A novel DNA repair enzyme containing RNA recognition, G-patch and specific splicing factor 45-like motifs in the protozoan parasite Toxoplasma gondii Najoua Dendouga1, Isabelle Callebaut
Trang 1A novel DNA repair enzyme containing RNA recognition, G-patch and specific splicing factor 45-like motifs in the protozoan parasite
Toxoplasma gondii
Najoua Dendouga1, Isabelle Callebaut2and Stanislas Tomavo1
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
630 bp, and encodes a protein with a predicted molecular
mass of 49.6 kDa The native TgDRE protein, with a
molecular mass of 60 kDa, is only detected in the virulent
tachyzoite stage of T gondii However, the transcript is
present in both asexual parasite stages, virulent tachyzoite
and avirulent encysted bradyzoite When an Escherichia coli
mutant lacking ruvC endonuclease and recG helicase was
transformed with TgDRE cDNA, a significant increase in
resistance to DNA-damaging agents, such as UV light and
mitomycin C, was observed Moreover, database searches
revealed that TgDRE orthologues were present in the genome sequences of the related apicomplexa parasites Plasmodium falciparumand Plasmodium yoelii, as well as in those of Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegansand Homo sapiens This novel family
of proteins is characterized by the presence of human splicing factor SF45-like, RNA recognition (RRM) and glycine-rich (G-patch) motifs The presence of these motifs suggests that T gondii TgDRE might also be involved
in other biological functions such as RNA metabolism in addition to DNA-repair
Keywords: Toxoplasma gondii; DNA repair enzyme; RRM; G-patch; splicing factor motif
Among protozoan parasites of the Apicomplexa phylum,
are numerous pathogens such as Toxoplasma gondii (an
important opportunistic pathogen associated with AIDS
and congenital birth defects), Plasmodium falciparum
(causative agent of malaria), Eimeria (agent of coccidiosis)
and Cryptosporidium (opportunistic intestinal agent) In
mammalian nonfeline hosts, T gondii is found in two
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 mechanisms and the proteins involved in apicomplexan parasites such as T gondii and Plasmodia However, preliminary characterization of some DNA repair proteins have been described in Plasmodium falciparum [6,7] In addition, two expressed sequence tags (EST) homologous to DNA repair proteins have also been identified in T gondii [8] Independently, we identified a T gondii cDNA fragment
Correspondence to S Tomavo, Equipe de Parasitologie Mole´culaire,
Laboratoire de Chimie Biologique, CNRS UMR 8576,
B atiment C9, Universite´ des Sciences et Technologies de Lille,
59655 Villeneuve d’Ascq, France.
Fax: + 33 3 20 43 65 55, Tel.: + 33 3 20 43 69 41,
E-mail: Stan.Tomavo@univ-lille1.fr
Abbreviations: TgDRE, Toxoplasma gondii DNA repair enzyme;
RRM, RNA recognition motif; HCA, hydrophobic cluster analysis;
MMC, DNA-damaging agent mitomycin C; G-patch, glycine rich
motif; SF45, splicing factor 45; DRT111, DNA repair/tolerance 111
enzyme; EST, expressed sequence tag; IPTG, isopropyl thio-b- D
-galactoside.
Note: the nucleotide sequences reported in this paper has been
sub mitted to GenBank with accession numb ers AF402310
(genomic DNA) and AF402311 (cDNA).
(Received 19 March 2002, revised 2 May 2002, accepted 13 May 2002)
Trang 2encoding 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 gondiithat displays strong similarities with the P
falci-parum, P yoeliiand 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
E X P E R I M E N T A L P R O C E D U R E S
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 mM glutamine (Sigma) and 0.05 mgÆmL)1
gentamicin (Sigma)
Southern blotting, Library screening and sequencing
For Southern blots, genomic DNA was extracted from
T gondii tachyzoites according to standard procedures
[12] The specific probes for Southern blots were generated
using the PCR DIG labelling mix (Roche Diagnostics)
Detection was performed using the chemiluminescence
method (Roche Diagnostics) A T gondii genomic library
(kindly provided by D Roos, University of Pennsylvania,
Philadelphia, USA) constructed in lambda DASHII
vector (Stratagene) using DNA fragment obtained after
BamHI digestion was screened by plaque hybridization
using a 380-bp digoxigenin-labeled probe [9] Two
fragments, 7000- and 1700-bp, were subcloned into
pBluescript-SK+ and sequenced using an ALFexpress
automated sequencer (Amersham Pharmacia Biotech)
Rapid amplification of cDNA ends (RACE) and sequencing
To obtain full-length cDNAs, a RACE-PCR technique was
performed using a Marathon cDNA Amplification Kit
(Clontech) with the adaptor primer 2 and specific
oligonu-cleotides N34AS 5¢-CTTCACCTGGAGGAGATTTCC
AAA-3¢ for 5¢ RACE and N34S 5¢-GGGAGGGTC
TCGGCGTCAACAAAC-3¢ for 3¢ RACE Nested PCR
was performed using internal oligonucleotides S10 5¢-GT
CGAGATGTTGGTTGTCGGAGACC-3¢ for 5¢ RACE
and PR2AS 5¢-GACCGTTACCACTGATTGCGGCTG
3¢ for 3¢ RACE The PCR products were cloned into the TA
cloning vector (Invitrogen) which were used to transform
E coli strain DH5a cells The purified plasmids were
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 programNIH IMAGE(http://rsb.info.nih.gov/nih-image/)
Expression of glutathionS-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 preScissionTM protease, according to manufacturer’s instructions (Pharmacia) Polyclonal antiserum was gen-erated in Balb/c mice using the purified recombinant protein IgG specific to TgDRE was purified from this antiserum by incubating the polyclonal antibodies to recombinant TgDRE blotted onto nitrocellulose After, one hour of incubation at room temperature and washing with NaCl/Pi, the bound-IgG was eluted with 3.5M potassium thiocyanate (KSCN), dialyzed and stored at )20 C with 20% BSA Western blots were performed using the purified anti-TgDRE IgG diluted at 1 : 100 or with a monoclonal antibody specific to T gondii actin diluted at 1 : 100 and rabbit secondary antibodies con-jugated to peroxidase diluted at 1 : 10 000 followed by cheminilescence detection
Functional complementation inE coli mutants lacking ruvC endonuclease recG helicase
E coli RuvC/RecG-deficient N3398 strain [16,17] and AB1157 wild-type strain (generously provided by J.B Hays, Oregon State University, USA) were transformed with the pGEX-6P-3 containing the TgDRE ORF or pGEX-6P-3 alone After isopropyl thio-b-D-galactoside (IPTG)-induc-tion, the bacteria were plated onto Luria–Bertani plates containing 0.1 lgÆmL)1of DNA-damaging agent mitomy-cin C (MMC) and incubated at 30C for 3 days 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
30C
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 cluster analysis (HCA) was also used in order to detect
Trang 3distant but significant sequence similarities between the
members of the SF45 family [19]
R E S U L T S
Cloning of the gene encodingT gondii DNA repair
protein
A 380-bp cDNA fragment encoding a putative DNA
repair protein (designated TgDRE) isolated from a
T gondii subtractive library [9] was used to probe
Southern blots performed using genomic DNA digested
with several restriction enzymes Figure 1A shows the blot
washed under stringent conditions Most enzymes tested
gave a single band except those with a predicted internal
restriction site The hybridization data is consistent with a
single copy TgDRE gene in the nuclear genome In order
to determine TgDRE genomic organization, a T gondii
genomic library was screened using the 380-bp cDNA
probe Two BamHI-digested fragments of 7000- and
1700-bp were isolated, subcloned and sequenced The
nucleo-tide sequence analysis of the genomic DNA revealed the
presence of two introns and three exons, which was
confirmed by the isolation and sequencing of the
full-length TgDRE cDNA The comparison of cDNA and
genomic DNA nucleotide sequences allowed the
deter-mination of the exact position of the two introns which
were 780- and 630-bp long (Fig 1B) These introns
contained the typical GT/AG consensus splicing signal
The excision of the two introns from the primary
transcript results in a mature TgDRE mRNA of
1401 nucleotides, which probably represents most, if not
all, of the entire full-length cDNA This was confirmed by
the reconstruction of full-length cDNA achieved by 5¢ and
3¢ RACE, which gave a nucleotide sequence of 2002 bp
with an ORF of 1401 bp (nucleotides 325–1725) This
2002-bp cDNA contains a ORF of 1401 bp that encodes
a protein of 466 amino acids with a calculated molecular
mass of 49.6 kDa In addition, RT-PCR performed with
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 mRNAlevel 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 104parasites 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.
Trang 4Developmental expression of TgDRE gene measured
by RT-PCR
In order to determine the transcript level of TgDRE in
both virulent tachyzoite and avirulent encysted bradyzoite,
RT-PCR was performed To ensure that equal quantities
of each mRNA were being compared, the amplification of
a-tubulin ORF was used as control (Fig 2A,B) The
RT-PCR revealed a band of 1.4 kb corresponding to
TgDRE in both tachyzoite and bradyzoite stages of
T gondii The cloning and sequencing of this RT-PCR
product confirmed its identity In addition, no RT-PCR
products could be amplified from the negative controls
corresponding to cDNA prepared from human and
uninfected mice brain cells (Fig 2A, lanes H and C) It
appears that TgDRE transcript is slightly more
predom-inant in encysted bradyzoites than in tachyzoites We used
semiquantitative RT-PCR to show that TgDRE transcript
in encysted bradyzoites represents twofold to threefold
higher level than that in tachyzoites as evaluated by
densitometry (Fig 2B), suggesting that TgDRE gene is
either overexpressed at the transcriptional level or its
mRNA is more stable in encysted bradyzoites than in
tachyzoites of T gondii
Characterization of TgDRE protein expressed in the two
developmental stages ofT gondii
To investigate whether these mRNA abundances increase
the expression of TgDRE protein, a GST fusion protein
consisting of amino-acid residues 1–466 of the ORF fused
to the C-terminus of the 30-kDa GST protein was
constructed SDS/PAGE analysis of the IPTG-induced
E colirevealed a predominant 90-kDa full-length protein
in both total SDS-lysate and sonicated soluble extract
(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 recombin-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
to obtain an equivalent amount of protein in encysted bradyzoites, we were unable to identify the TgDRE protein in this developmental parasite stage We conclu-ded that the higher level of transcript detected in RT-PCR
in encysted bradyzoites does not correspond to an increased level of TgDRE protein, and that the protein level is very low in the virulent tachyzoite This conclusion was supported by the control experiments where we measured the quality/quantity of total protein extracts by immunoblots using monoclonal antibodies directed against actin, the tachyzoite-specific protein SAG1 and the bradyzoite-specific antigen (data not shown) Collec-tively, our data demonstrate that a 60-kDa protein can be recognized by the anti-TgDRE IgG in the virulent tachyzoite of T gondii
Fig 3 Developmental expression of TgDRE protein in T gondii (A) SDS/PAGE analysis of the GST fusion recombinant TgDRE protein Lane 1, molecular mass markers; lane 2, total SDS lysate from uninduced E coli containing the pGEX-6P-3 vector alone; lane 3, total SDS lysate of the same E coli containing pGEX-6P-3 vector with TgDRE ORF after induction by IPTG for 3 h; lane 4, soluble fraction obtained after centrifu-gation of induced E coli disrupted by sonication; lane 5, affinity purified recombinant TgDRE after cleavage with the preScission protease and elution from the glutathione column; lane 6, immunoblot of anti-TgDRE serum against the purified specific recombinant TgDRE (B) Western blot analysis of protein extracts corresponding to 5 · 10 7 (1), 1 · 10 8 (2) and 6 · 10 8 (3) equivalent tachyzoites of T gondii 76K strain.
Trang 5TgDRE cDNA is capable of functionally complementating
E coli 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
RuvC–RecG–mutant after UV light treatment because it
has been previously shown that this bacteria mutant is also
partially sensitive to this treatment The transfection with
TgDRE cDNA also increased the resistance to UV light
treatment in this mutant as compared to that transformed
with the pGEX alone (data not shown) Taken together,
these data show convincingly that TgDRE cDNA encode a
functional DNA repair protein which can partially correct
the E coli RuvC–RecG–-sensitive phenotype
TgDRE belongs to a large family of proteins sharing
G-patch, RNA recognition motifs and a specific SF45
motif An initial similarity search revealed a clear similarity
( 30% sequence identity) with DNA repair/toleration
protein (DRT111) of A thaliana, in addition to that
observed with two proteins from the related apicomplexa
parasites, Plasmodium falciparum and Plasmodium yoelii (Fig 5) No similarity with other known DNA repair enzymes could be found in the databases, suggesting that these proteins may represent a novel family of DNA repair enzymes Moreover, searches against domain databases (Pfam, Smart) revealed the presence of two well-defined motifs at the C-terminus of the TgDRE sequence: a G-patch 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 usingPSI-BLASTcombined 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 melanogasterand 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 small conserved motifs were also detected (A, B and C in Fig 7), but none were found in all of the sequences of the SF45 family Based on the similarity to the human splicing factor 45 and their cellular localization, we speculate that the SF45 family, whose members are schematically repre-sented in Fig 7, may also have functions in the nuclear spliceosome and thus in RNA metabolism
D I S C U S S I O N
In the present study, we report the isolation and expression
of a cDNA encoding a novel DNA repair enzyme in
T gondii designated TgDRE TgDRE-like sequences are also present in the genomes of two related apicomplexan,
P falciparum and P yoelii, and belongs to the SF45 family,
a large family of proteins found in several genomes, from human (SF45 protein) to plants (DRT111) The highest similarities of apicomplexan TgDRE sequences are ob-served with those of A thaliana, suggesting that they could
be of algal origin This assumption is supported by the recent discovery of a nonphotosynthetic remnant plastid
apicoplast in apicomplexan parasites [23–25] Moreover, cDNAs encoding glucose-6-phosphate isomerase (Glc6PI), enolases and other enzymes isolated from T gondii and
P falciparum were shown to have significant identity to enzymes from non or photosynthetic organisms [13,25]
We also showed that TgDRE mRNA is encoded by a single copy gene and several attempts to disrupt the gene by knockout strategy were unsuccessful (D Dendouga &
S Tomavo unpublished data), suggesting that TgDRE
Fig 4 Resistance of bacteria expressing TgDRE to the
DNAdam-aging agent mitomycin C The wild-type E coli strain AB 1157
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)1of mitomycin C.
Trang 6might 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
the protein seems to be unusually low compare to that
reported for other Toxoplasma proteins studied [13,14] In
addition, the native TgDRE protein detected in the parasite
migrates as 60 kDa protein instead of 50 kDa At this time,
we have no explanation for the discrepancy between the
apparent molecular mass and the native size of TgDRE in
the parasite
According to the functional similarities that can be
predicted from the sequence similarities observed between
TgDRE and A thaliana DRT111 enzyme, we have been
able to partially complement RuvC–RecG–E colimutants
Similar results have been described for the A thaliana DRT111 enzyme [8] The fact that TgDRE corrects only partially the E coli mutant phenotype can be interpreted as the result of phylogenetical distance between bacteria and the protozoan parasite T gondii Alternatively, other bio-logical functions can be ascribed to TgDRE because sequence analysis revealed the presence of some striking motifs such as G-patch and RRM motifs, which are presumed to be involved in RNA metabolism or splicing occurring in the spliceosome The G-patch domain has been described as a short conserved region of about 40 amino acids, and is present in a number of putative RNA-binding proteins including a tumor suppressor (human LUCA15) [27], many RNA-processing proteins (45-kDa splicing factor) [11], the type-D retroviral polyproteins [20], and 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).
Trang 7surprisingly 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 DNA repair proteins in medically relevant parasites, and thus may be helpful in the identification of other molecules which are critically important for parasite survival
Fig 6 Alignment of the different domains of the SF45 family (A) The G-patch motifs of the SF45 family (sequences above the line), aligned with those of the human RNA-binding protein 5 [GenBank identifier (gi) 13124794] and of the human DNA-binding SON protein (gi 15320061) (B) The RRM motifs of the SF45 family, aligned with those of the human Ro ribonucleoprotein-binding protein 1 (gi 6684440) (C) The SF45 motifs, specific of members of the SF45 family The members of this family correspond to the here-described TgDRE, two Plasmodium sequences (chr14–
1180 (P falciparum) and chrPy1 c275 (P yoelii), A thaliana DRT111 and F26G16.10 proteins (gi 1169200 and 15220757), D melanogaster CG17540 protein (gi 7289585), C elegans F58B3.7 protein (gi 7504595) and H sapiens SF45 protein (gi 5454082) Sequence identities are indicated white on a black background, whereas sequence similarities are shaded (white and black letters for hydrophobic and nonhydrophobic positions, respectively).
Trang 8A C K N O W L E D G E M E N T S
We would like to thank Drs Michael Kibe and Kim Binderup for
critical reading of the manuscript This work was supported by grants
to S Tomavo by the Centre National de la Recherche Scientifique
(CNRS) through the Action The´matique Incitative sur Programme et
Equipe (ATIPE) N D was supported by fellowships from the
Ministe`re de l’Enseignement Supe´rieur et de la Recherche (MESR).
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