:C interaction (Figures 1D)
interaction of full length mutant ARTEMIS and affect V(D)J recombination activity in the
(Figure 5, lanes 3 and 11). context of C-terminally truncated ARTEMIS
(Table 1). We were interested to see, which
Mutation sensitivity in the C-terminal protein effect point mutations of ARTEMIS-deficient
binding domain is more extended, if tested in the patients and located at various positions within
D
context of the non-overlapping N- and C- the catalytic domain, have on the N:C o
w
terminal fragments (aa 448 – 462, Figure 1E, interaction. Therefore we tested the mutations in nlo
a
lanes 5 - 10), as compared to the full length the context of the amino acid exchanges de
d
protein (aa 454 – 458, Figure 4, lanes 11 and 12). N456A+S457A+E458Q (Figure 8). According to fro
m
Therefore we wanted to characterize the N:C our working hypothesis, we would expect h
interaction of full length protein in more detail, additional mutations, which destroy the N- ttp
in particular with respect to mutations in the N- terminal protein binding domain, to result in loss ://w
w
terminal domain. In the context of the C-terminal of co-immunoprecipitation by the isolated C- w.jb
mutation N456A+S457A+E458Q deletion of terminal fragment. Co-immunoprecipitation of c.o
amino acids 1-7 abolishes the interaction, full length ARTEMIS is impaired in the case of brg/
whereas the single amino acid exchanges E10Q proteins which contain in addition to the amino y g
u
and P12A have no influence with respect to the acid exchange N456A+S457A+E458Q either es
interaction with the C-terminal fragment (Figure mutations corresponding to null alleles (G135E, t on
A
6, lanes 1 – 5). Wild type sequence in the C- D165V and H228N (7, 28)) or amino acid p
terminal domain in all cases is incompatible with exchange G211V (29) (Figure 8, compare lane 2 ril 6
, 2
co-immunoprecipitation by the C-terminus to lanes 3, 5, 7 and 8). Co-immunoprecipitation 0
1
9
(Figure 6, lanes 6 – 8). of full length proteins containing in addition the
hypomorphic mutations G153R, P171R (10, 11)
Binding of the C-terminal Fragment to Full or the polymorphism H243R (NCBI-dbSNP
Length Mutant ARTEMIS is DNA-independent – entry rs12768894) is clearly visible (Figure 8,
Since ARTEMIS is a nuclease, we were compare lane 2 to lanes 4, 6 and 9). Co-
interested to see, whether the N:C interaction is immunoprecipitation of endogenous DNA-PKcs
DNA-independent. We therefore performed is observed in all cases. Thus, specific mutations
experiments in which prior to observed in ARTEMIS-deficient patients affect
immunoprecipitation, the precleared lysate was the N:C interaction in full length proteins.
incubated with ethidium bromide. By its Comparable results were obtained with the non-
intercalation into the DNA ethidium bromide overlapping fragments (data not shown).
distorts the conformation, resulting in loss of
protein-DNA interactions. The interactions of Mutations in ARTEMIS-deficient Patients do
DNA-PKcs with ART-WT (17) and with KU80 not Affect the N:N Interaction of Full Length
(27) were used as negative and positive controls, ARTEMIS – Having demonstrated that mutations
respectively. As expected, the DNA-PKcs found in ARTEMIS-deficient patients have an
interaction with ART-WT (Figure 7, lanes 4 – 6) impact on the N:C interaction (Figure 8), we
is DNA-independent, whereas DNA-PKcs co- wanted to see, whether the N:N interaction (see
immunoprecipitation by KU80 is diminished in Figure 1B) also is affected. We made use of the
the presence of ethidium bromide, indicating its observation that additional amino acids added to
6
Autoinhibition of the Nuclease ARTEMIS

the N-terminus of ARTEMIS drastically reduce relevance of the protein interaction data
the N:C interaction capability (Figure 9A, presented above we performed cellular V(D)J
compare the intensities of the IP, Co-IP and recombination assays in ARTEMIS-deficient
input signals). Therefore, we tested the N:N human primary fibroblasts. First, we established
interaction of full length ARTEMIS proteins as the relative expression levels of the different
co-immunoprecipitation by Max/ARM ARTEMIS constructs in the absence of
1-382/myc, which contains at its N-terminus the endogenous ARTEMIS protein. We compared
Xpress epitope. We observe co- the expression of ART-WT and full length
immunoprecipitation of all full length proteins ARTEMIS containing either the double amino
analyzed (Figure 9B). Neither amino acid acid exchange F446A+T447A or the triple
exchanges N456A+S457A+E458Q (Figure 9B, mutation N456A+S457A+E458Q. In the co-
lane 2), nor additional deletion of the 7 N- immunoprecipitation experiments the former
terminal amino acids (Figure 9B, lane 3), nor mutant behaved like ART-WT, whereas the latter
single amino acid exchanges found in was shown to have an unmasked N-terminal
ARTEMIS-deficient patients (Figure 9B, lanes domain (Figure 4). Expression levels of ARM
4-10) interfere with the N:N interaction. Full 1-692+F446A+T447A and ART-WT are
length ARTEMIS protein runs at 100 kDa in comparable (Figure 11A, compare lanes 1-2 and
SDS-PAGE. In addition we observe a band at 6-7). However, ARM
approximately 75 kDa, corresponding to a 1-692+N456A+S457A+E458Q is dramatically
protein presumably derived from an internal unstable as compared to ART-WT (Figure 11A,
D
translational start at position M121. In agreement compare lanes 1-5 to lane 8). As estimated from ow
n
with the differences in mutation sensitivity of the titration of the amount of expression plasmid lo
a
d
N:C versus N:N interactions (see Figure 1B) this transfected, the expression of ARM e
d
N-terminally truncated protein is also co- 1-692+N456A+S457A+E458Q is reduced about fro
m
immunoprecipitated strongly via N:N interaction 20-fold compared to ART-WT and ARM h
(Figure 9B lanes 1-10). 1-692+F446A+T447A. In the subsequent ttp://w
cellular V(D)J recombination assay, it was w
w
Expression levels of the different mutant important to supply ARTEMIS in limiting .jb
proteins vary. In particular proteins that have concentrations, in order to detect potential c.o
impaired N:C interaction show reduced differences in the V(D)J recombination rates brg/
expression levels. This holds true also for amino between wild type and mutant proteins. We y g
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aRcSid-S CexIcDh apnagtieesn tcso (rGre1s3p5oEnd, iDng1 6t5oV n aunlld aHll2el2e8sN i)n, tphreimreafroyr e fibrotrbalnasstfse ctwedit h diAffRerTeEntM aISm-oduenfitcsi eonft est on A
a9sB ,w ceollm apsa raem sihnoor ta ceixdp oexsucrhea nogfe i nGp2u1t 1lVan e(sF i1g,u r4e, AcoRmTpEoMneInSt se xcpornesstsainotn avnedc tourssin, gk eveepcintogr awll itohtohuert pril 6, 2
6, 8 and 9). In order to evaluate the effect of the insert to fill up to identical amounts of DNA. 01
9
different patient mutations on protein stability in Table 2 summarizes the results of four
the absence of endogenous ARTEMIS we independent V(D)J recombination assays, each
transiently transfected the corresponding comparing the V(D)J recombination rates of
expression plasmids in ARTEMIS-deficient ART-WT and the two mutants as well as
human primary fibroblasts and analyzed protein relevant controls (- ART, - RAG1/2). The
levels by Western blot analysis (Figure 10). recombination rates at the different amounts of
Again, there is a dramatic reduction in input ART/ARM expression vector (range 5 –
expression of ARTEMIS proteins that contain 1000 ng) are similar for all three ARTEMIS
amino acid exchanges corresponding to null proteins (Table 2 and Figure 11B). Taking into
alleles in RS-SCID patients, as well as mutation account the difference in expression levels
G211V (Figure 10, lanes 1, 3, 5 and 6). Since (Figure 11A) and presuming that a similar ratio
transcription in all cases is regulated by the same is also present in the cellular V(D)J
CMV promoter, we assume that differences in recombination assay, ARM
protein levels as analyzed by Western blot reflect 1-692+N456A+S457A+E458Q is much more
variations in protein stability. active than ART-WT and mutant ARM
1-692+F446A+T447A. Figure 11C illustrates the
ARTEMIS with Amino Acid Exchanges
relative V(D)J recombination activities if a 10-
N546Q+S457A+E458E is More Active than
fold difference in expression levels at
ART-WT in a Cellular V(D)J Recombination
ARTEMIS-limiting conditions is assumed. In
Assay – To address the question of functional
this case ARM 1-692+N456A+S457A+E458Q is
7
Autoinhibition of the Nuclease ARTEMIS

4.5-fold more active in V(D)J recombination terminal portions of ARTEMIS and the N:N
than ART-WT. Expression levels at 5 and 50 ng interaction in which the N-terminal domain binds
input of the corresponding expression vectors are to itself. Systematic analyses with different
far below the detection limit of Western blot combinations of full length ARTEMIS proteins
analyses, precluding this control. The results of and N- or C-terminal fragments allow the
the cellular V(D)J recombination assays, following conclusions: (a) In wild type
comparing ART-WT to full length ARTEMIS ARTEMIS the N-terminus is engaged with
with specific mutations within the C-terminal binding to the C-terminal domain. (b) Specific
protein binding domain (Table 2 and Figure 11), mutations in the C-terminal domain of full length
are in support of the hypothesis that ARTEMIS protein (aa 454 – 458) abolish this interaction,
function is regulated by autoinhibition via the C- leaving the N-terminus free to bind the isolated
terminal domain. C-terminal fragment. These results suggest that
mutant ARM 1-692+N456A+S457A+E458Q
Phosphomimetic Amino Acid Exchanges in C- adopts a different conformation than ART-WT.
terminal ATM and DNA-PKcs Phosphorylation Evidence is strong that the N:C interaction of full
Sites do not Unmask the Catalytic Domain – length ARTEMIS corresponds to the interaction
ARTEMIS with amino acid exchanges observed between the non-overlapping N- and C-
N546Q+S457A+E458E which abolish the terminal fragments, since deletion of the 7 N-
intrinsic N:C interaction is a useful experimental terminal amino acids abolishes binding in either
tool, mimicking activation of ARTEMIS case. Importantly, the N:C interaction is DNA-
D
function. We wanted to assess whether independent. In the N-terminal protein binding ow
phosphorylation of the C-terminus affects the domain amino acid exchanges in conserved nlo
a
interaction between the N- and C-terminal residues distributed throughout the metallo-β- ded
domains. In full length ARTEMIS we introduced lactamase and β-CASP domains are important fro
m
phosphomimetic amino acid exchanges (S to E) for the N:C interaction. This resembles the h
at one or more previously described ttp
phosphorylation sites (16, 18, 19, 25). As mutation sensitivity observed in V(D)J ://w
recombination assays performed to define the w
w
depicted in Figure 12 only the positive control catalytic center of the nuclease (5, 6). In the .jb
(ARM 1-692+N546Q+S457A+E458E, lane 2), current study we show that in the context of C- c.o
but none of the other full length ARTEMIS terminally truncated ARTEMIS deletion of and brg/
proteins, can be co-immunoprecipitated by the missense mutations in the N-terminus result in y g
u
Cch-aterrgme inparol vfridagedm ebnyt . gTlhuutasm, cico nasctiitdu tiavte pnoesgiatitoivnes loss of both V(D)J recombination activity and est on
N:C interaction. Interestingly, amino acid A
503, 516, 534, 538, 562, 645 and 656 in the p
ARTEMIS C-terminal tail does not affect the exchange P12A allows a separation of functions, ril 6
since it impairs V(D)J recombination activity but , 2
accessibility of the N-terminus in full length 0
retains N:C interaction capability. 1
9
ARTEMIS, as monitored by co-
immunoprecipitation. The majority of missense mutations found in
patients diagnosed with an ARTEMIS defect lie
DISCUSSION
in the N-terminal catalytic domain (7). In this
study, we show that mutations found in
Protein crystallography is a potent technique
ARTEMIS-deficient patients affect the N:C but
for the analysis of conformational changes in
not the N:N interaction. These results also
proteins and has been successfully employed for
suggest that in full length ARTEMIS the N:C
DNA-PKcs and other enzymes of the NHEJ
interaction and the concomitant masked
pathway (30). However up to now, no structural
conformation are not a prerequisite for the N:N
data are available for full length ARTEMIS. We
interaction. Amino acid exchanges
therefore conducted co-immunoprecipitation
corresponding to null alleles (G135E, D165V
experiments to test whether ARTEMIS is
and H228N) and G211V but not the
capable of self-interaction, a prerequisite for
hypomorphic mutations G153R and P171R
autoinhibition mediated by protein:protein
impair N:C interaction. Notably, in full length
interaction (31). Our experiments with
context mutations corresponding to null alleles in
ARTEMIS fragments identify protein binding
ARTEMIS-deficient patients result in reduced
domains which mediate two independent modes
expression of the respective proteins as
of self-interaction: The N:C interaction between
compared to ART-WT. The coincidence of
non-overlapping fragments from the N- and C-
reduced expression levels and loss of N:C
8
Autoinhibition of the Nuclease ARTEMIS

interaction of full length proteins may point to a actin assembly (32). Loss of WASP function
causal relationship. Therefore we tentatively results in immunodeficiency (33). Autoinhibition
suggest that mutations which inactivate the N:C is mediated by an intramolecular interaction and
interaction destabilize the protein, and this is also activation involves binding of regulatory proteins
contributing to the RS-SCID phenotype. In to the inhibitory domain and phosphorylation
principle the N:C interaction of full length (32, 34). In ARTEMIS, DNA-Ligase IV binds to
protein and protein stabilization, could occur the C-terminal tail (22) in the vicinity of the
either as an inter- or intramolecular event and protein binding domain described in this work.
both possibilities are compatible with the data Mutation of the DNA-Ligase IV binding site in
presented. Unfortunately, the overlapping ARTEMIS did not affect the N:C interaction,
association via the N:N interaction, as well as the indicating that DNA Ligase IV is not a mediator.
fact that additional amino acids positioned at the Specific mutations in the N-terminal fragment
N-terminus impair the N:C interaction, preclude impair both N:C interaction and V(D)J
simple experiments to answer this important recombination activity. Therefore we assume
question. Up to now, we have not been able to that the activation step most likely affects either
identify any single amino acid mutation affecting the C-terminal domain itself or amino acids
the N:N interaction and analyses with many which link it to the N-terminal catalytic domain.
different deletion constructs suggest, that this The first possibility is underscored by our
interaction is mediated by several redundant finding, that the N:C interactions of full length
interfaces (data not shown). protein and of non-overlapping fragments are
D
comparable with respect to mutation sensitivity ow
C-terminally truncated ARTEMIS is in both protein binding domains. Mutations nlo
a
sufficient to confer V(D)J recombination activity N456A+S457A+E458Q hit residues that can de
d
(5, 21). Here we show that amino acid exchanges form salt- and amide bridges and this may either fro
m
N456A+S457A+E458Q which abolish N:C distort protein folding or affect directly the h
irnetceormacbtiinoant,i orens ualctt iivni tay 4a.s5 c-foomldp ainrecdre taos eA iRn TV-W(DT)J. ibnitnedriancgti odno msauinrf.a c e in the C-terminal protein ttp://ww
w
Similar results were shown for ARM 1-382 and .jb
were also obtained with C-terminally truncated Phosphorylation by DNA-PKcs is not a c.o
ARM 1-413 (data not shown). Therefore we prerequisite for ARTEMIS self-interaction, since brg/
would like to conclude that a conformational our initial experiments were conducted in CHO- y g
u
change occurs during activation of ARTEMIS, V3 cells. Also, an intact DNA-PKcs binding es
involving loss of physical association between domain in ARTEMIS is dispensable. The C- t on
A
the N- and C-terminal parts of the protein. This terminal domain (aa 454 – 458) contains a p
provides conclusive evidence for the formerly stretch of predominantly acidic residues and in ril 6
, 2
postulated autoinhibition model (15, 21). In vitro addition three serines which upon 0
1
9
nuclease assays with purified components are phosphorylation could add to the overall
potent experiments for the analysis of ARTEMIS negative charge. However, these serines do not
function. It will be of interest to elucidate the correspond to consensus phosphorylation sites
effect of amino acid exchanges (35). In vitro S503, S516 and S645 in ARTEMIS
N456A+S457A+E458Q on the different endo- have been defined as major DNA-PKcs and/or
and exonucleolytic activities of ARTEMIS, and ATM phosphorylation sites (16) but other serines
in particular their dependence on the presence of and threonines are also phosphorylated (15, 17).
and the phosphorylation by DNA-PKcs. Importantly, S645 in ARTEMIS is
However, mutations N456A+S457A+E458Q are phosphorylated in vivo in an ATM-dependent
only an experimental model imitating the manner and induction of DNA DSB by IR leads
activation process and it will be of utmost to hyperphosphorylation at this site (16, 19).
interest to identify the physiological stimuli, Phosphorylation at S645 and T656 is important
inducing the postulated conformational change. for ATM-dependent association between
Posttranslational modifications and the ARTEMIS and PTIP (25). Here we show that
displacement of inhibitory domains by phosphomimetic mutations at seven sites
intermolecular interactions with activator (S503E, S516E, S534E, S538E, S562E, S645E
proteins are frequent mechanisms for the relief of and T656E) do not affect N:C interaction in full
autoinhibition (31). A well-studied example is length ARTEMIS. Although it cannot be
the Wiskott-Aldrich Syndrome protein (WASP) excluded that glutamic acid was insufficient in
family which comprises proteins that regulate mimicking phosphorylation, our results have not
9
Autoinhibition of the Nuclease ARTEMIS

provided evidence, that phosphorylation at the small molecular compounds that specifically
sites tested is the in vivo trigger, which leads to block this activation.
unmasking of the N-terminal catalytic domain.
DNA-PKcs autophosphorylation has been In summary, we have presented evidence that
implicated in the regulation of ARTEMIS a physical interaction between the N-terminal
endonucleolytic activities and it is assumed, that catalytic domain and the C-terminal tail of the
the precise positioning of the nuclease on ARTEMIS protein mediates the previously
different DNA substrates is crucially regulated postulated autoinhibition of the nuclease.
by the phosphorylation status of the kinase (16, Specific amino acid exchanges in the C-terminal
36). The importance of the ARTEMIS:DNA- protein binding domain result in a
PKcs interplay is apparent also from the first conformational change that unmasks the N-
DNA-PKcs deficient RS-SCID patient described terminus and results in increased V(D)J
(37). The patient phenotype closely resembles recombination activity. Phosphomimetic
that of RS-SCID patients with ARTEMIS null mutations in the C-terminal domain did not
mutations possibly reflecting impairment of induce the postulated conformational change, so
ARTEMIS activation by DNA-PKcs. Additional that the exact role of DNA-PKcs in the relief of
experiments will have to be performed to address autoinhibition still needs to be elucidated.
the question how ARTEMIS self-interaction and Mutations found in ARTEMIS-deficient patients
the relief of autoinhibition is related to and contained in the catalytic domain affect the
phosphorylation by DNA-PKcs. Clearly, C- interaction with the C-terminal domain and
D
terminally truncated ARTEMIS is a very potent possibly as a consequence, also protein stability. ow
n
interaction partner of DNA-PKcs (21). lo
EXPERIMENTAL PROCEDURES ad
e
d
The involvement of DNA repair factors in Cells and Transfections – HEK293T cells from
tumorigenesis is well established for some and h
speculative for others (38). In case of ARTEMIS were cultured in DMEM supplemented with 10% ttp
a hypomorphic allele found in CID patients and FCS and transfected using the calcium phosphate ://w
w
precipitation method. Human primary w
resulting in a C-terminally truncated protein was ARTEMIS-deficient skin fibroblasts were .jb
suspected to predispose to lymphoma (8). c.o
Aconrarelysspeosn diinn gm icmeu tpartoiovnid edle aedvsi dentoc e gtheanto mthice c1(Au0l%mtua rxeFadCT MiSn CIeaMlnlDd l iMnter amNnesudfceilucetmoefd,e cstubopyrp Klenimtu cVelne,t oeLfdoe cnwtziiaot)hn. by gurg/
instability and, in the absence of p53, to es
enhanced tumorigenesis (39, 40). Intriguingly, The DNA-PKcs negative CHO cell line V-3 was t on
cultured in IMDM supplemented with 10% FCS A
this mutation results in a frameshift at position p
D451, followed by translational termination 10 and transfected by nucleofection (AmaxaTM Cell ril 6
line Nucleofector Kit T, Lonza). In transfections , 2
residues to the C-terminus. Thus the C-terminal 0
1
with DNA titrations the total amount of DNA 9
truncation leaves the DNA-PKcs binding region
was kept constant with vector DNA.
intact but destroys the C-terminal self-interaction
domain described in this study (aa 454 – 458).
Protein Expression Constructs – Human wild
Overexpression of wild type ARTEMIS in mice
type (ART-WT) and mutant (ARM) ARTEMIS
does not lead to lymphopoietic malignancies
expression plasmids were cloned via KpnI /NotI
(41). Therefore we cautiously speculate, that loss
restriction sites into pcDNA6myc-His and
of the N:C interaction, resulting in an unmasked
pcDNA6V5-His vector (Invitrogen, Breda,
catalytic domain may possibly cause DNA
Netherlands). The nomenclature refers to the
instability. In diffuse large B cell lymphomas,
amino acids included in the respective
DNA repair genes are selectively mutated and
ARTEMIS mutant proteins (ARM). Construct
the somatic mutation identified in ARTEMIS
ARM 8-692 corresponds to ARM29 in (9)
also resulted in a C-terminally truncated protein
constructs ARM 1-413 and ARM 1-382
(42). DNA repair factors have been in special
correspond to ARM37 and ARM23 in (21).
focus for the development of novel strategies for
Supplemental Table 1 shows the forward and
the treatment of drug resistant tumors (26). The
reverse primers used for amplification of the
findings presented in this paper, describing a
ARTEMIS cDNAs of the deletion mutants. In
protein interaction interphase involved in the
expression plasmids Max/ARM 348-692 and
activation of the structure specific nuclease
Max/ARM 1-382 the ARTEMIS cDNAs were
ARTEMIS, may help to refine the search for
cloned via Kpn I and Not I into
10