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Stripped-down DNA repair in a highly reduced parasite Gill, Erin E; Fast, Naomi M Mar 20, 2007

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ralssBioMed CentBMC Molecular BiologyOpen AcceResearch articleStripped-down DNA repair in a highly reduced parasiteErin E Gill* and Naomi M FastAddress: Department of Botany, University of British Columbia, Vancouver, British Columbia, CanadaEmail: Erin E Gill* - egill@interchange.ubc.ca; Naomi M Fast - nfast@interchange.ubc.ca* Corresponding author    AbstractBackground: Encephalitozoon cuniculi is a member of a distinctive group of single-celled parasiticeukaryotes called microsporidia, which are closely related to fungi. Some of these organisms,including E. cuniculi, also have uniquely small genomes that are within the prokaryotic range. Thus,E. cuniculi has undergone a massive genome reduction which has resulted in a loss of genes fromdiverse biological pathways, including those that act in DNA repair.DNA repair is essential to any living cell. A loss of these mechanisms invariably results inaccumulation of mutations and/or cell death. Six major pathways of DNA repair in eukaryotesinclude: non-homologous end joining (NHEJ), homologous recombination repair (HRR), mismatchrepair (MMR), nucleotide excision repair (NER), base excision repair (BER) and methyltransferaserepair. DNA polymerases are also critical players in DNA repair processes.Given the close relationship between microsporidia and fungi, the repair mechanisms present in E.cuniculi were compared to those of the yeast Saccharomyces cerevisiae to ascertain how the processof genome reduction has affected the DNA repair pathways.Results: E. cuniculi lacks 16 (plus another 6 potential absences) of the 56 DNA repair genes soughtvia BLASTP and PSI-BLAST searches. Six of 14 DNA polymerases or polymerase subunits are alsoabsent in E. cuniculi. All of these genes are relatively well conserved within eukaryotes. The absenceof genes is not distributed equally among the different repair pathways; some pathways lack onlyone protein, while there is a striking absence of many proteins that are components of both doublestrand break repair pathways. All specialized repair polymerases are also absent.Conclusion: Given the large number of DNA repair genes that are absent from the double strandbreak repair pathways, E. cuniculi is a prime candidate for the study of double strand break repairwith minimal machinery. Strikingly, all of the double strand break repair genes that have beenretained by E. cuniculi participate in other biological pathways.BackgroundDNA repair in eukaryotesDNA repair processes are vital to all living organisms.accumulate, leading to total genome degradation and lossof vital genetic information. DNA lesions take manyforms, including single strand and double strand breaks,Published: 20 March 2007BMC Molecular Biology 2007, 8:24 doi:10.1186/1471-2199-8-24Received: 16 February 2007Accepted: 20 March 2007This article is available from: http://www.biomedcentral.com/1471-2199/8/24© 2007 Gill and Fast; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 14(page number not for citation purposes)Without appropriate mechanisms to remove and replacedamaged bases and nucleotides, multiple lesions wouldin addition to inter- and intra-strand crosslinks and mod-ified bases. Several pathways operate in a concerted man-BMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24ner to minimize information loss each time a DNA lesionoccurs. Many of these fundamental pathways have beenconserved throughout eukaryotes, and many eukaryoticenzymes have homologues in prokaryotes [1].Eukaryotic DNA repair can be divided into six primarypathways, all of which are conserved [2]. Some types ofDNA lesions (such as double stranded breaks) can be rec-ognized and repaired by more than one pathway. There-fore there is some overlap in function between pathways[2]. Mismatch repair (MMR), base excision repair (BER)and nucleotide excision repair (NER) all operate to repairaberrant bases or nucleotides from one strand of the dou-ble helix, using the other strand as a template for newDNA synthesis. In contrast, methyltransferase repair doesnot require the synthesis of new DNA; anomalous methylgroups are removed without causing any breaks in thedouble helix. Non-homologous end joining repair(NHEJ) and homologous recombination repair (HRR) aredouble strand break repair pathways [2]. Double strandbreaks are one of the most detrimental forms of DNAlesions, as they can cause genome fragmentation andapoptosis if they are not properly repaired [3]. (See Figure1 for a comparison of DNA repair processes.)DNA polymerases are key players in DNA repair, as theyare required to fill in gaps created by repair enzymes orincurred from damage [4]. Eukaryotic cells have a widerange of polymerases that are specialized in function; cer-tain polymerases act almost solely in genome replication,others are only active at DNA lesions, and a few have dualroles in repair and replication [4].For consistency, the names of the genes and proteinsinvolved in these pathways will be referred to using Sac-charomyces cerevisiae nomenclature.Genome reduction in Encephalitozoon cuniculiEncephalitozoon cuniculi belongs to a group of obligateintracellular parasites known as microsporidia. Theseorganisms infect a variety of animals including fish,insects and mammals. Various microsporidia have gainedthe attention of the medical community in the past fewdecades due to their infection of immuno-compromisedhumans, such as AIDS and chemotherapy patients [5].Microsporidian genomes range in size from 2.3 Mbp to19.5 Mbp. The only completely sequenced microsporid-ian genome, that of E. cuniculi, is a mere 2.9 Mbp in size[6]. The E. cuniculi genome is smaller than that of othereukaryotes for several reasons: it has fewer and shortergenes that are separated by tiny intergenic spaces and areonly interrupted by a few short introns [6]. Given theThe precise phylogenetic position of microsporidia is notyet known, but a large body of evidence indicates that theyare closely related to the fungi [6–10 and others]. There-fore, E. cuniculi's DNA repair systems have been comparedprimarily to those of another fungus, the yeast S. cerevi-siae. S. cerevisiae's repair pathways have been well studiedat the functional level, making this organism ideal forcomparative purposes. In order to gain an accurate per-spective of what genes have been lost from E. cuniculi dur-ing the process of genome reduction (i.e., genes that werepresent in the common ancestor of microsporidia andfungi), only genes that have homologues in animals wereexamined.ResultsDNA repair inventoryComparison of DNA repair proteins in S. cerevisiae to E.cuniculi's genome and proteome via BLAST and PSI-BLASTsearches has revealed that E. cuniculi appears to contain areduced set of proteins in all major repair pathways. Ofthe 56 repair genes that were sought in E. cuniculi, 16 areabsent, with another 6 potentially absent. Six out of 14DNA polymerases or polymerase subunits are absent (SeeTable 1). Although all repair pathways have been reduced,the loss of genes is not distributed evenly among path-ways. Each process has been affected differently bygenome reduction. A detailed discussion of the compo-nents of each pathway is presented below.Base excision repair (BER)BER is one of the least complex of the DNA repair mecha-nisms, and involves only a small number of proteins.When a base becomes damaged, it is recognized by a DNAglycosylase that is specific for the particular base and/orthe type of damage (methylation, oxidation, etc.). S. cere-visiae contains four types of glycosylases, although farmore have been found in other organisms (animals, bac-teria, etc.) [11]. Glycosylases cleave the glycosylic bondbetween the base and the deoxyribose to remove the dam-aged base, at which point a non-specific apurinic/apyri-midinic (AP) endonuclease (Apn1 or Apn2) removes theremaining deoxyribose phosphate to create a gap [12]. Inshort patch BER (which replaces a single nucleotide), thegap is filled by DNA polymerase β. In long patch BER(which replaces two or more nucleotides), the DNApolymerases β, or δ and ε in concert with proliferating cellnuclear antigen (PCNA) synthesize several nucleotideswhich displace the original DNA strand. Rad27 thenremoves the displaced DNA. The ligase Cdc9 (or DNAligase III and Xrcc1 in other eukaryotes) is used to seal thenick [13,14].The Rad1-Rad10 and Mus81-Mms4 endonucleases arePage 2 of 14(page number not for citation purposes)degree of genome reduction, the effects are evident inmost cellular processes, including DNA repair.also believed to play minor roles in BER by processing theBMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/243' ends of the DNA once an incision has been made intothe sugar-phosphate backbone [12].E. cuniculi's BER pathway appears to be nearly complete,but lacks DNA polymerase β, the Cdc9 DNA ligase (butpossesses Xrcc1, the cofactor of the ligase used in thisprocess in some eukaryotes) and part of a 3' endonucle-ase, Mms4. (See Table 1) Deletion of either polymerase βor Mms4 is not a lethal mutation in yeast, however S. cer-evisiae cannot survive in the absence of Cdc9 [15].Another ligase is likely utilized for BER in E. cuniculi, assharing non-specialized enzymes between pathways is notuncommon in S. cerevisiae (see discussion), and there isno reason to believe that this is not the case in E. cuniculi.Nucleotide excision repair (NER)NER is used primarily to remove bulky lesions from DNA,such as inter- and intra-strand crosslinks. NER is a morecomplex process than BER, and utilizes a large number ofgenome repair (GGR) and transcription-coupled repair(TCR). As is suggested by their names, the subpathwaysact on different types of DNA: DNA that is not transcribed(or the non-transcribed strands of expressed genes), andactively transcribed DNA, respectively. In both GGR andTCR, DNA damage recognition is the first step to occur,followed by DNA unwinding. Next, incisions are made oneither side of the aberrant base(s), and a total of 25–30nucleotides on either side are removed as a single strand.The gap is then filled by DNA polymerase and sealed byDNA ligase. Recruitment of five multi-protein complexes,nucleotide excision repair factors (NEFs) 1 through 4 andthe replication protein A (RPA) complex, is believed totake place in a stepwise manner to complete this process.The RPA complex is composed of Rpa1 and Rpa2 and rec-ognizes damaged DNA.In GGR, the first protein complex to arrive at the damagedsite is NEF4, which recognizes damage and is composed ofA comparison of the five major DNA repair pathwaysFigure 1A comparison of the five major DNA repair pathways. (See text for explanation.) Newly synthesized DNA is indicated in grey.6LQJOH6WUDQG5HSDLU'RXEOH6WUDQG5HSDLUGn\e^hmb]^>q\blbhgK^iZbk!G>K"FblfZm\aK^iZbk!FFK"HKAhfheh`hnlK^\hf[bgZmbhg!AKK" Ghg&Ahfheh`hnl>g]Chbgbg`!GA>C"8***&;Zl^>q\blbhgK^iZbk!;>K"Page 3 of 14(page number not for citation purposes)proteins that are evolutionarily conserved among eukary-otes. NER is comprised of two subpathways, globalthe proteins Rad7 and Rad16. Rad7 binds the NEF2 com-plex (Rad4/Rad23), recruiting it to the damaged site andBMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24Table 1: S. cerevisiae DNA polymerases and proteins that participate in the five primary DNA repair pathways.Base Excision Repair (BER) Nucleotide Excision Repair (NER)Gene Name Present in E. cuniculiS. cerevisiae Protein IDE. cuniculi Protein IDGene Name Present in E. cuniculiS. cerevisiae Protein IDE. cuniculi Protein IDApn1 Y CAA81954 CAD26065 Rad1 (XPF) Y P06777 CAD26381Apn2 Y NP_009534 NP_585892 Rad2 (XPG) Y CAA97287 CAD27314/XP_955715Mag1 Y NP_011069 CAD25924/CAD26679Rad3 (Ercc2/XPD) Y CAA46255 NP_585776Mus81 Y NP_010674 NP_584664 Rad4 (XPC) Y CAA39375 CAD24914Ntg1 Y NP_009387 CAD26394 Rad10 (Ercc1) Y CAA86642 CAD25852/NP_586248Ogg1 Y NP_013651 CAD26383 Rad14 (XPA) Y P28519 NP_586232PCNA Y AAS56041 NP_597446 Rad25 (Ssl2/XPB) Y Q00578 CAD24977Rad1 (XPF) Y P06777 CAD26381 Rad26 (Ercc6/CSB) Y CAA57290 CAD27013/XP_955594Rad10 (Ercc1) Y CAA86642 CAD25852/NP_586248Rpa1 Y NP_009404 CAD25779Rad27 (Fen1) Y CAA81953 CAD26252 Rpa2 Y NP_014087 CAD25396Ung1 Y CAA86634 CAD26772 Ssl1 Y CAA97527 CAD25215*Xrcc1 (Cut5) Y P32372 NP_584657 Tfb2 Y AAB40628 CAD24937Cdc9 (DNA lig I) N CAA48158 - Tfb3 Y AAB64899 CAD25932Ddc1 (Rad9) N NP_015130 - Tfb4 Y NP_015381 CAD25620Mec3 (Hus1) N NP_013391 - Rad7 ? CAA85071 -Mms4 N NP_009656 - Rad16 ? CAA89580 -Rad17 (Rad1) N CAA99699 - Rad23 (HR23B) N AAB28441 -Tfb1 N AAB64747 -Methyltransferase Repair DNA PolymerasesGene Name Present in E. cuniculiS. cerevisiae Protein IDE. cuniculi Protein IDGene Name Present in E. cuniculiS. cerevisiae Protein IDE. cuniculi Protein IDMgt1 N CAA42920 - α Pol1 Y AAZ22505 CAD26619α Pol12 Y NP_009518 CAD25827Mismatch Repair (MMR) α Pri1 Y AAT92878 CAD26368Gene Name Present in E. cuniculiS. cerevisiae Protein IDE. cuniculi Protein IDα Pri2 Y NP_012879 CAD26641Page 4 of 14(page number not for citation purposes)BMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24δ Pol3 Y CAA43922 CAD27015Exo1 Y NP_014676 CAD25986 δ Hys2 Y NP_012539 CAD27050Mlh1 Y NP_013890 NP_597370 ε Dpb2 Y NP_015501 NP_597373Msh2 Y CAA99102 CAD26200/NP_597565ε Pol2 Y NP_014137 CAD25840Msh6 Y NP_010382 NP_586186 β Pol4 N NP_009940 -PCNA Y AAS56041 NP_597446 γ Mip1 N NP_014975 -Pms1 Y P14242 NP_586432 η Rad30 N NP_010707 -Msh3 N CAA42247 - ζ Rev3 N NP_015158 -ζ Rev7 N AAA98667 -Rev1 N CAA99674 -Homologous Recombination (HRR) Non-Homologous End Joining (NHEJ)Gene Name Present in E. cuniculiS. cerevisiae Protein IDE. cuniculi Protein IDGene Name Present in E. cuniculiS. cerevisiae Protein IDE. cuniculi Protein IDMre11 (Rad32) Y BAA02017 CAD26648/NP_597471Mre11 (Rad32) Y BAA02017 CAD26648/NP_597471Rad50 Y CAA65494 CAD25593/NP_585989Rad27 (Fen1) Y CAA81953 CAD26252Rad51 Y CAA45563 CAD25992/NP_586388Rad50 Y CAA65494 CAD25593/NP_585989Rad52 Y CAA86623 XP_955647 Lif1 (Xrcc4) ? NP_011425 -Rpa1 Y NP_009404 CAD25779 *Xrs2 (Nbs1) ? BAC80248 -Rpa2 Y NP_014087 CAD25396 Ddc1 (Rad9) N NP_015130 -Sgs1 Y NP_013915 CAD25646 Dnl4 N CAA99193 -Rad55 (Rad51C) ? BAA01284 - Mec3 (Hus1) N NP_013391 -Rad57 (Xrcc3) ? NP_010287 - Ku70 N NP_014011 -*Xrs2 (Nbs1) ? BAC80248 - Ku80 N NP_013824 -Ddc1 (Rad9) N NP_015130 - Rad17 (Rad1) N CAA99699 -Hpr5 (Srs2) N CAA89385 -Mec3 (Hus1) N NP_013391 -Rad17 (Rad1) N CAA99699 -Rad24 (Rad17) N P32641 -Rad54 N CAA88534 -Rdh54 (Rad54B) N CAA85017 -Presence or absence in E. cuniculi is indicated (as defined in Methods), along with the genbank accession numbers for both S. cerevisiae and E. cuniculi proteins. Absent proteins are presented in bold type. Italicized accession numbers indicate that the presence or absence of these proteins in E. cuniculi was unclear. (See results for more information.) When S. pombe proteins appeared to be more conserved among eukaryotes than S. cerevisiae homologues (or where S. cerevisiae homologues do not exist), they were used to conduct the BLAST and PSI-BLAST searches. These proteins are marked with asterixes. S. cerevisiae nomenclature is used, with S. pombe or animal homologues given in brackets. Pathway components were largely compiled from the following sources: MMR from Marti et al. [22], BER from Boiteux and Guillet [12], NER from Prakash and Prakash [17], NHEJ from Daley et al. [57], HRR from Aylon and Kupiec [24], DNA polymerases from Burgers [58] and Hubscher et al., [4].Table 1: S. cerevisiae DNA polymerases and proteins that participate in the five primary DNA repair pathways. (Continued)Page 5 of 14(page number not for citation purposes)BMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24increasing DNA binding efficiency. The presence of NEF4is not strictly required for the recruitment of NEF2 to theDNA lesion, but facilitates the process. The above proteinsdo not act in the other sub-pathway; in TCR, initiation ofrepair takes place when an RNA polymerase stalls. Twoproteins involved specifically in TCR, Rad26 and Rad28,also participate in the beginning of this process [16].In both GGR and TCR, NEF1 and NEF3 are the next com-ponents to be recruited, and are held at the damage site byNEF2. NEF1 is composed of Rad1, Rad10 and Rad14,while NEF3 is composed of Rad2 and transcription elon-gation factor IIH (TFIIH). TFIIH contains the Rad3,Rad25, SSL1, TFB1, TFB2 and TFB3 proteins, and providesthe single strand DNA helicases required for repair pro-teins to access the damaged site. Rad1 and Rad10 form aheterodimer that acts as a single strand endonuclease atthe 5' end of the stretch of damaged DNA and Rad2 is asingle strand endonuclease that cuts at the 3' end. RPA isthought to be the last player to arrive at the scene.Many of these proteins also have roles in other cellularprocesses, such as recombination and transcription, there-fore mutants express defects in several pathways. For acomprehensive review of NER, see Prakash and Prakash[17].Most proteins participating in NER are present in E.cuniculi, with two exceptions. Half of a GGR het-erodimeric damage sensor complex (Rad23) and the Tfb1subunit of TFIIH appear to be absent (See Table 1). Rad23appears to have diverse functions within the cell, rangingfrom DNA repair to the regulation of a cell-cycle check-point and protein degradation. Specifically, this proteinhelps to prevent the degradation of Rad4, as well as serv-ing a role with the 26S proteasome in regulating the NERpathway [18,19]. Deletion of Tfb1 in S. cerevisiae is lethal[20], likely due to loss of function in transcription.The presence or absence of Rad7 and Rad16 were not con-firmed, as BLAST and PSI-BLAST searches using S. cerevi-siae and S. pombe sequences as queries did not returnhomologues from most animals or other eukaryotesbesides fungi.Methyltransferase RepairMethyltransferases are present in both eukaryotes andprokaryotes and remove certain DNA lesions involvingmethylation (O6-methylguanine, O4-methylthymine).These proteins irreversibly relocate methyl groups fromDNA to their own cysteine residues, and are therefore sui-cide enzymes [21].E. cuniculi does not possess the methyltransferase foundin other eukaryotes, Mgt1. Deletion of this gene is notlethal in S. cerevisiae [15].Mismatch repair (MMR)In MMR, mismatches are recognized by the heterodimersMutSα (Msh2/Msh6) and MutSβ (Msh2/Msh3). Singlebase mismatches are recognized by MutSα and insertion/deletion loops (IDLs) less than about 9 nucleotides inlength are recognized by MutSβ [22]. Both MutSα andMutSβ can recognize a single unpaired nucleotide. PCNAis also involved in MMR, perhaps assisting in these initialrecognition steps. MutLα (Mlh1/Pms1) binds MutSα andβ and allows them to efficiently bind to IDLs and mis-matches. The exonuclease Exo1 then excises the mis-matched base(s) and a DNA polymerase and DNA ligasefill and seal the gap.It should be noted that the proteins required for the MMRprocess differ among eukaryotes. For instance, Drosophilaand Caenorhabditis lack Msh3 homologues, and thereforedo not require them for the removal of IDLs [22].Schizosaccharomyces does possess a Msh3 homologue, butit appears to play a different role within the cell, insteadparticipating in recombination [23].The majority of S. cerevisiae MMR proteins are present inE. cuniculi. The sole missing protein is Msh3, a subunit ofthe MutSβ heterodimer that recognizes small IDLs (SeeTable 1). Deletion of this gene in S. cerevisiae is not lethal(See discussion) [20].Homologous recombination repair (HRR)HRR is the major form of double strand break repair uti-lized in yeast. A double stranded break is recognized bydamage recognition proteins, and single stranded over-hangs are generated at both sides of the break. A region ofthe genome that is homologous to the single strandedoverhangs is then found. Strand invasion follows, and thehomologous (non-damaged) DNA is used as a templatefor synthesis on the broken strand. HRR is completedthrough re-annealing of the broken DNA strand and liga-tion. See figure 2 for an overview of this process.The Rad51, 52, 54, 55 and 57 proteins perform most stepsof the HRR process. Rad51 is a homologue of the bacterialenzyme RecA, and is well conserved within eukaryotes.When a double strand break is formed, the MRX complex(which is composed of Mre11, Rad50 and Xrs2, and alsoacts in NHEJ) is involved in damage recognition. TheDNA ends on either side of the break are then chewedback in the 5' to 3' direction by an unknown nuclease.Rad24 (which is a checkpoint protein as well) is alsoPage 6 of 14(page number not for citation purposes)involved in end processing. The results of this process areshort 3' overhangs on either strand. RPA (which also actsBMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24Page 7 of 14(page number not for citation purposes)The homologous recombination repair pathwayFigure 2The homologous recombination repair pathway. (See text for explanation.) Blue proteins are present in E. cuniculi; all others are absent. Newly synthesized DNA is indicated in grey. Although the MRX complex (Mre11/Rad50/Xrs2) acts in dam-age recognition in this pathway, it is not shown. (Modified from Aylon and Kupiec [24].)$OUBLESTRANDBREAKCREATED2AD2AD-EC$DC20!.UCLEASE2AD 2AD2AD2AD2AD$.!POLYMERASE3RS2ESECTIONANDCHECKPOINTACTIVIATION&ILAMENTFORMATIONANDHOMOLOGYSEARCH)NVASION$.!SYNTHESISANDGENECONVERSION2ELIGATION4HE0LAYERSBMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24in NER, as described above) then coats the overhangs.RPA is later replaced by Rad51, with the aid of Rad52,Rad55/Rad57, and very likely Rad54 as a genome-widesearch for homologous sequences takes place. Strandinvasion then occurs while the helicase Hpr5 removesRad51 from the DNA. DNA is synthesized by an undeter-mined polymerase based on the donor template strands,and then ligated. Although the mechanism is not clear, itis evident that the Rad55/Rad57 complex is somehowinvolved in this last step. The Sgs1 helicase plays a specificrole in the repair of double strand breaks generated by thestalling of a replication fork. For a review of HRR, seeAylon and Kupiec [24].Three other signaling and damage sensor proteins are alsoinvolved in the HRR pathway, as well as the BER pathwayand the NHEJ pathways. The Rad17/Med3/Ddc1 (9-1-1)complex triggers DNA damage checkpoints [25], andstimulates repair pathways [26] as well as various individ-ual repair proteins, including DNA polymerase β [27],Rad51 [28] (HRR), Rad27 [29] (BER, NHEJ) and Cdc9[30] (BER).E. cuniculi lacks more than half of the proteins involved inthe HRR pathway. Almost all steps of the process areaffected by these losses (see discussion). Missing proteinsinclude the Hpr5 helicase, Rad54 and Rdh54 (See Figure2, Table 1). Rad24 and the 9-1-1 complex are all absentfrom the cell signaling pathways. S. cerevisiae singlemutants lacking these proteins are viable [20], likely dueto yeast's ability to use either double strand break repairpathway (HRR or NHEJ) to fix damaged DNA.The presence or absence of Rad55 and Rad57 was notdetermined. Rad55 and Rad57 are paralogs of Rad51. PSI-BLAST searches using S. cerevisiae Rad55 and Rad57 pro-teins retrieve Rad51 in other fungi, therefore making it dif-ficult to discern the presence of these proteins in E.cuniculi, which is related to fungi.Non-homologous end joining repair (NHEJ)NHEJ is the second form of double strand break repairthat is a separate, though not completely independentpathway from HRR. In S. cerevisiae this method of doublestrand break repair plays a minor role compared to theHRR pathway. Upon double strand break formation,damage is recognized and both ends of the lesion arebrought together through the action of several proteins. Aminimal amount of DNA synthesis occurs, which is fol-lowed by ligation. As DNA on either side of the break maybe degenerated before the break is repaired, the potentialfor information loss in this case is substantial [24].3). These proteins are DNA-dependent protein kinasesthat also have a role in telomere maintenance. Oncebound to the damaged site, the Ku complex is responsiblefor recruiting the MRX complex for the next stage in therepair process. The MRX complex is composed of Rad50(an ATP binding protein), Mre11 (a 5'-3' exonuclease)and Xrs2 (responsible for aligning the MRX complex withthe break site) [31]. Dnl4/Lif1 (a DNA ligase complex) istethered to the break site by Xrs2 and the Ku complex. TheDNA polymerase Pol4 and the structure-specific nucleaseRad27 are the last players to arrive at the scene, thus com-pleting the repair complex.All of the yeast NHEJ proteins are present in most eukary-otes, and the core of Ku70 and 80 is homologous to asmaller bacterial protein that performs the same function,thus indicating a large degree of conservation. For a reviewof this process, see Hefferin and Tomkinson [32].E. cuniculi is missing nearly all NHEJ proteins. Absent pro-teins include Ku70, Ku80, Xrs2, Dnl4, and Pol4 (See Fig 3,Table 1). As is the case with single S. cerevisiae mutants forgenes involved in the HRR pathway, most are viable [20]due to yeast's ability to rely on the other (HRR) doublestrand break repair pathway.Although there are animal homologues of Lif1 and Xrs2(Xrcc4 and Nbs1, respectively), BLASTP and PSI-BLASTsearches using yeast proteins did not retrieve homologuesin any organisms other than fungi. The presence orabsence of these proteins is therefore not known.DNA polymerasesDNA polymerases are essential for both genome replica-tion and repair. There are several polymerases present ineukaryotic cells, all of which serve particular functionswithin the cell. The polymerases α, δ and ε act in the proc-ess of genome replication, but also play roles in certainrepair processes, notably NER and HRR. Polymerase γ actssolely within mitochondria, while all other polymerasesare nuclear. Polymerase β is a specialized repair polymer-ase that is involved in BER and NHEJ. The polymerases ζ,η and Rev1 help prevent double stranded DNA breaksfrom forming during replication due to their ability tosynthesize DNA through a lesion, where polymerases α, δand ε stall and dissociate from the replication fork [4].Of the 8 polymerases identified in S. cerevisiae that havehuman counterparts (confirming that they are not fungalor ascomycete specific), E. cuniculi possesses 3: α, δ and ε(See Table 1). All three of these polymerases are necessaryfor viability in S. cerevisiae. All of the polymerases that areabsent in E. cuniculi are utilized solely for repair or lesionPage 8 of 14(page number not for citation purposes)The NHEJ process begins when the Ku complex (Ku70/Ku80) binds either end of the double strand break (See Figbypass and are not essential for viability, likely becausetheir function is replaced by other polymerases [20].BMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24Page 9 of 14(page number not for citation purposes)The non-homologous end joining repair pathwayFigure 3The non-homologous end joining repair pathway. (See text for explanation.) Blue proteins are present in E. cuniculi; all others are absent. Newly synthesized DNA is indicated in grey. (Modified from Hefferin and Tomkinson [32].)+52AD 8RS$NL ,IF0OL 2AD-RE#REATIONOFDOUBLESTRANDBREAKIN$.!+UCOMPLEXBINDS$.!-28COMPLEXISRECRUITED$.!LIGASECOMPLEXISRECRUITED$.!POLYMERASEAND2ADNUCLEASEARRIVE,ESIONISREPAIRED4HE0LAYERSBMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24DiscussionIn general, the consequences of genome reduction onDNA repair in E. cuniculi are most evident in the doublestrand break pathways. The single strand repair pathwayshave been less affected, but some are operating at a levelof reduced complexity compared to S. cerevisiae.Reduction in complexity of DNA repairE. cuniculi's BER pathway lacks the DNA ligase Cdc9, DNApolymerase β and Mms4. Although deletion of Cdc9 islethal in S. cerevisiae [20], the role of this protein is likelyfilled by another ligase. This is not unusual in S. cerevisiae,as several enzymes are sometimes able to act on the samesubstrate. For example, in the HRR pathway, the polymer-ase and nuclease have not yet been defined, likely becausedifferent combinations of polymerases and nucleases arecapable of performing the required functions of this path-way [24]. The absence of DNA polymerase β could indi-cate that most BER in E. cuniculi is carried out via the longpatch pathway, where DNA is synthesized by the polymer-ases δ and ε. The use of one BER pathway over another iscommon in eukaryotes; studies have indicated that inyeast, long patch BER is carried out preferentially insteadof short patch BER, whereas in humans, the reverse is true[13]. The absence of Mms4 is not likely to have seriousramifications for BER in E. cuniculi. The Mus81-Mms4endonuclease processes 3' ends of nicked DNA to preparefor DNA synthesis. However, its role is predicted to beminor, and somewhat overlapping with that of the Rad1-Rad10 endonuclease, which is present [12].The NER pathway is missing a core TFIIH component andthe Rad23 subunit of the Rad4/Rad23 damage recogni-tion complex. TFIIH is composed of a ring containing thethree Tfb proteins (Tfb1, Tfb2, and Tfb3), which serve totether the functional parts of the complex: the helicasesRad3 and Rad25 [33]. Since transcription must occur in E.cuniculi, it is difficult to predict exactly how the absence ofthese proteins would affect this organism, as deletion ofTfb1 is lethal in S. cerevisiae. Complete absence of this pro-tein is difficult to reconcile with the Tfb ring's essentialfunctions, as well as the presence of the two other ringcomponents (See below). However, it is not unreasonableto assume that the absence of Tfb1 would likely lead to areduction in the efficiency of this repair process, particu-larly when Rad23 also appears to be absent.E. cuniculi also lacks Msh3, which interacts with Msh2 toform MutSβ, which recognizes insertion or deletion loops(IDLs) in the MMR pathway. In S. cerevisiae, deletion ofMsh3 is not lethal, but mutants are slightly more prone toframeshift mutations [15]. Although the MutSβ het-erodimer is present in S. cerevisiae, Schizosaccharomyces,Msh3, where it appears that the MutSα complex is able torecognize both mismatches and insertion or deletionloops [22].Drosophila and Caenorhabditis are able to effectively per-form MMR in the absence of Msh3, which is the sole miss-ing protein in E. cuniculi. Therefore, it is very likely thatthis pathway operates in E. cuniculi in a similar manner tothese organisms, whose MMR systems are fully functional.The absence of the DNA methyltransferase Mgt1 suggeststhat E. cuniculi is able to employ other methods to removeO6-methylguanine from its DNA. In the bacteriumEscherichia coli, O6-methylguanine can be removed byboth the NER and the methyltransferase mechanisms[34], therefore it is likely that E. cuniculi has simply dis-pensed with one of two parallel pathways.DNA polymerases and repairEukaryotes and prokaryotes possess many specializedDNA polymerases to accomplish specific tasks within thecell. Some of these polymerases are involved in genomereplication, while others act solely in repair processes.E. cuniculi possesses only three DNA polymerases (α, δand ε) of the 8 present in S. cerevisiae. All of thesepolymerases are involved in standard genome replication,while polymerase δ also plays a role in BER, NER, MMRand in bypassing DNA lesions [4]. Polymerase ε isrequired for BER and probably NER. Polymerases α, δ andε are all likely utilized in HRR [4]. E. cuniculi lackspolymerase β, which is utilized in a variety of repair path-ways and polymerases ζ and η, which are used for error-prone and error-free DNA synthesis across lesions, respec-tively [4]. E. cuniculi also lacks the mitochondrial DNApolymerase γ.S. cerevisiae mutants lacking polymerase β display a highfrequency of recombination and sensitivity to methylmethanesulfonate (MMS). Rev1 mutants displaydecreased revertibility, while polymerase η mutants havea heightened sensitivity to UV radiation. Conversely,polymerase ζ deletion mutants resist UV mutagenesis.Cells lacking polymerase γ lose their mitochondrial DNA[15], however microsporidian mitochondria (mitosomes)are highly reduced and it is unlikely that they possessautonomous DNA [35]. The phenotype of a S. cerevisiaecell lacking several polymerases is not known, but onecould speculate that such cells would display a higher fre-quency of double stranded DNA breaks generated duringreplication due to a lack of translesion polymerases.Double strand break repair in E. cuniculiPage 10 of 14(page number not for citation purposes)humans and Arabidopsis, its presence is not ubiquitousamong eukaryotes. Drosophila and Caenorhabditis lackThe fact that most of the NHEJ repair proteins appear tobe absent in E. cuniculi is perhaps not overly surprising, asBMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24this method of double strand break repair appears to be aback-up method in yeast [24]. (Note that this preferenceis not strictly maintained throughout eukaryotic life.Humans, for example, use NHEJ as the primary pathway[1].) E. cuniculi's genome is known to be highly reducedcompared to that of S. cerevisiae. Therefore, it seems logi-cal that the first genes to be deleted from a genome under-going reduction would be those encoding proteins thatact in back-up pathways.Of key interest is the lack of Ku proteins (Ku70 and Ku80)in E. cuniculi. These proteins play a pivotal role in NHEJ;they are involved in recognizing double strand break sitesand in recruiting other repair factors to the break site. Notonly is their function key, but they are present in archae-bacteria, bacteria and eukaryotes. The core of the Ku pro-teins is largely conserved from prokaryotes to eukaryotes[32]. However, the absence of these proteins in E. cuniculiis not entirely unique, as we were also unable to identifyKu proteins in the genome of the human parasite Plasmo-dium, nor has it been recognized in Trichomonas [36].Dispensing with a backup double strand break repairpathway during genome reduction would stand to reasonif the primary repair pathway was retained, however, thisis also highly questionable. E. cuniculi also lacks over halfof the HRR proteins that are present in yeast (See Table 1).The DNA helicase Hpr5, Rad54, Rdh54 and the check-point/DNA end-processing Rad24 are among the proteinsthat appear to be absent from the HRR pathway. Hpr5plays a cryptic role in HRR, as S. cerevisiae deletionmutants have hyperrecombination phenotypes [37], andthe protein was therefore assumed to be a negative regula-tor of the process. However, recent work by Aylon et al.[38] has shown that Hpr5 is intimately involved in com-mitment to gene conversion, which must take placebefore recombination can occur. Rdh54 is a Rad54homolog that participates in interhomologue gene con-version and meiosis [39], while Rad54 is a chromatinremodeling protein that has been implicated in strandinvasion and the removal of repair proteins from DNAafter HRR has taken place [40]. In addition to functioningas a checkpoint protein, Rad24 also plays a role in theresection and recombination processes [41].It is possible that the functions of Rad55 and Rad57(which are potentially absent) are carried out by Rad51, asall three proteins are homologues of the bacterial proteinRecA. This is a distinct possibility, as Rad55 and Rad57appear to act in concert with Rad51 during the HRR proc-ess [24].in the absence of all other HRR components. It is difficultto imagine this process occurring in the absence of DNAresection (Rad24), strand invasion (Rad54) and gene con-version (Hpr5 and Rdh54).Therefore, E. cuniculi appears to have drastically reducedboth mechanisms for double strand break repair.Although E. cuniculi's genome contains very few duplicategenes (regions of homologous sequence) to use as tem-plates for DNA synthesis in HRR, both S. cerevisiae [42]and mammals [43] prefer to use sister chromatids ratherthan homologous sequences (on the same or differentchromosomes) for this process. As E. cuniculi is likely dip-loid [44] (as are yeast and mammals), it is reasonable toassume that this preference would exist in this organismas well.Given that such a large number of genes involved in bothdouble strand break repair pathways are absent, it is curi-ous that some of these genes have been retained. Whenone looks closely at the functions of these genes, it is evi-dent that they all play roles in other critical biologicalprocesses. Mre11 and Rad50, both members of the MRXcomplex (found in both double strand break repair path-ways), are also involved in telomere maintenance and thegeneration of meiotic double strand breaks [45,46].Rad27 is a nuclease that is implicated in the processing ofOkazaki fragments during replication [47].All of the proteins belonging to the HRR pathway that arepresent in E. cuniculi are also involved in meiosis [45].Although sexual reproduction has not been observed in E.cuniculi, it does contain three of the seven core meiosis-specific genes (Hop2, Mnd1 and Spo11), as discussed inRamesh et al. [48], and there is evidence that it may pos-sess a mating type locus [49]. Sexual reproduction has alsobeen observed in numerous other microsporidia [5],therefore there is little reason to suspect that E. cuniculi isan exception. As a large number of proteins involved inthe HRR pathway are absent in E. cuniculi, the repair func-tions of the remaining proteins are unknown. It is possi-ble that they have been retained because of their role inmeiosis.Potential consequences for E. cuniculiReductions within the DNA repair pathways have led totwo fundamentally different outcomes: reduced complex-ity by loss of a few proteins (NER, MMR, BER) and drasticlosses of half or more proteins involved in a pathway(methyltransferase repair, HRR and NHEJ). Although anorganism may be able to tolerate a somewhat sloppyrepair system, it is difficult to imagine how the organismcould exist without any means to mend double-strandPage 11 of 14(page number not for citation purposes)Although the Rad51, Rad52 and Sgs1 proteins are presentin E. cuniculi, it is not known whether HRR can take placeDNA breaks, especially given their frequency during mei-osis and mitosis. E. cuniculi must, therefore, utilize someBMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24other form of double strand break repair, or contain suchhighly divergent copies of most NHEJ and HRR proteinsthat they were impossible to identify in this study.Along with many of the proteins that carry out the workof repair, E. cuniculi has lost several proteins that partici-pate in cell signaling and cycle control. The 9-1-1 signal-ing complex is absent, which has been proposed to play arole in the signaling cascade leading to cell cycle arrest andapoptosis [50]. Both the Ku and MRX complexes are alsoinvolved in cell cycle control, although their roles are notwell defined [51]. Loss of coordination of cellular activi-ties could result from the absence of these proteins.In addition to their role in repair, the Ku proteins protecttelomeres from degradation and help to control telomer-ase activity [52]. As E. cuniculi houses eleven chromo-somes that contain telomeres [44], and encodes thecatalytic subunit of the telomerase enzyme [6], this organ-ism must have developed an alternate method to main-tain its telomeres, or it would suffer extreme telomereattrition.Like the Ku proteins, the DNA ligase Cdc9 performs sev-eral functions as well. It plays a role in recombination andin the ligation of Okazaki fragments during replication[53], therefore, it is possible that these processes are some-what impaired.The reduction that is observed within the DNA repairpathways is similar to that observed throughout E.cuniculi's genome, as this organism lacks many proteinsthat participate in diverse biosynthetic pathways. In thisway, the genome of E. cuniculi is very similar to those ofmany endosymbiotic and parasitic bacteria. Buchneraaphidicola has also lost many DNA repair genes during theprocess of genome reduction; indeed it has been proposedthat it was this lack of DNA repair genes that allowedBuchnera's genome to become so small in the first place[54].We cannot rule out the possibility that our bioinformaticstools were unsuccessful in locating highly divergent pro-teins that act in the DNA repair processes. It is also possi-ble that in some cases, other non-homologous proteinscarry out essential functions to replace absent proteins (ie.Tfb1 in NER). Such proteins may still be identified, asroughly half of E. cuniculi's genome consists of hypotheti-cal proteins [6]. Another potential explanation for thislack of biosynthetic machinery is that E. cuniculi is able toimport many of the products of these pathways from thehost's cytoplasm (ie., ATP) [5]. However, it seems unlikelythat this would be the case for DNA repair proteins, asthe nucleus in order for them to function. For themoment, double strand break repair in E. cuniculi willremain a mystery.ConclusionOur survey of E. cuniculi's DNA repair genes indicates thatthe process of genome reduction has affected all majorDNA repair pathways. All of the single strand repair path-ways (BER, NER and MMR) have lost at least one compo-nent, indicating that these pathways are less complex thanin S. cerevisiae, and could be less efficient. All replicativeDNA polymerases are present in E. cuniculi, although thespecialized repair polymerases are absent. The absence ofthese enzymes could lead to inefficient DNA damagerepair and creation of double stranded DNA breaks thatare not easily repaired. Surprisingly, more than half of theproteins participating in both double strand break repairpathways (HRR and NHEJ) and the sole componentinvolved in methyltransferase repair are absent in E.cuniculi. The proteins that remain are all involved in addi-tional cellular functions (such as meiosis).MethodsIdentification of DNA repair pathway components in S. cerevisiae and data mining in E. cuniculiComponents of the six major DNA repair pathways weregathered from recent literature and supplemented withdata from the Saccharomyces genome database [15]. Referto Table 1 for a list of genes involved in each pathway andthe DNA polymerase subunits and references.Amino acid sequences of DNA repair proteins from S. cer-evisiae (Table 1) were collected from NCBI GENBANK,and compared to E. cuniculi's protein and nucleotide datausing BLASTP and TBLASTN [55]. In instances where aSchizosaccharomyces pombe homologue to an S. cerevisiaeprotein existed that was more conserved among eukaryo-tes than the S. cerevisiae protein itself, the S. pombesequence was used in the BLAST searches. (These proteinsare indicated with asterixes in Table 1 and S. pombe pro-tein ID numbers are given).In most instances, BLASTP searches were sufficient toidentify putative E. cuniculi homologues. In cases whereno significant results (significance was defined arbitrarilyas an e-value of 10-5 or less) were produced from the ini-tial BLASTP analysis, the PSI-BLAST algorithm was used.Homologues of the S. cerevisiae protein were identified inall available eukaryotic protein data to construct a posi-tion-specific scoring matrix [55]. Up to six iterations wererun in cases where no significant E. cuniculi alignment wasfound. In order to rule out similarity by chance, the iden-tities of putative homologues detected in E. cuniculi werePage 12 of 14(page number not for citation purposes)protein uptake has not been documented in micro-sporidia, and these proteins would have to be targeted toconfirmed by comparing them to GENBANK's S. cerevisiaeprotein database using BLASTP. Homology was inferredBMC Molecular Biology 2007, 8:24 http://www.biomedcentral.com/1471-2199/8/24when this search recovered the S. cerevisiae protein thatwas used for the initial E. cuniculi search as the top hit.In many instances, BLAST searches in E. cuniculi con-firmed annotations of DNA repair genes and polymerasesby Katinka et al. [6].A brief examination of the number of interaction partnersof each protein in S. cerevisiae was conducted using datafrom the online Database of Interacting Proteins (DIP)[56]. Proteins that are absent in E. cuniculi do not have asignificantly different number of interaction partnersfrom proteins that are present. (Data not shown.)Authors' contributionsEEG conceived of the study, performed data mining andanalyses and drafted the manuscript. NMF assisted inanalyses and made critical contributions to the manu-script. Both authors read and approved the final manu-script.AcknowledgementsWe would like to thank P. Keeling and R. 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