Skip to content


  • Research article
  • Open Access

Taking U out, with two nucleases?

BMC Bioinformatics20067:305

  • Received: 04 April 2006
  • Accepted: 16 June 2006
  • Published:



REX1 and REX2 are protein components of the RNA editing complex (the editosome) and function as exouridylylases. The exact roles of REX1 and REX2 in the editosome are unclear and the consequences of the presence of two related proteins are not fully understood. Here, a variety of computational studies were performed to enhance understanding of the structure and function of REX proteins in Trypanosoma and Leishmania species.


Sequence analysis and homology modeling of the Endonuclease/Exonuclease/Phosphatase (EEP) domain at the C-terminus of REX1 and REX2 highlights a common active site shared by all EEP domains. Phylogenetic analysis indicates that REX proteins contain a distinct subfamily of EEP domains. Inspection of three-dimensional models of the EEP domain in Trypanosoma brucei REX1 and REX2, and Leishmania major REX1 suggests variations of previously characterized key residues likely to be important in catalysis and determining substrate specificity.


We have identified features of the REX EEP domain that distinguish it from other family members and hence subfamily specific determinants of catalysis and substrate binding. The results provide specific guidance for experimental investigations about the role(s) of REX proteins in RNA editing.


  • Hide Markov Model
  • Trypanosoma
  • Hydrophobic Pocket
  • Exonuclease Activity
  • Leishmania Species


Most mitochondrial mRNAs in trypanosomatid parasites such as Trypanosoma, and Leishmania species undergo RNA editing [13]. This post-transcriptional process produces mature and functional mRNAs through a series of coordinated steps catalysed by a multi-protein complex that inserts and deletes uridylates (Us) specified by guide RNAs (gRNAs). One hypothesis posits a structural and functional subdivision of the editosome into insertion and deletion subcomplexes [48]. Editosome proteins with endonuclease (REN1, REN2) [9, 10], terminal uridylyl transferase (TUTase; RET1, RET2) [6, 11, 12], 3' exouridylylase (exoUase; REX1, REX2[5, 13], Ernst et al., unpublished), ligase (REL1, REL2) [5, 8, 14, 15], and helicase (REH1) [16] activities have been identified and functionally characterized. Sets of proteins related by sequence similarity exhibit both unique and common functions. For instance, REN1 is an endoribonuclease that is specific for RNA editing deletion sites whereas REN2 is specific for RNA editing insertion sites. RET1 is implicated in the addition of the non-encoded 3'-oligo U tails to gRNAs but RET2 adds Us to pre-edited mRNAs. REL1 may be involved in U-deletion editing and REL2 in U-insertion editing. Six additional editosome proteins, KREPA1-A6, have varying degrees of sequence relatedness with each protein containing a C-terminal motif associated with an oligonucleotide-binding (OB) fold [5, 1720]. Recent results point to both REX1 and REX2 as candidates for the RNA editing exoUase responsible for deletion of the 3' overhanging U residues from the mRNA 5' cleavage fragment. A U-specific exonuclease, REX1, has been partially purified from L. tarentolae [13]. The reconstitution of precleaved U-deletion in vitro with recombinant L. tarentolae REX1 and REL1 proteins and the in vivo RNAi down-regulation of REX1 expression in T. brucei suggest that REX1 is the exoUase. However, the closely related REX2 protein (28% overall identity and 46% similarity in T. brucei) may be the putative exoUase since tagged T. brucei REL1 sub-complex consisting of REX2, REL1 and KREPA2 catalyze accurate U removal and ligation (i.e. pre-cleaved deletion editing) [5]. Thus, the exact roles of REX1 and REX2 in the editosome complex are unclear and the consequences of the presence of two related proteins are not fully understood.

Comparative sequence analysis indicates that both REX1 and REX2 contain a putative C-terminal Endonuclease/Exonuclease/Phosphatase (EEP) domain as well as a region exhibiting subtle, but significant similarity to a known 5'->3' exonuclease domain (L. major REX2 lacks an EEP domain because of a truncation at the C-terminus) [17]. Whether REX1 and REX2 have 5'->3' exonuclease activity in the editing complex is unknown.

In this study, we extend our previous analysis of the REX1 and REX2 EEP domains [17]. We use sequence analysis, homology modeling and phylogenetic analysis to enhance understanding of the structure and functions of REX proteins, as well as the relationships amongst EEP family members. Our results suggest that while these enzymes have diverged at the sequence level, the EEP domains share a common catalytic site. Our three-dimensional modeling studies suggest that the REX EEP domains fold in much the same way as other EEP domains whose structures have been determined by X-ray crystallography. We identify features of the REX EEP domain that distinguish it from other family members and hence subfamily specific determinants of catalysis and substrate binding.

Results and discussion

Trypanosomal REX proteins

The REX1 and REX2 proteins from three trypanosomatids show considerable sequence similarity suggesting they are encoded by paralogous genes (Fig. 1). Since the genes are present on non syntenic chromosomal regions in L. major and T. brucei (chromosome assignments not having been made for the T. cruzi genes) it is likely that the ancestral genes diverged prior to the fission/fusion events which resulted in the modern day trypanosomatid genomes.
Figure 1
Figure 1

Schematic diagram of the REX1 and REX2 editosome proteins from Trypanosoma brucei (REX1_Tbrucei, REX2_Tbrucei), T. cruzi (REX1_Tcruzi, REX2_Tcruzi), and Leishmania major (REX1_Lmajor, REX2_Lmajor). Each protein is represented as a horizontal bar and the number of amino acids (aa) is given. The percent identity and similarity for each pair of putative paralogs is indicated. The regions of similarity are in blue whereas segments that are added or deleted in one paralog (Indels) are in red. In L. major, the C-terminal portion of REX1 which contains an EEP domain (gray) has no counterpart in REX2 because of a truncation.

Sequence and phylogenetic analysis of EEP domains

The EEP domains in proteins from a variety of Eucarya and Bacteria were modelled and analyzed using an HMM-based approach. Members of the EEP domain family include magnesium dependent endonucleases (L1-EN, DNaseI, APE1, APE2) [2126], exonucleases (ExoIII, REX1, REX2) [5, 13, 17, 27], and phosphatases of lipid second messengers (I5PP) [28] (Fig. 2). Although these proteins have diverse substrate specificities, REX EEP domains possess the conserved sequence motifs that have been used to characterize other EEP domains (I to VI, Fig. 2). A phylogenetic tree of EEP domains indicates that REX and APE proteins form distinct subfamilies (Fig. 3). EEP domains in I5PP proteins are more REX- than APE-like.
Figure 2
Figure 2

HMM-generated multiple sequence alignment of the EEP domain from a variety of Bacteria and Eucarya. The EEP domain at the C-terminus of REX2 and REX1 is the putative exonuclease component of the editosome. Residues that are conserved in >50% of the sequences are in black and numbers indicate the number of amino acids not shown explicitly. Six EEP domains of particular interest are in yellow. The Roman numerals above the alignment mark the six conserved sequence motifs that have been used to characterize EEP domain (see also Table 1). Columns in pink indicate the putative substrate specificity active-site hydrophobic pocket. The column in green marks the location of the essential Glu in DNaseI (DNaseI_Bt_1DNK, the last sequence highlighted in yellow). The proteins shown are REX1 (RNA editing exonuclease 1), REX2 (RNA editing exonuclease 2), I5PP (inositol polyphosphate 5'-phosphatase), APE1 (apurinic/apyrimidinic endonuclease 1), and APE2 (apurinic/apyrimidinic endonuclease 2); these sequences are from T. brucei (Tbrucei), T. gambiense (Tbgambiense), T. congolense (Tbcongolense), T. cruzi (Tcruzi), T. vivax (Tvivax), L. major (Lmajor), L. infantum (Linfantum), L. braziliensis (Lbraziliensis), Schizosaccharomyces pombe (Sp), Homo sapiens (Hs), Escherichia coli (Ec), and Bos taurus (Bt). EEP domains whose structures have been determined experimentally are I5PP_Sp_1I9Z (S. pombe phosphatidylinositol phosphate phosphatase, RCSB code1I9Z), APE1_Hs_1HD7 (H. sapiens AP endonuclease 1, 1HD7), ExoIII_Ec_1AKO, (E. coli exonuclease III, 1AKO); DNaseI_Bt_1DNK (B. taurus deoxyribonuclease I, 1DNK) and L1EN_Hs_1VYB: (H. sapiens L1 endonuclease, 1VYB). Cylinders and arrows denote the α-helices and β-strands given in the RCSB entries 1AKO and 1HD7. The protein sequences, EEP domain HMM and alignment are available as Supplementary material.

Figure 3
Figure 3

A maximum likelihood tree estimated from an HMM-generated multiple sequence alignment of EEP protein domains (Fig. 2 provides information on the proteins shown).

Table 1

Conserved sequence motifs found in EEP domains (labeled I through VI in Fig. 2) and the roles of specific amino acids in two EEP domains of known three-dimensional structure (APE1_Hs_1HD7, ExoIII_Ec_1AKO in Fig. 5–7).





Asn forms a hydrogen bond to catalytic Asp in motif IV

Asn interacts with 5' phosphate group


Glu coordinates Mg2+ ion

Glu coordinates Mg2 ion


His is substituted by Tyr in APE1 but is the catalytic His in DNase I family

His is substituted by Tyr


Catalytic Asp deprotonates the water molecule; Asn forms a hydrogen bond to scissile phosphate

Asn forms a hydrogen bond to scissile phosphate

V. GRXD (X is S in REX1 and A in REX2)

Asp forms a hydrogen bond to His in motif VI

Asp forms a hydrogen bond to His in motif VI


His forms a hydrogen bond to Asp in motif V and to scissile phosphate

Catalytic His deprotonates the water molecule and forms a hydrogen bond to Asp in motif V

Homology modelling of T. brucei REX1 and REX2 and L. major REX1

The X-ray crystal structures of the EEP domains in two DNA repair enzymes (Fig. 2, APE1_Hs_1HD7, ExoIII_Ec_1AKO) [25, 27] were used as the templates to build homology models of the EEP domains in three REX proteins (REX1_Tbrucei, REX2_Tbrucei, REX1_Lmajor). H. sapiens APE1 and E. coli ExoIII are functional homologs that possess apurinic/apyrimidinic (AP) endonuclease activity and which hydrolyze the phosphodiester bond of DNA at the AP sites by cleaving the DNA in intact strands [29]. Although ExoIII also has 3'->5' exonuclease activity, its biological role remains unclear. Following cleavage by ExoIII or APE1 the bacterium E. coli uses DNA polymerase I (pol I) to fill in the single-nucleotide gap whereas the eucaryote H. sapiens uses DNA polymerase β (pol β). Pol I has also a 3'->5' proof-reading activity which allows the removal of misincorporated nucleotides [30]. Although pol β is prone to high error (one mistake per 4000 bases inserted) [31], it lacks the proof reading mechanism found in pol I [32]. Instead, APE1 acts as an exonuclease that trims off nucleotides from DNA ends that do not terminate in correct basepairs [33].

As would be expected, the four-layer α/β fold observed in the crystal structures of the templates (APE1/1HD7; ExoIII/1AKO) is reflected in the three-dimensional models of the target proteins (Fig. 4). The roles of amino acids in the conserved sequence motifs found in EEP domains (I to VI, Fig. 2) were examined in APE1/1HD7 and ExoIII/1AKO (Table 1). Based on high resolution X-ray crystal structures, the similar overall catalytic mechanism of APE1 and ExoIII involves the abstraction of a proton from a water molecule by a residue acting as a general base [23, 27]. The resultant nucleophilic hydroxide ion attacks a scissile phosphate. The major difference between APE1 and ExoIII is that the catalytic residue which deprotonates the water molecule in APE1 is Asp in motif IV whereas in ExoIII it is the His in motif VI (Table 1 and Fig. 5, 6, 7). In addition, ExoIII appears to be a relatively more powerful 3'-exonuclease than APE1 [34, 35]. This enhanced activity has been attributed to the fewer hydrophobic residues in the active site of ExoIII [23, 24] (and see below).
Figure 4
Figure 4

Three-dimensional models of trypanosomal REX EEP domains built using EEP domains whose structures have been determined by X-ray crystallography. The upper panel shows ribbon diagrams for two experimentally determined structures: 1AKO is E. coli exonuclease III (Fig. 2, ExoIII_Ec_1AKO) and 1HD7 is H. sapiens APE1 (APE1_Hs_1HD7). The lower panel shows ribbon diagrams of the homology-built structures: T. brucei REX2 (REX2_Tbrucei), T. brucei REX1 (REX1_Tbrucei) and L. major REX1 (REX1_Lmajor). The four-layered α, β sandwich fold in each EEP domain is shown in the same orientation with the substrate binding surface at the top.

Figure 5
Figure 5

The active sites of APE1_Hs_1HD7 (green) superposed onto REX2_Tbrucei (yellow) with the side chains of critical amino acids shown explicitly. The APE1 (REX2) catalytic residues are in white (green) and the hydrophobic residues are in red (orange).

Figure 6
Figure 6

The active sites of ExoIII_Ec_1AKO (grey) superposed on REX1_Tbrucei (green) with the side chains of critical amino acids shown explicitly. The ExoIII (REX1) catalytic residues are in yellow (orange) and the hydrophobic residues are in red (white).

Figure 7
Figure 7

The active sites of ExoIII_Ec_1AKO (grey) superposed on REX1_Lmajor (blue) with the side chains of critical amino acids shown explicitly. The ExoIII (REX1) catalytic residues are in yellow (white) and the hydrophobic residues are in red (green).

Putative activity and mechanisms of action of REX proteins

To gain insights into the enzymatic activity of the REX proteins, we measured the rmsd values of the superposed models of each of REX EEP domain model to APE1/1HD7 and ExoIII/1AKO. The REX2_Tbrucei model is closer to APE1/1HD7 than to ExoIII/1AKO (2.54 Å versus 4.19 Å). In contrast, both the REX1_Lmajor and REX1_Tbrucei models are closer to ExoIII (2.17 Å, 1.97 Å, respectively) than to APE1/1HD7 (3.41 Å, 3.91 Å, respectively). These results suggest that REX2_Tbrucei may have more in common with APE1/1HD7 whereas REX1_Lmajor and REX1_Tbrucei may be more related to ExoIII/1AKO. The latter data suggest that REX1 exoribonuclease activity in both Leishmania and Trypanosoma species may share a similar catalytic mechanism with ExoIII [13]. These results also raise the possibility that if REX2_Tbrucei is similar to APE1, the potential proof-reading function of Trypanosoma REX2 may remove (i) the extraneous U residues added during TUTase activity [3638] and (ii) the Us that result from TUTase function within a U-deletion site [39]. Current data indicate that Leishmania REX2 lack a C-terminal EEP domain (Fig. 1, Fig. 2) and hence potential proof-reading activity. Although this function could be compensated for by the related REX1 protein (see below), the absence of an EEP domain could be explained by less extensive editing in Leishmania species compared to Trypanosoma family members. While both APE1 and ExoIII are known to act as endonucleases, such activity has not yet been demonstrated for the REX family of proteins.

In addition to the primary catalytic residues, the active site of APE1/1HD7 and ExoIII/1AKO contains a bulky hydrophobic pocket that has been proposed to act as a sequence specific "gate-keeper" able to accommodate only abasic sites [23, 24]. In APE1/1HD7, the hydrophobic pocket is composed of Phe266, Trp280, and Leu282 (Fig. 2, Fig. 5). The equivalent pocket in ExoIII/1AKO is larger and consists of Trp212, Leu226, and Ile228 (Fig. 2, Fig. 6, 7). Mutations of the APE1 hydrophobic pocket that results in smaller residues (e.g. Phe266 to Ala/Cys, Trp280 to Ile/Leu/Ser), can enhance its 3'-exonuclease activity [24]. ExoIII possesses a hydrophobic pocket containing only one aromatic residue and the enzyme is a better 3' exonuclease than APE1. These two findings support the idea that the hydrophobic pocket in EEP domains plays a significant role in nucleotide binding and specificity. Our sequence and structural analysis suggests that REX1 and REX2 do not have a bulky hydrophobic pocket but instead share a pocket composed of smaller residues. The pocket is formed by Arg834, Gly848, and Ala850 in REX2_T.brucei, Thr825, Gly839, and Ser841 in REX1_Tbrucei, and by Ser916, Gly930, and Ser932 in REX1_Lmajor (Fig. 2, Fig. 5, 6, 7). The equivalent of Trp280 in APE1/1HD7 or Leu226 in ExoIII/1AKO is Gly839 in REX1_Tbrucei, Gly848 in REX2_Tbrucei, and Gly930 in REX1_Lmajor. Thus, we predict that REX1 and REX2 have the potential to accommodate an extrahelical residue (i.e. uridine) downstream (3') of the scissile bond. The conserved polar Ser/Thr in REX1, and positively charged Arg in REX2 may form hydrogen bonds with the extrahelical base (column in pink between conserved motifs IV and V in Fig. 2). In REX1, a Ser in Leishmania is a Thr in Trypanosoma (Fig. 6, 7). This suggests altered substrate specificity for Leishmania species, which may partially compensate for the absence of an EEP domain in REX2.

Comparison of the REX subfamily of EEP domains with that of the B. taurus DNase I (DnaseI_Bt_1DNK, Fig. 2) and inositol polyphosphate 5'-phosphatase (I5PP, Fig. 2) family members reveals a conserved His in motif III (Fig. 2 and Table 1) [22, 28, 40]. In DNase I, this residue is part of the essential His-Glu catalytic pair located within the active site and is proposed to act as a general acid acting to stabilize the leaving group [22]. Mutagenesis of Glu in DNase I has also shown the importance of this residue for catalytic activity. However, the sequence analysis of the REX and other family members of EEP domains does not reveal a conserved Glu or other negatively charged residue that could pair with the His residue (Fig. 2) [41]. Therefore, we predict that in the REX EEP domains, the His residue forms a hydrogen bond to and further polarizes the scissile phosphate, as previously proposed for the I5PP family of proteins [41].


Using a variety of computational approaches, we have identified conserved motifs and a critical substrate binding pocket in the REX subfamily of EEP domains. Our results suggest experiments that could be performed to examine the distinct catalytic roles of REX proteins in the editosome.


Trypanosomal proteins

The genomic locations of the trypanosomal proteins discussed in this work were determined using the GeneDB Artemis interface to data from the TriTryp genomes sequencing consortium [42]. Putative orthologs were initially identified using BLAST searches of the unfinished genome sequences. These findings were later confirmed through mutual best BlastP analysis amongst the unique portions of the three essentially complete trypanosomatid genomes. The sequences of these homologous genes were also confirmed against the high coverage sequences available from the genome projects. The available, unfinished genome sequences for the remaining trypanosomatids discussed in this manuscript have been made available through GeneDB, and were searched using the blast algorithm to identify putative orthologues in these species. In a number of instances, the matches were not full length, and the gene was present in more than a single contig, thereby requiring assembly of the sequence to obtain full-length genes.

Sequence and phylogenetic analysis of EEP domains

The sequence and phylogenetic analysis of EEP domains was performed using a hidden Markov model (HMM)-based approach that has been employed successfully elsewhere (see, for example, the following refs [4347]).

Previously, we estimated an HMM of the EEP domain using the SAM software suite version 3.3.1 [48] and a limited number of protein sequences [17]. For this work, the parameters of this initial HMM were updated using an expanded training set that included additional eukaryotic (including trypanosomatid) and bacterial sequences. The ensuing EEP domain HMM was used to generate a multiple sequence alignment of all the EEP domains in the training set and the alignment was annotated with known structural information for some members of the EEP domain family (Fig. 2).

A phylogenetic tree for EEP domains was estimated using an HMM-generated multiple sequence alignment of the training set and ProtML in the MOLPHY software suite version 2.3b3s. Since insert states in the HMM are uninformative, the alignment consisted only of residues aligned to match states of the EEP HMM. ProtML infers an evolutionary tree from amino acid sequences using the Maximum Likelihood (ML) method. The tree with the maximum likelihood was used to understand the relationships between EEP domains.

Homology modeling

Three-dimensional models of selected REX EEP domains were built as described previously [49] using the MODELLER program [50] using software programs from Accelrys Inc., DS Modeling 1.1 and an alignment of a domain of unknown structure against a domain of known structure (Fig. 2). The sequences/structures of APE1_Hs_1HD7 and ExoIII_Ec_1AKO were used as the templates for constructing models of three targets, REX1_Tbrucei, REX2_Tbrucei and REX1_Lmajor. This particular choice was based on (i) the functional homology and multiple sequence alignment (Fig. 2), (ii) a statistically significant PSI-BLAST score between the target and an EEP family protein (E-value = 5e-12 REX1_Tbrucei, 6e-08 REX1_Lmajor, and 2e-08 REX2_Tbrucei), and (iii) a statistical significant score produced by 3D-Jury (120–156, well above the cutoff value of 50). The 3D-Jury metaserver [51] selects the most abundant models from the set of 3D models generated by various independent prediction providers. To measure the r.m.s. deviation of the superposed template and the target, the complete sequences of the predicted EEP domains aligned in Figure 2 were used to measure the r.m.s. deviation values. The quality of predicted modeled structures were checked with the Profiles_3D program [52] in DS Modeling 1.1.



RNA editing exouridylylase




RNA editing endonuclease


RNA editing terminal uridylyl transferase


terminal uridylyl transferase




RNA editing ligase


RNA editing helicase, APE, apurinic/apyrimidinic endonuclease


oligonucleotide binding


L1 endonuclease


exonuclease III


inositol polyphosphate 5'-phosphatase

pol I: 

DNA polymerase I

pol β: 

DNA polymerase β.



The authors thank the TriTryp Genome Consortium for their considerable effort that made this work possible. This work was supported by NIH grant 1R21AI053784-01 to R.S and funds from the National Institute on Aging, National Institute of Environmental Health Sciences, U.S. Department of Energy (OBER) and California Breast Cancer Research Program to I.S.M.

Authors’ Affiliations

Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720-8265, USA
Seattle Biomedical Research Institute, Seattle, Washington 98109, USA
McGill University, Institute of Parasitology, Ste.-Anne-De-Bellevue, Quebec, H9X 3V9, Canada


  1. Madison-Antenucci S, Grams J, Hajduk SL: Editing machines: the complexities of trypanosome RNA editing. Cell 2002, 108: 435–438. 10.1016/S0092-8674(02)00653-0View ArticlePubMedGoogle Scholar
  2. Simpson L, Aphasizhev R, Gao G, Kang X: Mitochondrial proteins and complexes in Leishmania and Trypanosoma involved in U-insertion/deletion RNA editing. RNA 2004, 10: 159–170. 10.1261/rna.5170704PubMed CentralView ArticlePubMedGoogle Scholar
  3. Stuart KD, Schnaufer A, Ernst NL, Panigrahi AK: Complex management: RNA editing in trypanosomes. Trends Biochem Sci 2005, 30: 97–105. 10.1016/j.tibs.2004.12.006View ArticlePubMedGoogle Scholar
  4. Aphasizhev R, Aphasizheva I, Nelson RE, Gao G, Simpson AM, Kang X, Falick AM, Sbicego S, Simpson L: Isolation of a U-insertion/deletion editing complex from Leishmania tarentolae mitochondria. EMBO J 2003, 22: 913–924. 10.1093/emboj/cdg083PubMed CentralView ArticlePubMedGoogle Scholar
  5. Schnaufer A, Ernst NL, Palazzo SS, O'Rear J, Salavati R, Stuart K: Separate insertion and deletion subcomplexes of the Trypanosoma brucei RNA editing complex. Mol Cell 2003, 12: 307–319. 10.1016/S1097-2765(03)00286-7View ArticlePubMedGoogle Scholar
  6. Ernst NL, Panicucci B, Igo RPJ, Panigrahi AK, Salavati R, Stuart K: TbMP57 is a 3' terminal uridylyl transferase (TUTase) of the Trypanosoma brucei editosome. Mol Cell 2003, 11: 1525–1536. 10.1016/S1097-2765(03)00185-0View ArticlePubMedGoogle Scholar
  7. Huang CE, O'Hearn SF, Sollner-Webb B: Assembly and function of the RNA editing complex in Trypanosoma brucei requires band III protein. Mol Cell Biol 2002, 22: 3194–3203. 10.1128/MCB.22.9.3194-3203.2002PubMed CentralView ArticlePubMedGoogle Scholar
  8. Cruz-Reyes J, Zhelonkina AG, Huang CE, Sollner-Webb B: Distinct functions of two RNA ligases in active Trypanosoma brucei RNA editing complexes. Mol Cell Biol 2002, 22: 4652–4660. 10.1128/MCB.22.13.4652-4660.2002PubMed CentralView ArticlePubMedGoogle Scholar
  9. Trotter JR, Ernst NL, Carnes J, Panicucci B, Stuart K: A deletion site editing endonuclease in Trypanosoma brucei. Mol Cell 2005, 20: 403–412. 10.1016/j.molcel.2005.09.016View ArticlePubMedGoogle Scholar
  10. Carnes J, Trotter JR, Ernst NL, Steinberg A, Stuart K: An essential RNase III insertion editing endonuclease in Trypanosoma brucei. Proc Natl Acad Sci U S A 2005, 102: 16614–16619. 10.1073/pnas.0506133102PubMed CentralView ArticlePubMedGoogle Scholar
  11. Aphasizhev R, Sbicego S, Peris M, Jang SH, Aphasizheva I, Simpson AM, Rivlin A, Simpson L: Trypanosome mitochondrial 3' terminal uridylyl transferase (TUTase): the key enzyme in U-insertion/deletion RNA editing. Cell 2002, 108: 637–648. 10.1016/S0092-8674(02)00647-5View ArticlePubMedGoogle Scholar
  12. Aphasizhev R, Aphasizheva I, Simpson L: A tale of two TUTases. Proc Natl Acad Sci U S A 2003, 100: 10617–10622. 10.1073/pnas.1833120100PubMed CentralView ArticlePubMedGoogle Scholar
  13. Kang X, Rogers K, Gao G, Falick AM, Zhou S, Simpson L: Reconstitution of uridine-deletion precleaved RNA editing with two recombinant enzymes. Proc Natl Acad Sci U S A 2005, 102: 1017–1022. 10.1073/pnas.0409275102PubMed CentralView ArticlePubMedGoogle Scholar
  14. Huang CE, Cruz-Reyes J, Zhelonkina AG, O'Hearn S, Wirtz E, Sollner-Webb B: Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U-deletion and RNA repair. EMBO J 2001, 20: 4694–4703. 10.1093/emboj/20.17.4694PubMed CentralView ArticlePubMedGoogle Scholar
  15. Drozdz M, Palazzo SS, Salavati R, O'Rear J, Clayton C, Stuart K: TbMP81 is required for RNA editing in Trypanosoma brucei. EMBO J 2002, 21: 1791–1799. 10.1093/emboj/21.7.1791PubMed CentralView ArticlePubMedGoogle Scholar
  16. Missel A, Souza AE, Norskau G, Goringer HU: Disruption of a gene encoding a novel mitochondrial DEAD-box protein in Trypanosoma brucei affects edited mRNAs. Mol Cell Biol 1997, 17: 4895–4903.PubMed CentralView ArticlePubMedGoogle Scholar
  17. Worthey EA, Schnaufer A, Mian IS, Stuart K, Salavati R: Comparative analysis of editosome proteins in trypanosomatids. Nucleic Acids Res 2003, 31: 6392–6408. 10.1093/nar/gkg870PubMed CentralView ArticlePubMedGoogle Scholar
  18. Law JA, Huang CE, O'Hearn SF, Sollner-Webb B: In Trypanosoma brucei RNA editing, band II enables recognition specifically at each step of the U insertion cycle. Mol Cell Biol 2005, 25: 2785–2794. 10.1128/MCB.25.7.2785-2794.2005PubMed CentralView ArticlePubMedGoogle Scholar
  19. Brecht M, Niemann M, Schluter E, Muller UF, Stuart K, Goringer HU: TbMP42, a protein component of the RNA editing complex in African trypanosomes, has endo-exoribonuclease activity. Mol Cell 2005, 17: 621–630. 10.1016/j.molcel.2005.01.018View ArticlePubMedGoogle Scholar
  20. Panigrahi AK, Schnaufer A, Carmean N, Igo RPJ, Gygi SP, Ernst NL, Palazzo SS, Weston DS, Aebersold R, Salavati R, Stuart KD: Four related proteins of the Trypanosoma brucei RNA editing complex. Mol Cell Biol 2001, 21: 6833–6840. 10.1128/MCB.21.20.6833-6840.2001PubMed CentralView ArticlePubMedGoogle Scholar
  21. Weichenrieder O, Repanas K, Perrakis A: Crystal structure of the targeting endonuclease of the human LINE-1 retrotransposon. Structure 2004, 12: 975–986. 10.1016/j.str.2004.04.011View ArticlePubMedGoogle Scholar
  22. Suck D, Oefner C: Structure of DNase I at 2.0 A resolution suggests a mechanism for binding to and cutting DNA. Nature 1986, 321: 620–625. 10.1038/321620a0View ArticlePubMedGoogle Scholar
  23. Mol CD, Izumi T, Mitra S, Tainer JA: DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination [corrected]. Nature 2000, 403: 451–456. 10.1038/35000249View ArticlePubMedGoogle Scholar
  24. Hadi MZ, Ginalski K, Nguyen LH, Wilson DMIII: Determinants in nuclease specificity of Ape1 and Ape2, human homologues of Escherichia coli exonuclease III. J Mol Biol 2002, 316: 853–866. 10.1006/jmbi.2001.5382View ArticlePubMedGoogle Scholar
  25. Beernink PT, Segelke BW, Hadi MZ, Erzberger JP, Wilson DMIII, Rupp B: Two divalent metal ions in the active site of a new crystal form of human apurinic/apyrimidinic endonuclease, Ape1: implications for the catalytic mechanism. J Mol Biol 2001, 307: 1023–1034. 10.1006/jmbi.2001.4529View ArticlePubMedGoogle Scholar
  26. Hadi MZ, Wilson DMIII: Second human protein with homology to the Escherichia coli abasic endonuclease exonuclease III. Environ Mol Mutagen 2000, 36: 312–324. 10.1002/1098-2280(2000)36:4<312::AID-EM7>3.0.CO;2-KView ArticlePubMedGoogle Scholar
  27. Mol CD, Kuo CF, Thayer MM, Cunningham RP, Tainer JA: Structure and function of the multifunctional DNA-repair enzyme exonuclease III. Nature 1995, 374: 381–386. 10.1038/374381a0View ArticlePubMedGoogle Scholar
  28. Tsujishita Y, Guo S, Stolz LE, York JD, Hurley JH: Specificity determinants in phosphoinositide dephosphorylation: crystal structure of an archetypal inositol polyphosphate 5-phosphatase. Cell 2001, 105: 379–389. 10.1016/S0092-8674(01)00326-9View ArticlePubMedGoogle Scholar
  29. Mol CD, Hosfield DJ, Tainer JA: Abasic site recognition by two apurinic/apyrimidinic endonuclease families in DNA base excision repair: the 3' ends justify the means. Mutat Res 2000, 460: 211–229.View ArticlePubMedGoogle Scholar
  30. Tabor S, Huber HE, Richardson CC: Escherichia coli thioredoxin confers processivity on the DNA polymerase activity of the gene 5 protein of bacteriophage T7. J Biol Chem 1987, 262: 16212–16223.PubMedGoogle Scholar
  31. Osheroff WP, Jung HK, Beard WA, Wilson SH, Kunkel TA: The fidelity of DNA polymerase beta during distributive and processive DNA synthesis. J Biol Chem 1999, 274: 3642–3650. 10.1074/jbc.274.6.3642View ArticlePubMedGoogle Scholar
  32. Kunkel TA: The mutational specificity of DNA polymerase-beta during in vitro DNA synthesis. Production of frameshift, base substitution, and deletion mutations. J Biol Chem 1985, 260: 5787–5796.PubMedGoogle Scholar
  33. Chou KM, Cheng YC: An exonucleolytic activity of human apurinic/apyrimidinic endonuclease on 3' mispaired DNA. Nature 2002, 415: 655–659. 10.1038/415655aView ArticlePubMedGoogle Scholar
  34. Demple B, Harrison L: Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem 1994, 63: 915–948. 10.1146/ ArticlePubMedGoogle Scholar
  35. Seki S, Hatsushika M, Watanabe S, Akiyama K, Nagao K, Tsutsui K: cDNA cloning, sequencing, expression and possible domain structure of human APEX nuclease homologous to Escherichia coli exonuclease III. Biochim Biophys Acta 1992, 1131: 287–299.View ArticlePubMedGoogle Scholar
  36. Byrne EM, Connell GJ, Simpson L: Guide RNA-directed uridine insertion RNA editing in vitro. EMBO J 1996, 15: 6758–6765.PubMed CentralPubMedGoogle Scholar
  37. McManus MT, Adler BK, Pollard VW, Hajduk SL: Trypanosoma brucei guide RNA poly(U) tail formation is stabilized by cognate mRNA. Mol Cell Biol 2000, 20: 883–891. 10.1128/MCB.20.3.883-891.2000PubMed CentralView ArticlePubMedGoogle Scholar
  38. Igo RPJ, Weston DS, Ernst NL, Panigrahi AK, Salavati R, Stuart K: Role of uridylate-specific exoribonuclease activity in Trypanosoma brucei RNA editing. Eukaryot Cell 2002, 1: 112–118. 10.1128/EC.1.1.112-118.2002PubMed CentralView ArticlePubMedGoogle Scholar
  39. Zhelonkina AG, O'Hearn SF, Law JA, Cruz-Reyes J, Huang CE, Alatortsev VS, Sollner-Webb B: T. brucei RNA editing: action of the U-insertional TUTase within a U-deletion cycle. RNA 2006, 12: 476–487. 10.1261/rna.2243206PubMed CentralView ArticlePubMedGoogle Scholar
  40. Matsuo Y, Yamada A, Tsukamoto K, Tamura H, Ikezawa H, Nakamura H, Nishikawa K: A distant evolutionary relationship between bacterial sphingomyelinase and mammalian DNase I. Protein Sci 1996, 5: 2459–2467.PubMed CentralView ArticlePubMedGoogle Scholar
  41. Whisstock JC, Romero S, Gurung R, Nandurkar H, Ooms LM, Bottomley SP, Mitchell CA: The inositol polyphosphate 5-phosphatases and the apurinic/apyrimidinic base excision repair endonucleases share a common mechanism for catalysis. J Biol Chem 2000, 275: 37055–37061. 10.1074/jbc.M006244200View ArticlePubMedGoogle Scholar
  42. Hertz-Fowler C, Peacock CS, Wood V, Aslett M, Kerhornou A, Mooney P, Tivey A, Berriman M, Hall N, Rutherford K, Parkhill J, Ivens AC, Rajandream MA, Barrell B: GeneDB: a resource for prokaryotic and eukaryotic organisms. Nucleic Acids Res 2004, 32: D339-D343. 10.1093/nar/gkh007PubMed CentralView ArticlePubMedGoogle Scholar
  43. Dalgaard JZ, Klar AJ, Moser MJ, Holley WR, Chatterjee A, Mian IS: Statistical modeling and analysis of the LAGLIDADG family of site-specific endonucleases and identification of an intein that encodes a site-specific endonuclease of the HNH family. Nucleic Acids Res 1997, 25: 4626–4638. 10.1093/nar/25.22.4626PubMed CentralView ArticlePubMedGoogle Scholar
  44. Dalgaard JZ, Moser MJ, Hughey R, Mian IS: Statistical modeling, phylogenetic analysis and structure prediction of a protein splicing domain common to inteins and hedgehog proteins. J Comput Biol 1997, 4: 193–214.View ArticlePubMedGoogle Scholar
  45. Mian IS: Sequence, structural, functional, and phylogenetic analyses of three glycosidase families. Blood Cells Mol Dis 1998, 24: 83–100.PubMedGoogle Scholar
  46. Mian IS, Moser MJ, Holley WR, Chatterjee A: Statistical modelling and phylogenetic analysis of a deaminase domain. J Comput Biol 1998, 5: 57–72.View ArticlePubMedGoogle Scholar
  47. Moser MJ, Holley WR, Chatterjee A, Mian IS: The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains. Nucleic Acids Res 1997, 25: 5110–5118. 10.1093/nar/25.24.5110PubMed CentralView ArticlePubMedGoogle Scholar
  48. System SAM: 2005. Scholar
  49. Salavati R, Ernst NL, O'Rear J, Gilliam T, Tarun SJR, Stuart K: KREPA4, an RNA binding protein essential for editosome integrity and survival of Trypanosoma brucei. RNA 2006, 12: 819–31. 10.1261/rna.2244106PubMed CentralView ArticlePubMedGoogle Scholar
  50. Sali A, Blundell TL: Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 1993, 234: 779–815. 10.1006/jmbi.1993.1626View ArticlePubMedGoogle Scholar
  51. Ginalski K, Elofsson A, Fischer D, Rychlewski L: 3D-Jury: a simple approach to improve protein structure predictions. Bioinformatics 2003, 19: 1015–1018. 10.1093/bioinformatics/btg124View ArticlePubMedGoogle Scholar
  52. Bowie JU, Luthy R, Eisenberg D: A method to identify protein sequences that fold into a known three-dimensional structure. Science 1991, 253: 164–170.View ArticlePubMedGoogle Scholar


© Mian et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.