A novel DNA repair enzyme containing RNA recognition, G-patch
and specific splicing factor 45-like motifs in the protozoan parasite
Toxoplasma gondii
Najoua Dendouga
1
, Isabelle Callebaut
2
and Stanislas Tomavo
1
1
Equipe de Parasitologie Mole
´
culaire, Laboratoire de Chimie Biologique, CNRS UMR 8576, Universite
´
des Sciences et Technologies
de Lille, France;
2
Equipe Syste
`
mes Mole
´
culaires et Biologie Structurale, Laboratoire de Mine
´
ralogie-Cristallographie,
CNRS UMR 7590, Universite
´
Paris, France
We report the cloning and functional charaterization of the
full-length cDNA and gene encoding a Toxoplasma gondii
DNA repair enzyme designated TgDRE. The gene is com-
posed of three exons separated by two introns of 780 and

haploid asexual forms, the replicating tachyzoite and the
slowly dividing quiescent encysted bradyzoites. T. gondii
infection is asymptomatic in most adults, but the parasite
persists during the lifetime of infected hosts as slowly
dividing encysted bradyzoites. When the protective immu-
nity fails, as in AIDS or in transplanted patients [1], the
encysted bradyzoites can transform into actively replicating
and virulent tachyzoites. The tachyzoites differentiate into
encysted bradyzoites in response to the immune system
attack during disease progression. They will remain in the
brain and other organs during the lifetime of infected hosts.
The reactivation of encysted bradyzoites into actively
replicating and cytolytic tachyzoites can trigger a disease
with severe clinical syndromes leading in many cases to the
death of the patients. While the actively replicating tachy-
zoites can be successfully treated by different chemothera-
peutic agents, none of these drugs are capable of inhibiting
the encysted bradyzoites which is the source of toxoplas-
mosis reactivation. Therefore, a better understanding of the
biology of this obligate intracellular parasite and the
molecular mechanisms underlying T. gondii differentiation
is useful in controlling the infection.
DNA repair proteins have been well characterized in
human, yeast, plant and bacteria and play important roles
in preserving the genetic information that ensure the normal
cellular function and development [2–4]. Although all
organisms contain various DNA repair mechanisms, the
repair of a specific DNA damage is often conserved from
bacteria to human, and in many cases the proteins are
highly similar [5]. Little is known about DNA repair

(Received 19 March 2002, revised 2 May 2002, accepted 13 May 2002)
Eur. J. Biochem. 269, 3393–3401 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02993.x
encoding a polypeptide homologous to the DNA repair
enzyme (DRT111) of Arabidopsis thaliana [9,10]. Here, we
report on the isolation of the gene locus, full-length cDNA
and the functional analysis of the corresponding protein of
T. gondii that displays strong similarities with the P. falci-
parum, P. yoelii and A. thaliana proteins. We demonstrate
that T. gondii DNA repair protein is capable of comple-
menting an Escherichia coli mutant lacking ruvC endonuc-
lease and recG helicase. In addition, analyses of primary
structure indicate that a T. gondii DNA repair protein,
designated TgDRE (Tg DNA repair enzyme) for consis-
tency with the Arabidopsis thaliana enzyme, belongs to a
large family of proteins containing RNA recognition motifs
(RRM), glycine-rich motifs (G-patch) and a specific motif
named SF45 because of its similarity to the human splicing
factor 45 protein, which was described as a component of
the spliceosome [11]. The presence of the two first motifs
suggests that TgDRE may also be involved in RNA
metabolism.
EXPERIMENTAL PROCEDURES
Parasite and host cell cultures
The T. gondii 76K strain was used throughout this study.
Tachyzoites were grown in human foreskin fibroblasts
(HFF) using Dulbecco’s modified Eagle’s medium (Bio-
whittaker) supplemented with 10% fetal bovine serum
(Dutscher), 2 m
M
glutamine (Sigma) and 0.05 mgÆmL

sequenced as described above.
Measurement of mRNA levels
Total RNA was isolated from encysted bradyzoites, tach-
yzoites, uninfected mice brain cells and HFF cells, as
previously described [13,14]. cDNA was synthesized using
reverse transcriptase and serial dilutions of the cDNA were
used to amplify ORFs of T. gondii a-tubulin [15] or that of
TgDRE. Primers were as follows: a-tubulin 5¢-ATGAGAG
AGGTTATCAGCATC-3¢ and 5¢-TTAGTACTCGTCAC
CATAGCC-3¢; for TgDRE, 34S13 sens: 5¢-ATGCTGGA
CTCTCTCTACGGGGAT-3¢ and 34AS15 antisens: 5¢-TT
AGTCGAGGGGTTTGTCTGC-3¢. PCR products were
electrophoresed on agarose gels, stained with ethidium
bromide, scanned and quantified by densitometry using the
program
NIH IMAGE
( />Expression of glutathion
S
-transferase (GST)-TgDRE
fusion protein and immunological analysis
The cDNA fragment of TgDRE was cloned into the
expression vector pGEX-6P-3 (Pharmacia). After induc-
tion of transformed E. coli (BL21 strain) using isopro-
pylthio-b-
D
-galactoside, recombinant proteins were
purified on glutathione–Sepharose 4B column using
preScission
TM
protease, according to manufacturer’s

)1
of DNA-damaging agent mitomy-
cin C (MMC) and incubated at 30 °Cfor3days.The
transformed E. coli RuvC/RecG-deficient N3398 strain
and AB1157 wild-type strain were subjected to UV light
after IPTG-induction. All experiments were performed in
darkness and the plates were then incubated overnight at
30 °C.
Database searches and sequence analysis
Searches within nonredundant databases were performed
using
PSI
-
BLAST
[18] at NCBI. The Smart and Pfam
databases were searched using
RPS
-
BLAST
. Hydrophobic
clusteranalysis(HCA)wasalsousedinordertodetect
3394 N. Dendouga et al. (Eur. J. Biochem. 269) Ó FEBS 2002
distant but significant sequence similarities between the
members of the SF45 family [19].
RESULTS
Cloning of the gene encoding
T. gondii
DNA repair
protein
A 380-bp cDNA fragment encoding a putative DNA

primers encompassing the start and stop codons demon-
strates a predominant mRNA species of  1.4 kb
(Fig. 2A,B).
Fig. 1. Genomic organization of T. gondii TgDRE locus. (A) Southern blot analysis using genomic DNA from tachyzoites of T. gondii 76K strain
digested with several restriction enzymes indicated at the top. The blot was hybridized with a 380-bp DNA probe indicated by the letter P in (B).
(B) Genomic organization of TgDRE locus and its comparison with cDNA map revealed two introns designated i
1
and i
2
. Boxes correspond to
exons deduced from cDNAs sequences. The positions of the start and stop codons are indicated by an arrow and asterisk, respectively.
Fig. 2. Comparative analysis of TgDRE mRNA level in the two devel-
opmental stages of T. gondii. (A) Results of RT-PCR with the primers
corresponding to the full-length ORF of T. gondii TgDRE by using
total RNA from tachyzoites, T, and in vivo encysted bradyzoites (B),
RNA from uninfected human fibroblasts, H, or from naive mice brain
cells (C). H and C represent the negative controls. The quantity of
cDNA used corresponds to the equivalent of 10
4
parasites for both
tachyzoite and bradyzoite stages. RT-PCR were also performed by
using the tubulin primers used as positive controls. (B) Semi-quanti-
tative RT-PCR was used for the amplification of the ORFs of TgDRE
and the housekeeping gene a-tubulin using serial dilutions of cDNAs
prepared from tachyzoites and bradyzoites isolated from brain of
infected mice.
Ó FEBS 2002 A novel T. gondii DNA repair enzyme (Eur. J. Biochem. 269) 3395
Developmental expression of TgDRE gene measured
by RT-PCR
In order to determine the transcript level of TgDRE in

(Fig. 3A, lanes 3 and 4). The size of this recombinant
fused protein appears higher in both total extract lysate
than expected (a difference of 10 kDa) from the apparent
molecular mass of 49.6 kDa predicted by TgDRE
primary structure plus the 30-kDa GST protein. The
recombinant TgDRE protein without the fused GST was
also obtained by affinity chromatography on a GST
column followed by the preScission protease digestion
(Fig. 3A, lane 5). Again, the pure recombinant TgDRE
migrated as a 55-kDa protein instead of the apparent
molecular mass of  50 kDa. One additional lower
molecular mass protein species is present in the purified
preparation. We believe this to be due to proteolytic
degradation of the 90-kDa protein as a result of
contaminating cellular proteases. The purified recombin-
ant protein was used to raise polyclonal antisera in mice.
The Western blot (Fig. 3A, lane 6) shows the strong
immunoreactivity of the affinity-purified IgG from the
polyclonal antiserum against the electroeluted pure
recombinant TgDRE of 55 kDa, demonstrating that a
specific anti-TgDRE serum was generated. However,
Western blots of this purified antiserum specific to
TgDRE revealed a faint specific native protein of
60 kDa in the tachyzoites (data not shown). This
60-kDa protein can be readily detected when 20- to
200-fold amount of proteins from tachyzoites were loaded
in the SDS/PAGE (Fig. 3B, lanes 1–3). It is interesting to
note that a native protein of 60-kDa was detected in the
parasite instead of 55-kDa as expected for the recombin-
ant protein (Fig. 3A, lanes 5 and 6). Because it is difficult

mutant lacking ruvC endonuclease
and recG helicase
Similarity searches revealed that TgDRE displays signifi-
cant homology to DNA repair/toleration protein (DRT111)
of A. thaliana [10]. As the DRT111 protein was previously
shown to partially complement E. coli mutants lacking
RuvC and RecG helicase activities, we decided to investi-
gate whether TgDRE was capable of correcting these
activities in E. coli mutant phenotype. The transformation
of this E. coli mutant lacking RuvC and RecG enzyme
activities with the pGEX-6P-3 alone or pGEX containing
TgDRE ORF was performed in the presence of mitomycin
C. The in vivo effect of TgDRE activity on E. coli RuvC
–
RecG
–
revealed a significant increased resistance of trans-
formed mutants to the DNA-crosslinking agent mitomy-
cin C (Fig. 4). The resistance was increased by as much as
fourfold relative to mutants transformed with the pGEX
alone and treated with mitomycin C under the same
experimental conditions (Fig. 4). However, it appears that
TgDRE did not fully correct the mutant phenotype because
the surviving bacteria in the wild type E. coli is higher in the
presence of this chemical DNA-damaging agent (Fig. 4).
The partial correction of the E. coli mutant by TgDRE
cDNA is estimated at 40% less than that of the wild-type
bacteria in the presence of mitomycin C. In addition, we
also investigated whether TgDRE is capable of correcting
the DNA-damaging sensitive phenotype in the same E. coli

motif, ranging from amino acid 296–340 (Fig. 6A) followed
by a RRM module in the amino acid range 362–441
(Fig. 6B). G-Patch is a predicted glycine-rich nucleic
binding domain found in the splicing factor 45 and other
DNA-binding proteins [11,20], whereas RRM (RNA
recognition motif) was already described in numerous
proteins [21,22].
Further analyses of the N-terminal moiety of the TgDRE
sequence using
PSI
-
BLAST
combined with HCA, revealed a
conserved and as yet undescribed motif upstream these two
domains (Fig. 6C). This motif is present in a limited set of
proteins in addition to the DNA repair/toleration DRT111
protein from A. thaliana [8] and the two above mentioned
proteins of Plasmodium, the human splicing factor 45 [11]
and several hypothetical proteins from sequenced genomes
of D. melanogaster and C. elegans. All these proteins share
the same architecture with TgDRE as they also possess
C-terminal G-patch and RRM domains (Fig. 7). However,
the conserved motif appears specific for this family of
orthologous proteins here named SF45 motif (the SF45-
family-specific motif), is located at different distances from
the N-terminus and from the G-patch/RRM couple
(Fig. 7). The N-terminal parts of the SF45 family, located
before the G-patch and RRM domains, are rich in low
complexity sequences, typical of nonglobular regions and
could not be well aligned (see also Fig. 5). Several other

RuvC
+
RecG
+
was transformed with the pGEX expression vector
alone (WT). The E. coli mutant strain N 3398 RuvC
–
RecG
–
was
transformed with the pGEX expression vector alone (mutant) or with
the pGEX vector containing the TgDRE ORF (complemented). All
bacteria were transformed with 10 ng of plasmids and then spread
onto agar-plates containing 0.1 lgÆmL
)1
of mitomycin C.
Ó FEBS 2002 A novel T. gondii DNA repair enzyme (Eur. J. Biochem. 269) 3397
might be essential for parasite growth as expected for a
protein involved in DNA repair and conservation of
genome integrity. It is interesting that a similar gene
knock-out approach to disrupt a T. brucei splicing enzyme
containing RRM motif also failed [26]. TgDRE and
orthologous sequences do not show obvious similarities
with other proteins specialized in DNA repair. Interestingly,
they also contain motifs typical of proteins involved in
RNA metabolism. TgDRE mRNA and protein expression
have been evaluated using RT-PCR and Western blotting,
respectively. Although the TgDRE transcript was over-
expressed in encysted bradyzoites, the protein could only be
detected in the virulent tachyzoite. In this case, the level of

several hypothetical proteins from S. cerevisae, A. thaliana
and C. elegans [20]. The RRMs are also found in a variety
of RNA binding proteins, including heterogeneous nuclear
ribonucleoproteins (hnRNPs), proteins implicated in regu-
lation of alternative splicing, and components of small
nuclear ribonucleoproteins (snRNPs) [28]. In the protozoan
parasite Entamoeba histolytica, database searches for
enhancer-binding proteins did not detect any motifs
commonly associated with DNA-binding proteins, but
Fig. 5. Alignment of the TgDRE sequence of T. gondii and Plasmodium with that of the A. thaliana DRT11. Sequences can be aligned in four distinct
regions, corresponding to a small conserved domain present at the N-terminus (overlined, A), a SF45 motif, specific of members of the splicing
factor 45 family (overlined and boxed), a G-patch motif (shaded box) and a RRM domain (boxed). The automatic alignment first obtained was
refined in the N-terminal moiety as the SF45 motif, which is located at variable distances from the conserved motifs A and G-patch, was not aligned
correctly. The SF45 motif was identified after refined analysis using PSI-BLAST and HCA (see Fig. 6). The symbol (*) indicates identical residues;
(:) indicates well conserved residues. T. gondii DNA repair (TgDRE, this study), P. falciparum (90938047 chr14–1180), P. yoelii (chrPy1 °c275),
A. thaliana (DRT111, M98455).
3398 N. Dendouga et al. (Eur. J. Biochem. 269) Ó FEBS 2002
surprisingly revealed sequences containing regions of simi-
larities with RRM [29]. The specific contribution that the
different motifs of TgDRE perform in the maintenance of
the genome and/or in the RNA metabolism in T. gondii
remains to be determined. Further work is needed to
identify how T. gondii DNA repair proteins and their
constitutive motifs (SF45, G-patch and RRM) function at
the molecular level. The identification of the SF45 family
opens many new avenues of future investigations into the
unexplored molecular mechanisms involved in DNA repair
and RNA metabolism in the protozoan parasites such as
T. gondii and the Plasmodia. Further analyses should
contribute to our understanding of the biochemistry of

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domains, but none were found in all of the
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The total lengths of the proteins are indicated
at the end of each sequence.
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