BACKGROUND
dna repair in eukaryotes
dna repair processes are vital to all living organisms.
without appropriate mechanisms to remove and replace damaged bases and nucleotides, multiple lesions would accumulate, leading to total genome degradation and loss of vital genetic information.
dna lesions take many forms, including single strand and double strand breaks, in addition to inter- and intra-strand crosslinks and modified bases.
several pathways operate in a concerted manner to minimize information loss each time a dna lesion occurs.
many of these fundamental pathways have been conserved throughout eukaryotes, and many eukaryotic enzymes have homologues in prokaryotes  <cit> .
eukaryotic dna repair can be divided into six primary pathways, all of which are conserved  <cit> .
some types of dna lesions  can be recognized and repaired by more than one pathway.
therefore there is some overlap in function between pathways  <cit> .
mismatch repair , base excision repair  and nucleotide excision repair  all operate to repair aberrant bases or nucleotides from one strand of the double helix, using the other strand as a template for new dna synthesis.
in contrast, methyltransferase repair does not require the synthesis of new dna; anomalous methyl groups are removed without causing any breaks in the double helix.
non-homologous end joining repair  and homologous recombination repair  are double strand break repair pathways  <cit> .
double strand breaks are one of the most detrimental forms of dna lesions, as they can cause genome fragmentation and apoptosis if they are not properly repaired  <cit> .
dna polymerases are key players in dna repair, as they are required to fill in gaps created by repair enzymes or incurred from damage  <cit> .
eukaryotic cells have a wide range of polymerases that are specialized in function; certain polymerases act almost solely in genome replication, others are only active at dna lesions, and a few have dual roles in repair and replication  <cit> .
for consistency, the names of the genes and proteins involved in these pathways will be referred to using saccharomyces cerevisiae nomenclature.
genome reduction in encephalitozoon cuniculi
encephalitozoon cuniculi belongs to a group of obligate intracellular parasites known as microsporidia.
these organisms infect a variety of animals including fish, insects and mammals.
various microsporidia have gained the attention of the medical community in the past few decades due to their infection of immuno-compromised humans, such as aids and chemotherapy patients  <cit> .
microsporidian genomes range in size from  <dig>  mbp to  <dig>  mbp.
the only completely sequenced microsporidian genome, that of e. cuniculi, is a mere  <dig>  mbp in size  <cit> .
the e. cuniculi genome is smaller than that of other eukaryotes for several reasons: it has fewer and shorter genes that are separated by tiny intergenic spaces and are only interrupted by a few short introns  <cit> .
given the degree of genome reduction, the effects are evident in most cellular processes, including dna repair.
the precise phylogenetic position of microsporidia is not yet known, but a large body of evidence indicates that they are closely related to the fungi .
therefore, e. cuniculi's dna repair systems have been compared primarily to those of another fungus, the yeast s. cerevisiae.
s. cerevisiae's repair pathways have been well studied at the functional level, making this organism ideal for comparative purposes.
in order to gain an accurate perspective of what genes have been lost from e. cuniculi during the process of genome reduction , only genes that have homologues in animals were examined.
RESULTS
dna repair inventory
comparison of dna repair proteins in s. cerevisiae to e. cuniculi's genome and proteome via blast and psi-blast searches has revealed that e. cuniculi appears to contain a reduced set of proteins in all major repair pathways.
of the  <dig> repair genes that were sought in e. cuniculi,  <dig> are absent, with another  <dig> potentially absent.
six out of  <dig> dna polymerases or polymerase subunits are absent .
although all repair pathways have been reduced, the loss of genes is not distributed evenly among pathways.
each process has been affected differently by genome reduction.
a detailed discussion of the components of each pathway is presented below.
base excision repair
ber is one of the least complex of the dna repair mechanisms, and involves only a small number of proteins.
when a base becomes damaged, it is recognized by a dna glycosylase that is specific for the particular base and/or the type of damage .
s. cerevisiae contains four types of glycosylases, although far more have been found in other organisms   <cit> .
glycosylases cleave the glycosylic bond between the base and the deoxyribose to remove the damaged base, at which point a non-specific apurinic/apyrimidinic  endonuclease  removes the remaining deoxyribose phosphate to create a gap  <cit> .
in short patch ber , the gap is filled by dna polymerase β. in long patch ber , the dna polymerases β, or δ and ε in concert with proliferating cell nuclear antigen  synthesize several nucleotides which displace the original dna strand.
rad <dig> then removes the displaced dna.
the ligase cdc <dig>  is used to seal the nick  <cit> .
the rad1-rad <dig> and mus81-mms <dig> endonucleases are also believed to play minor roles in ber by processing the 3' ends of the dna once an incision has been made into the sugar-phosphate backbone  <cit> .
e. cuniculi's ber pathway appears to be nearly complete, but lacks dna polymerase β, the cdc <dig> dna ligase  and part of a 3' endonuclease, mms <dig>   deletion of either polymerase β or mms <dig> is not a lethal mutation in yeast, however s. cerevisiae cannot survive in the absence of cdc <dig>  <cit> .
another ligase is likely utilized for ber in e. cuniculi, as sharing non-specialized enzymes between pathways is not uncommon in s. cerevisiae , and there is no reason to believe that this is not the case in e. cuniculi.
nucleotide excision repair
ner is used primarily to remove bulky lesions from dna, such as inter- and intra-strand crosslinks.
ner is a more complex process than ber, and utilizes a large number of proteins that are evolutionarily conserved among eukaryotes.
ner is comprised of two subpathways, global genome repair  and transcription-coupled repair .
as is suggested by their names, the subpathways act on different types of dna: dna that is not transcribed , and actively transcribed dna, respectively.
in both ggr and tcr, dna damage recognition is the first step to occur, followed by dna unwinding.
next, incisions are made on either side of the aberrant base, and a total of 25– <dig> nucleotides on either side are removed as a single strand.
the gap is then filled by dna polymerase and sealed by dna ligase.
recruitment of five multi-protein complexes, nucleotide excision repair factors   <dig> through  <dig> and the replication protein a  complex, is believed to take place in a stepwise manner to complete this process.
the rpa complex is composed of rpa <dig> and rpa <dig> and recognizes damaged dna.
in ggr, the first protein complex to arrive at the damaged site is nef <dig>  which recognizes damage and is composed of the proteins rad <dig> and rad <dig>  rad <dig> binds the nef <dig> complex , recruiting it to the damaged site and increasing dna binding efficiency.
the presence of nef <dig> is not strictly required for the recruitment of nef <dig> to the dna lesion, but facilitates the process.
the above proteins do not act in the other sub-pathway; in tcr, initiation of repair takes place when an rna polymerase stalls.
two proteins involved specifically in tcr, rad <dig> and rad <dig>  also participate in the beginning of this process  <cit> .
in both ggr and tcr, nef <dig> and nef <dig> are the next components to be recruited, and are held at the damage site by nef <dig>  nef <dig> is composed of rad <dig>  rad <dig> and rad <dig>  while nef <dig> is composed of rad <dig> and transcription elongation factor iih .
tfiih contains the rad <dig>  rad <dig>  ssl <dig>  tfb <dig>  tfb <dig> and tfb <dig> proteins, and provides the single strand dna helicases required for repair proteins to access the damaged site.
rad <dig> and rad <dig> form a heterodimer that acts as a single strand endonuclease at the 5' end of the stretch of damaged dna and rad <dig> is a single strand endonuclease that cuts at the 3' end.
rpa is thought to be the last player to arrive at the scene.
many of these proteins also have roles in other cellular processes, such as recombination and transcription, therefore mutants express defects in several pathways.
for a comprehensive review of ner, see prakash and prakash  <cit> .
most proteins participating in ner are present in e. cuniculi, with two exceptions.
half of a ggr heterodimeric damage sensor complex  and the tfb <dig> subunit of tfiih appear to be absent .
rad <dig> appears to have diverse functions within the cell, ranging from dna repair to the regulation of a cell-cycle checkpoint and protein degradation.
specifically, this protein helps to prevent the degradation of rad <dig>  as well as serving a role with the 26s proteasome in regulating the ner pathway  <cit> .
deletion of tfb <dig> in s. cerevisiae is lethal  <cit> , likely due to loss of function in transcription.
the presence or absence of rad <dig> and rad <dig> were not confirmed, as blast and psi-blast searches using s. cerevisiae and s. pombe sequences as queries did not return homologues from most animals or other eukaryotes besides fungi.
methyltransferase repair
methyltransferases are present in both eukaryotes and prokaryotes and remove certain dna lesions involving methylation .
these proteins irreversibly relocate methyl groups from dna to their own cysteine residues, and are therefore suicide enzymes  <cit> .
e. cuniculi does not possess the methyltransferase found in other eukaryotes, mgt <dig>  deletion of this gene is not lethal in s. cerevisiae  <cit> .
mismatch repair
in mmr, mismatches are recognized by the heterodimers mutsα  and mutsβ .
single base mismatches are recognized by mutsα and insertion/deletion loops  less than about  <dig> nucleotides in length are recognized by mutsβ  <cit> .
both mutsα and mutsβ can recognize a single unpaired nucleotide.
pcna is also involved in mmr, perhaps assisting in these initial recognition steps.
mutlα  binds mutsα and β and allows them to efficiently bind to idls and mismatches.
the exonuclease exo <dig> then excises the mismatched base and a dna polymerase and dna ligase fill and seal the gap.
it should be noted that the proteins required for the mmr process differ among eukaryotes.
for instance, drosophila and caenorhabditis lack msh <dig> homologues, and therefore do not require them for the removal of idls  <cit> .
schizosaccharomyces does possess a msh <dig> homologue, but it appears to play a different role within the cell, instead participating in recombination  <cit> .
the majority of s. cerevisiae mmr proteins are present in e. cuniculi.
the sole missing protein is msh <dig>  a subunit of the mutsβ heterodimer that recognizes small idls .
deletion of this gene in s. cerevisiae is not lethal   <cit> .
homologous recombination repair
hrr is the major form of double strand break repair utilized in yeast.
a double stranded break is recognized by damage recognition proteins, and single stranded overhangs are generated at both sides of the break.
a region of the genome that is homologous to the single stranded overhangs is then found.
strand invasion follows, and the homologous  dna is used as a template for synthesis on the broken strand.
hrr is completed through re-annealing of the broken dna strand and ligation.
see figure  <dig> for an overview of this process.
the rad <dig>   <dig>   <dig>   <dig> and  <dig> proteins perform most steps of the hrr process.
rad <dig> is a homologue of the bacterial enzyme reca, and is well conserved within eukaryotes.
when a double strand break is formed, the mrx complex  is involved in damage recognition.
the dna ends on either side of the break are then chewed back in the 5' to 3' direction by an unknown nuclease.
rad <dig>  is also involved in end processing.
the results of this process are short 3' overhangs on either strand.
rpa  then coats the overhangs.
rpa is later replaced by rad <dig>  with the aid of rad <dig>  rad55/rad <dig>  and very likely rad <dig> as a genome-wide search for homologous sequences takes place.
strand invasion then occurs while the helicase hpr <dig> removes rad <dig> from the dna.
dna is synthesized by an undetermined polymerase based on the donor template strands, and then ligated.
although the mechanism is not clear, it is evident that the rad55/rad <dig> complex is somehow involved in this last step.
the sgs <dig> helicase plays a specific role in the repair of double strand breaks generated by the stalling of a replication fork.
for a review of hrr, see aylon and kupiec  <cit> .
three other signaling and damage sensor proteins are also involved in the hrr pathway, as well as the ber pathway and the nhej pathways.
the rad17/med3/ddc <dig>  complex triggers dna damage checkpoints  <cit> , and stimulates repair pathways  <cit>  as well as various individual repair proteins, including dna polymerase β  <cit> , rad <dig>  <cit>  , rad <dig>  <cit>   and cdc <dig>  <cit>  .
e. cuniculi lacks more than half of the proteins involved in the hrr pathway.
almost all steps of the process are affected by these losses .
missing proteins include the hpr <dig> helicase, rad <dig> and rdh <dig> .
rad <dig> and the 9-1- <dig> complex are all absent from the cell signaling pathways.
s. cerevisiae single mutants lacking these proteins are viable  <cit> , likely due to yeast's ability to use either double strand break repair pathway  to fix damaged dna.
the presence or absence of rad <dig> and rad <dig> was not determined.
rad <dig> and rad <dig> are paralogs of rad <dig>  psi-blast searches using s. cerevisiae rad <dig> and rad <dig> proteins retrieve rad <dig> in other fungi, therefore making it difficult to discern the presence of these proteins in e. cuniculi, which is related to fungi.
non-homologous end joining repair
nhej is the second form of double strand break repair that is a separate, though not completely independent pathway from hrr.
in s. cerevisiae this method of double strand break repair plays a minor role compared to the hrr pathway.
upon double strand break formation, damage is recognized and both ends of the lesion are brought together through the action of several proteins.
a minimal amount of dna synthesis occurs, which is followed by ligation.
as dna on either side of the break may be degenerated before the break is repaired, the potential for information loss in this case is substantial  <cit> .
the nhej process begins when the ku complex  binds either end of the double strand break .
these proteins are dna-dependent protein kinases that also have a role in telomere maintenance.
once bound to the damaged site, the ku complex is responsible for recruiting the mrx complex for the next stage in the repair process.
the mrx complex is composed of rad <dig> , mre <dig>  and xrs <dig>   <cit> .
dnl4/lif <dig>  is tethered to the break site by xrs <dig> and the ku complex.
the dna polymerase pol <dig> and the structure-specific nuclease rad <dig> are the last players to arrive at the scene, thus completing the repair complex.
all of the yeast nhej proteins are present in most eukaryotes, and the core of ku <dig> and  <dig> is homologous to a smaller bacterial protein that performs the same function, thus indicating a large degree of conservation.
for a review of this process, see hefferin and tomkinson  <cit> .
e. cuniculi is missing nearly all nhej proteins.
absent proteins include ku <dig>  ku <dig>  xrs <dig>  dnl <dig>  and pol <dig> .
as is the case with single s. cerevisiae mutants for genes involved in the hrr pathway, most are viable  <cit>  due to yeast's ability to rely on the other  double strand break repair pathway.
although there are animal homologues of lif <dig> and xrs <dig> , blastp and psi-blast searches using yeast proteins did not retrieve homologues in any organisms other than fungi.
the presence or absence of these proteins is therefore not known.
dna polymerases
dna polymerases are essential for both genome replication and repair.
there are several polymerases present in eukaryotic cells, all of which serve particular functions within the cell.
the polymerases α, δ and ε act in the process of genome replication, but also play roles in certain repair processes, notably ner and hrr.
polymerase γ acts solely within mitochondria, while all other polymerases are nuclear.
polymerase β is a specialized repair polymerase that is involved in ber and nhej.
the polymerases ζ, η and rev <dig> help prevent double stranded dna breaks from forming during replication due to their ability to synthesize dna through a lesion, where polymerases α, δ and ε stall and dissociate from the replication fork  <cit> .
of the  <dig> polymerases identified in s. cerevisiae that have human counterparts , e. cuniculi possesses 3: α, δ and ε .
all three of these polymerases are necessary for viability in s. cerevisiae.
all of the polymerases that are absent in e. cuniculi are utilized solely for repair or lesion bypass and are not essential for viability, likely because their function is replaced by other polymerases  <cit> .
discussion
in general, the consequences of genome reduction on dna repair in e. cuniculi are most evident in the double strand break pathways.
the single strand repair pathways have been less affected, but some are operating at a level of reduced complexity compared to s. cerevisiae.
reduction in complexity of dna repair
e. cuniculi's ber pathway lacks the dna ligase cdc <dig>  dna polymerase β and mms <dig>  although deletion of cdc <dig> is lethal in s. cerevisiae  <cit> , the role of this protein is likely filled by another ligase.
this is not unusual in s. cerevisiae, as several enzymes are sometimes able to act on the same substrate.
for example, in the hrr pathway, the polymerase and nuclease have not yet been defined, likely because different combinations of polymerases and nucleases are capable of performing the required functions of this pathway  <cit> .
the absence of dna polymerase β could indicate that most ber in e. cuniculi is carried out via the long patch pathway, where dna is synthesized by the polymerases δ and ε. the use of one ber pathway over another is common in eukaryotes; studies have indicated that in yeast, long patch ber is carried out preferentially instead of short patch ber, whereas in humans, the reverse is true  <cit> .
the absence of mms <dig> is not likely to have serious ramifications for ber in e. cuniculi.
the mus81-mms <dig> endonuclease processes 3' ends of nicked dna to prepare for dna synthesis.
however, its role is predicted to be minor, and somewhat overlapping with that of the rad1-rad <dig> endonuclease, which is present  <cit> .
the ner pathway is missing a core tfiih component and the rad <dig> subunit of the rad4/rad <dig> damage recognition complex.
tfiih is composed of a ring containing the three tfb proteins , which serve to tether the functional parts of the complex: the helicases rad <dig> and rad <dig>  <cit> .
since transcription must occur in e. cuniculi, it is difficult to predict exactly how the absence of these proteins would affect this organism, as deletion of tfb <dig> is lethal in s. cerevisiae.
complete absence of this protein is difficult to reconcile with the tfb ring's essential functions, as well as the presence of the two other ring components .
however, it is not unreasonable to assume that the absence of tfb <dig> would likely lead to a reduction in the efficiency of this repair process, particularly when rad <dig> also appears to be absent.
e. cuniculi also lacks msh <dig>  which interacts with msh <dig> to form mutsβ, which recognizes insertion or deletion loops  in the mmr pathway.
in s. cerevisiae, deletion of msh <dig> is not lethal, but mutants are slightly more prone to frameshift mutations  <cit> .
although the mutsβ heterodimer is present in s. cerevisiae, schizosaccharomyces, humans and arabidopsis, its presence is not ubiquitous among eukaryotes.
drosophila and caenorhabditis lack msh <dig>  where it appears that the mutsα complex is able to recognize both mismatches and insertion or deletion loops  <cit> .
drosophila and caenorhabditis are able to effectively perform mmr in the absence of msh <dig>  which is the sole missing protein in e. cuniculi.
therefore, it is very likely that this pathway operates in e. cuniculi in a similar manner to these organisms, whose mmr systems are fully functional.
the absence of the dna methyltransferase mgt <dig> suggests that e. cuniculi is able to employ other methods to remove o6-methylguanine from its dna.
in the bacterium escherichia coli, o6-methylguanine can be removed by both the ner and the methyltransferase mechanisms  <cit> , therefore it is likely that e. cuniculi has simply dispensed with one of two parallel pathways.
dna polymerases and repair
eukaryotes and prokaryotes possess many specialized dna polymerases to accomplish specific tasks within the cell.
some of these polymerases are involved in genome replication, while others act solely in repair processes.
e. cuniculi possesses only three dna polymerases  of the  <dig> present in s. cerevisiae.
all of these polymerases are involved in standard genome replication, while polymerase δ also plays a role in ber, ner, mmr and in bypassing dna lesions  <cit> .
polymerase ε is required for ber and probably ner.
polymerases α, δ and ε are all likely utilized in hrr  <cit> .
e. cuniculi lacks polymerase β, which is utilized in a variety of repair pathways and polymerases ζ and η, which are used for error-prone and error-free dna synthesis across lesions, respectively  <cit> .
e. cuniculi also lacks the mitochondrial dna polymerase γ.
s. cerevisiae mutants lacking polymerase β display a high frequency of recombination and sensitivity to methyl methanesulfonate .
rev <dig> mutants display decreased revertibility, while polymerase η mutants have a heightened sensitivity to uv radiation.
conversely, polymerase ζ deletion mutants resist uv mutagenesis.
cells lacking polymerase γ lose their mitochondrial dna  <cit> , however microsporidian mitochondria  are highly reduced and it is unlikely that they possess autonomous dna  <cit> .
the phenotype of a s. cerevisiae cell lacking several polymerases is not known, but one could speculate that such cells would display a higher frequency of double stranded dna breaks generated during replication due to a lack of translesion polymerases.
double strand break repair in e. cuniculi
the fact that most of the nhej repair proteins appear to be absent in e. cuniculi is perhaps not overly surprising, as this method of double strand break repair appears to be a back-up method in yeast  <cit> .
e. cuniculi's genome is known to be highly reduced compared to that of s. cerevisiae.
therefore, it seems logical that the first genes to be deleted from a genome undergoing reduction would be those encoding proteins that act in back-up pathways.
of key interest is the lack of ku proteins  in e. cuniculi.
these proteins play a pivotal role in nhej; they are involved in recognizing double strand break sites and in recruiting other repair factors to the break site.
not only is their function key, but they are present in archaebacteria, bacteria and eukaryotes.
the core of the ku proteins is largely conserved from prokaryotes to eukaryotes  <cit> .
however, the absence of these proteins in e. cuniculi is not entirely unique, as we were also unable to identify ku proteins in the genome of the human parasite plasmodium, nor has it been recognized in trichomonas  <cit> .
dispensing with a backup double strand break repair pathway during genome reduction would stand to reason if the primary repair pathway was retained, however, this is also highly questionable.
e. cuniculi also lacks over half of the hrr proteins that are present in yeast .
the dna helicase hpr <dig>  rad <dig>  rdh <dig> and the checkpoint/dna end-processing rad <dig> are among the proteins that appear to be absent from the hrr pathway.
hpr <dig> plays a cryptic role in hrr, as s. cerevisiae deletion mutants have hyperrecombination phenotypes  <cit> , and the protein was therefore assumed to be a negative regulator of the process.
however, recent work by aylon et al.
<cit>  has shown that hpr <dig> is intimately involved in commitment to gene conversion, which must take place before recombination can occur.
rdh <dig> is a rad <dig> homolog that participates in interhomologue gene conversion and meiosis  <cit> , while rad <dig> is a chromatin remodeling protein that has been implicated in strand invasion and the removal of repair proteins from dna after hrr has taken place  <cit> .
in addition to functioning as a checkpoint protein, rad <dig> also plays a role in the resection and recombination processes  <cit> .
it is possible that the functions of rad <dig> and rad <dig>  are carried out by rad <dig>  as all three proteins are homologues of the bacterial protein reca.
this is a distinct possibility, as rad <dig> and rad <dig> appear to act in concert with rad <dig> during the hrr process  <cit> .
although the rad <dig>  rad <dig> and sgs <dig> proteins are present in e. cuniculi, it is not known whether hrr can take place in the absence of all other hrr components.
it is difficult to imagine this process occurring in the absence of dna resection , strand invasion  and gene conversion .
therefore, e. cuniculi appears to have drastically reduced both mechanisms for double strand break repair.
although e. cuniculi's genome contains very few duplicate genes  to use as templates for dna synthesis in hrr, both s. cerevisiae  <cit>  and mammals  <cit>  prefer to use sister chromatids rather than homologous sequences  for this process.
as e. cuniculi is likely diploid  <cit>  , it is reasonable to assume that this preference would exist in this organism as well.
given that such a large number of genes involved in both double strand break repair pathways are absent, it is curious that some of these genes have been retained.
when one looks closely at the functions of these genes, it is evident that they all play roles in other critical biological processes.
mre <dig> and rad <dig>  both members of the mrx complex , are also involved in telomere maintenance and the generation of meiotic double strand breaks  <cit> .
rad <dig> is a nuclease that is implicated in the processing of okazaki fragments during replication  <cit> .
all of the proteins belonging to the hrr pathway that are present in e. cuniculi are also involved in meiosis  <cit> .
although sexual reproduction has not been observed in e. cuniculi, it does contain three of the seven core meiosis-specific genes , as discussed in ramesh et al.
<cit> , and there is evidence that it may possess a mating type locus  <cit> .
sexual reproduction has also been observed in numerous other microsporidia  <cit> , therefore there is little reason to suspect that e. cuniculi is an exception.
as a large number of proteins involved in the hrr pathway are absent in e. cuniculi, the repair functions of the remaining proteins are unknown.
it is possible that they have been retained because of their role in meiosis.
potential consequences for e. cuniculi
reductions within the dna repair pathways have led to two fundamentally different outcomes: reduced complexity by loss of a few proteins  and drastic losses of half or more proteins involved in a pathway .
although an organism may be able to tolerate a somewhat sloppy repair system, it is difficult to imagine how the organism could exist without any means to mend double-strand dna breaks, especially given their frequency during meiosis and mitosis.
e. cuniculi must, therefore, utilize some other form of double strand break repair, or contain such highly divergent copies of most nhej and hrr proteins that they were impossible to identify in this study.
along with many of the proteins that carry out the work of repair, e. cuniculi has lost several proteins that participate in cell signaling and cycle control.
the 9-1- <dig> signaling complex is absent, which has been proposed to play a role in the signaling cascade leading to cell cycle arrest and apoptosis  <cit> .
both the ku and mrx complexes are also involved in cell cycle control, although their roles are not well defined  <cit> .
loss of coordination of cellular activities could result from the absence of these proteins.
in addition to their role in repair, the ku proteins protect telomeres from degradation and help to control telomerase activity  <cit> .
as e. cuniculi houses eleven chromosomes that contain telomeres  <cit> , and encodes the catalytic subunit of the telomerase enzyme  <cit> , this organism must have developed an alternate method to maintain its telomeres, or it would suffer extreme telomere attrition.
like the ku proteins, the dna ligase cdc <dig> performs several functions as well.
it plays a role in recombination and in the ligation of okazaki fragments during replication  <cit> , therefore, it is possible that these processes are somewhat impaired.
the reduction that is observed within the dna repair pathways is similar to that observed throughout e. cuniculi's genome, as this organism lacks many proteins that participate in diverse biosynthetic pathways.
in this way, the genome of e. cuniculi is very similar to those of many endosymbiotic and parasitic bacteria.
buchnera aphidicola has also lost many dna repair genes during the process of genome reduction; indeed it has been proposed that it was this lack of dna repair genes that allowed buchnera's genome to become so small in the first place  <cit> .
we cannot rule out the possibility that our bioinformatics tools were unsuccessful in locating highly divergent proteins that act in the dna repair processes.
it is also possible that in some cases, other non-homologous proteins carry out essential functions to replace absent proteins .
such proteins may still be identified, as roughly half of e. cuniculi's genome consists of hypothetical proteins  <cit> .
another potential explanation for this lack of biosynthetic machinery is that e. cuniculi is able to import many of the products of these pathways from the host's cytoplasm   <cit> .
however, it seems unlikely that this would be the case for dna repair proteins, as protein uptake has not been documented in microsporidia, and these proteins would have to be targeted to the nucleus in order for them to function.
for the moment, double strand break repair in e. cuniculi will remain a mystery.
CONCLUSIONS
our survey of e. cuniculi's dna repair genes indicates that the process of genome reduction has affected all major dna repair pathways.
all of the single strand repair pathways  have lost at least one component, indicating that these pathways are less complex than in s. cerevisiae, and could be less efficient.
all replicative dna polymerases are present in e. cuniculi, although the specialized repair polymerases are absent.
the absence of these enzymes could lead to inefficient dna damage repair and creation of double stranded dna breaks that are not easily repaired.
surprisingly, more than half of the proteins participating in both double strand break repair pathways  and the sole component involved in methyltransferase repair are absent in e. cuniculi.
the proteins that remain are all involved in additional cellular functions .

 dna repair in eukaryotes dna repair processes are vital to all living organisms. without appropriate mechanisms to remove and replace damaged bases and nucleotides, multiple lesions would accumulate, leading to total genome degradation and loss of vital genetic information. dna lesions take many forms, including single strand and double strand breaks, in addition to inter- and intra-strand crosslinks and modified bases. several pathways operate in a concerted manner to minimize information loss each time a dna lesion occurs. many of these fundamental pathways have been conserved throughout eukaryotes, and many eukaryotic enzymes have homologues in prokaryotes. eukaryotic dna repair can be divided into six primary pathways, all of which are conserved. some types of dna lesions can be recognized and repaired by more than one pathway. therefore there is some overlap in function between pathways. mismatch repair, base excision repair and nucleotide excision repair all operate to repair aberrant bases or nucleotides from one strand of the double helix, using the other strand as a template for new dna synthesis. in contrast, methyltransferase repair does not require the synthesis of new dna; anomalous methyl groups are removed without causing any breaks in the double helix. non-homologous end joining repair and homologous recombination repair are double strand break repair pathways. double strand breaks are one of the most detrimental forms of dna lesions, as they can cause genome fragmentation and apoptosis if they are not properly repaired. dna polymerases are key players in dna repair, as they are required to fill in gaps created by repair enzymes or incurred from damage. eukaryotic cells have a wide range of polymerases that are specialized in function; certain polymerases act almost solely in genome replication, others are only active at dna lesions, and a few have dual roles in repair and replication. for consistency, the names of the genes and proteins involved in these pathways will be referred to using saccharomyces cerevisiae nomenclature. genome reduction in encephalitozoon cuniculi encephalitozoon cuniculi belongs to a group of obligate intracellular parasites known as microsporidia. these organisms infect a variety of animals including fish, insects and mammals. various microsporidia have gained the attention of the medical community in the past few decades due to their infection of immuno-compromised humans, such as aids and chemotherapy patients. microsporidian genomes range in size from mbp to mbp. the only completely sequenced microsporidian genome, that of e. cuniculi, is a mere mbp in size. the e. cuniculi genome is smaller than that of other eukaryotes for several reasons: it has fewer and shorter genes that are separated by tiny intergenic spaces and are only interrupted by a few short introns. given the degree of genome reduction, the effects are evident in most cellular processes, including dna repair. the precise phylogenetic position of microsporidia is not yet known, but a large body of evidence indicates that they are closely related to the fungi. therefore, e. cuniculi's dna repair systems have been compared primarily to those of another fungus, the yeast s. cerevisiae. s. cerevisiae's repair pathways have been well studied at the functional level, making this organism ideal for comparative purposes. in order to gain an accurate perspective of what genes have been lost from e. cuniculi during the process of genome reduction, only genes that have homologues in animals were examined.  dna repair inventory comparison of dna repair proteins in s. cerevisiae to e. cuniculi's genome and proteome via blast and psi-blast searches has revealed that e. cuniculi appears to contain a reduced set of proteins in all major repair pathways. of the repair genes that were sought in e. cuniculi, are absent, with another potentially absent. six out of dna polymerases or polymerase subunits are absent. although all repair pathways have been reduced, the loss of genes is not distributed evenly among pathways. each process has been affected differently by genome reduction. a detailed discussion of the components of each pathway is presented below. base excision repair ber is one of the least complex of the dna repair mechanisms, and involves only a small number of proteins. when a base becomes damaged, it is recognized by a dna glycosylase that is specific for the particular base and/or the type of damage. s. cerevisiae contains four types of glycosylases, although far more have been found in other organisms. glycosylases cleave the glycosylic bond between the base and the deoxyribose to remove the damaged base, at which point a non-specific apurinic/apyrimidinic endonuclease removes the remaining deoxyribose phosphate to create a gap. in short patch ber, the gap is filled by dna polymerase β. in long patch ber, the dna polymerases β, or δ and ε in concert with proliferating cell nuclear antigen synthesize several nucleotides which displace the original dna strand. rad then removes the displaced dna. the ligase cdc is used to seal the nick. the rad1-rad and mus81-mms endonucleases are also believed to play minor roles in ber by processing the 3' ends of the dna once an incision has been made into the sugar-phosphate backbone. e. cuniculi's ber pathway appears to be nearly complete, but lacks dna polymerase β, the cdc dna ligase and part of a 3' endonuclease, mms deletion of either polymerase β or mms is not a lethal mutation in yeast, however s. cerevisiae cannot survive in the absence of cdc. another ligase is likely utilized for ber in e. cuniculi, as sharing non-specialized enzymes between pathways is not uncommon in s. cerevisiae, and there is no reason to believe that this is not the case in e. cuniculi. nucleotide excision repair ner is used primarily to remove bulky lesions from dna, such as inter- and intra-strand crosslinks. ner is a more complex process than ber, and utilizes a large number of proteins that are evolutionarily conserved among eukaryotes. ner is comprised of two subpathways, global genome repair and transcription-coupled repair. as is suggested by their names, the subpathways act on different types of dna: dna that is not transcribed, and actively transcribed dna, respectively. in both ggr and tcr, dna damage recognition is the first step to occur, followed by dna unwinding. next, incisions are made on either side of the aberrant base, and a total of 25– nucleotides on either side are removed as a single strand. the gap is then filled by dna polymerase and sealed by dna ligase. recruitment of five multi-protein complexes, nucleotide excision repair factors through and the replication protein a complex, is believed to take place in a stepwise manner to complete this process. the rpa complex is composed of rpa and rpa and recognizes damaged dna. in ggr, the first protein complex to arrive at the damaged site is nef which recognizes damage and is composed of the proteins rad and rad rad binds the nef complex, recruiting it to the damaged site and increasing dna binding efficiency. the presence of nef is not strictly required for the recruitment of nef to the dna lesion, but facilitates the process. the above proteins do not act in the other sub-pathway; in tcr, initiation of repair takes place when an rna polymerase stalls. two proteins involved specifically in tcr, rad and rad also participate in the beginning of this process. in both ggr and tcr, nef and nef are the next components to be recruited, and are held at the damage site by nef nef is composed of rad rad and rad while nef is composed of rad and transcription elongation factor iih. tfiih contains the rad rad ssl tfb tfb and tfb proteins, and provides the single strand dna helicases required for repair proteins to access the damaged site. rad and rad form a heterodimer that acts as a single strand endonuclease at the 5' end of the stretch of damaged dna and rad is a single strand endonuclease that cuts at the 3' end. rpa is thought to be the last player to arrive at the scene. many of these proteins also have roles in other cellular processes, such as recombination and transcription, therefore mutants express defects in several pathways. for a comprehensive review of ner, see prakash and prakash. most proteins participating in ner are present in e. cuniculi, with two exceptions. half of a ggr heterodimeric damage sensor complex and the tfb subunit of tfiih appear to be absent. rad appears to have diverse functions within the cell, ranging from dna repair to the regulation of a cell-cycle checkpoint and protein degradation. specifically, this protein helps to prevent the degradation of rad as well as serving a role with the 26s proteasome in regulating the ner pathway. deletion of tfb in s. cerevisiae is lethal, likely due to loss of function in transcription. the presence or absence of rad and rad were not confirmed, as blast and psi-blast searches using s. cerevisiae and s. pombe sequences as queries did not return homologues from most animals or other eukaryotes besides fungi. methyltransferase repair methyltransferases are present in both eukaryotes and prokaryotes and remove certain dna lesions involving methylation. these proteins irreversibly relocate methyl groups from dna to their own cysteine residues, and are therefore suicide enzymes. e. cuniculi does not possess the methyltransferase found in other eukaryotes, mgt deletion of this gene is not lethal in s. cerevisiae. mismatch repair in mmr, mismatches are recognized by the heterodimers mutsα and mutsβ. single base mismatches are recognized by mutsα and insertion/deletion loops less than about nucleotides in length are recognized by mutsβ. both mutsα and mutsβ can recognize a single unpaired nucleotide. pcna is also involved in mmr, perhaps assisting in these initial recognition steps. mutlα binds mutsα and β and allows them to efficiently bind to idls and mismatches. the exonuclease exo then excises the mismatched base and a dna polymerase and dna ligase fill and seal the gap. it should be noted that the proteins required for the mmr process differ among eukaryotes. for instance, drosophila and caenorhabditis lack msh homologues, and therefore do not require them for the removal of idls. schizosaccharomyces does possess a msh homologue, but it appears to play a different role within the cell, instead participating in recombination. the majority of s. cerevisiae mmr proteins are present in e. cuniculi. the sole missing protein is msh a subunit of the mutsβ heterodimer that recognizes small idls. deletion of this gene in s. cerevisiae is not lethal. homologous recombination repair hrr is the major form of double strand break repair utilized in yeast. a double stranded break is recognized by damage recognition proteins, and single stranded overhangs are generated at both sides of the break. a region of the genome that is homologous to the single stranded overhangs is then found. strand invasion follows, and the homologous dna is used as a template for synthesis on the broken strand. hrr is completed through re-annealing of the broken dna strand and ligation. see figure for an overview of this process. the rad and proteins perform most steps of the hrr process. rad is a homologue of the bacterial enzyme reca, and is well conserved within eukaryotes. when a double strand break is formed, the mrx complex is involved in damage recognition. the dna ends on either side of the break are then chewed back in the 5' to 3' direction by an unknown nuclease. rad is also involved in end processing. the results of this process are short 3' overhangs on either strand. rpa then coats the overhangs. rpa is later replaced by rad with the aid of rad rad55/rad and very likely rad as a genome-wide search for homologous sequences takes place. strand invasion then occurs while the helicase hpr removes rad from the dna. dna is synthesized by an undetermined polymerase based on the donor template strands, and then ligated. although the mechanism is not clear, it is evident that the rad55/rad complex is somehow involved in this last step. the sgs helicase plays a specific role in the repair of double strand breaks generated by the stalling of a replication fork. for a review of hrr, see aylon and kupiec. three other signaling and damage sensor proteins are also involved in the hrr pathway, as well as the ber pathway and the nhej pathways. the rad17/med3/ddc complex triggers dna damage checkpoints, and stimulates repair pathways as well as various individual repair proteins, including dna polymerase β, rad, rad and cdc. e. cuniculi lacks more than half of the proteins involved in the hrr pathway. almost all steps of the process are affected by these losses. missing proteins include the hpr helicase, rad and rdh. rad and the 9-1- complex are all absent from the cell signaling pathways. s. cerevisiae single mutants lacking these proteins are viable, likely due to yeast's ability to use either double strand break repair pathway to fix damaged dna. the presence or absence of rad and rad was not determined. rad and rad are paralogs of rad psi-blast searches using s. cerevisiae rad and rad proteins retrieve rad in other fungi, therefore making it difficult to discern the presence of these proteins in e. cuniculi, which is related to fungi. non-homologous end joining repair nhej is the second form of double strand break repair that is a separate, though not completely independent pathway from hrr. in s. cerevisiae this method of double strand break repair plays a minor role compared to the hrr pathway. upon double strand break formation, damage is recognized and both ends of the lesion are brought together through the action of several proteins. a minimal amount of dna synthesis occurs, which is followed by ligation. as dna on either side of the break may be degenerated before the break is repaired, the potential for information loss in this case is substantial. the nhej process begins when the ku complex binds either end of the double strand break. these proteins are dna-dependent protein kinases that also have a role in telomere maintenance. once bound to the damaged site, the ku complex is responsible for recruiting the mrx complex for the next stage in the repair process. the mrx complex is composed of rad, mre and xrs. dnl4/lif is tethered to the break site by xrs and the ku complex. the dna polymerase pol and the structure-specific nuclease rad are the last players to arrive at the scene, thus completing the repair complex. all of the yeast nhej proteins are present in most eukaryotes, and the core of ku and is homologous to a smaller bacterial protein that performs the same function, thus indicating a large degree of conservation. for a review of this process, see hefferin and tomkinson. e. cuniculi is missing nearly all nhej proteins. absent proteins include ku ku xrs dnl and pol. as is the case with single s. cerevisiae mutants for genes involved in the hrr pathway, most are viable due to yeast's ability to rely on the other double strand break repair pathway. although there are animal homologues of lif and xrs, blastp and psi-blast searches using yeast proteins did not retrieve homologues in any organisms other than fungi. the presence or absence of these proteins is therefore not known. dna polymerases dna polymerases are essential for both genome replication and repair. there are several polymerases present in eukaryotic cells, all of which serve particular functions within the cell. the polymerases α, δ and ε act in the process of genome replication, but also play roles in certain repair processes, notably ner and hrr. polymerase γ acts solely within mitochondria, while all other polymerases are nuclear. polymerase β is a specialized repair polymerase that is involved in ber and nhej. the polymerases ζ, η and rev help prevent double stranded dna breaks from forming during replication due to their ability to synthesize dna through a lesion, where polymerases α, δ and ε stall and dissociate from the replication fork. of the polymerases identified in s. cerevisiae that have human counterparts, e. cuniculi possesses 3: α, δ and ε. all three of these polymerases are necessary for viability in s. cerevisiae. all of the polymerases that are absent in e. cuniculi are utilized solely for repair or lesion bypass and are not essential for viability, likely because their function is replaced by other polymerases. discussion in general, the consequences of genome reduction on dna repair in e. cuniculi are most evident in the double strand break pathways. the single strand repair pathways have been less affected, but some are operating at a level of reduced complexity compared to s. cerevisiae. reduction in complexity of dna repair e. cuniculi's ber pathway lacks the dna ligase cdc dna polymerase β and mms although deletion of cdc is lethal in s. cerevisiae, the role of this protein is likely filled by another ligase. this is not unusual in s. cerevisiae, as several enzymes are sometimes able to act on the same substrate. for example, in the hrr pathway, the polymerase and nuclease have not yet been defined, likely because different combinations of polymerases and nucleases are capable of performing the required functions of this pathway. the absence of dna polymerase β could indicate that most ber in e. cuniculi is carried out via the long patch pathway, where dna is synthesized by the polymerases δ and ε. the use of one ber pathway over another is common in eukaryotes; studies have indicated that in yeast, long patch ber is carried out preferentially instead of short patch ber, whereas in humans, the reverse is true. the absence of mms is not likely to have serious ramifications for ber in e. cuniculi. the mus81-mms endonuclease processes 3' ends of nicked dna to prepare for dna synthesis. however, its role is predicted to be minor, and somewhat overlapping with that of the rad1-rad endonuclease, which is present. the ner pathway is missing a core tfiih component and the rad subunit of the rad4/rad damage recognition complex. tfiih is composed of a ring containing the three tfb proteins, which serve to tether the functional parts of the complex: the helicases rad and rad. since transcription must occur in e. cuniculi, it is difficult to predict exactly how the absence of these proteins would affect this organism, as deletion of tfb is lethal in s. cerevisiae. complete absence of this protein is difficult to reconcile with the tfb ring's essential functions, as well as the presence of the two other ring components. however, it is not unreasonable to assume that the absence of tfb would likely lead to a reduction in the efficiency of this repair process, particularly when rad also appears to be absent. e. cuniculi also lacks msh which interacts with msh to form mutsβ, which recognizes insertion or deletion loops in the mmr pathway. in s. cerevisiae, deletion of msh is not lethal, but mutants are slightly more prone to frameshift mutations. although the mutsβ heterodimer is present in s. cerevisiae, schizosaccharomyces, humans and arabidopsis, its presence is not ubiquitous among eukaryotes. drosophila and caenorhabditis lack msh where it appears that the mutsα complex is able to recognize both mismatches and insertion or deletion loops. drosophila and caenorhabditis are able to effectively perform mmr in the absence of msh which is the sole missing protein in e. cuniculi. therefore, it is very likely that this pathway operates in e. cuniculi in a similar manner to these organisms, whose mmr systems are fully functional. the absence of the dna methyltransferase mgt suggests that e. cuniculi is able to employ other methods to remove o6-methylguanine from its dna. in the bacterium escherichia coli, o6-methylguanine can be removed by both the ner and the methyltransferase mechanisms, therefore it is likely that e. cuniculi has simply dispensed with one of two parallel pathways. dna polymerases and repair eukaryotes and prokaryotes possess many specialized dna polymerases to accomplish specific tasks within the cell. some of these polymerases are involved in genome replication, while others act solely in repair processes. e. cuniculi possesses only three dna polymerases of the present in s. cerevisiae. all of these polymerases are involved in standard genome replication, while polymerase δ also plays a role in ber, ner, mmr and in bypassing dna lesions. polymerase ε is required for ber and probably ner. polymerases α, δ and ε are all likely utilized in hrr. e. cuniculi lacks polymerase β, which is utilized in a variety of repair pathways and polymerases ζ and η, which are used for error-prone and error-free dna synthesis across lesions, respectively. e. cuniculi also lacks the mitochondrial dna polymerase γ. s. cerevisiae mutants lacking polymerase β display a high frequency of recombination and sensitivity to methyl methanesulfonate. rev mutants display decreased revertibility, while polymerase η mutants have a heightened sensitivity to uv radiation. conversely, polymerase ζ deletion mutants resist uv mutagenesis. cells lacking polymerase γ lose their mitochondrial dna, however microsporidian mitochondria are highly reduced and it is unlikely that they possess autonomous dna. the phenotype of a s. cerevisiae cell lacking several polymerases is not known, but one could speculate that such cells would display a higher frequency of double stranded dna breaks generated during replication due to a lack of translesion polymerases. double strand break repair in e. cuniculi the fact that most of the nhej repair proteins appear to be absent in e. cuniculi is perhaps not overly surprising, as this method of double strand break repair appears to be a back-up method in yeast. e. cuniculi's genome is known to be highly reduced compared to that of s. cerevisiae. therefore, it seems logical that the first genes to be deleted from a genome undergoing reduction would be those encoding proteins that act in back-up pathways. of key interest is the lack of ku proteins in e. cuniculi. these proteins play a pivotal role in nhej; they are involved in recognizing double strand break sites and in recruiting other repair factors to the break site. not only is their function key, but they are present in archaebacteria, bacteria and eukaryotes. the core of the ku proteins is largely conserved from prokaryotes to eukaryotes. however, the absence of these proteins in e. cuniculi is not entirely unique, as we were also unable to identify ku proteins in the genome of the human parasite plasmodium, nor has it been recognized in trichomonas. dispensing with a backup double strand break repair pathway during genome reduction would stand to reason if the primary repair pathway was retained, however, this is also highly questionable. e. cuniculi also lacks over half of the hrr proteins that are present in yeast. the dna helicase hpr rad rdh and the checkpoint/dna end-processing rad are among the proteins that appear to be absent from the hrr pathway. hpr plays a cryptic role in hrr, as s. cerevisiae deletion mutants have hyperrecombination phenotypes, and the protein was therefore assumed to be a negative regulator of the process. however, recent work by aylon et al. has shown that hpr is intimately involved in commitment to gene conversion, which must take place before recombination can occur. rdh is a rad homolog that participates in interhomologue gene conversion and meiosis, while rad is a chromatin remodeling protein that has been implicated in strand invasion and the removal of repair proteins from dna after hrr has taken place. in addition to functioning as a checkpoint protein, rad also plays a role in the resection and recombination processes. it is possible that the functions of rad and rad are carried out by rad as all three proteins are homologues of the bacterial protein reca. this is a distinct possibility, as rad and rad appear to act in concert with rad during the hrr process. although the rad rad and sgs proteins are present in e. cuniculi, it is not known whether hrr can take place in the absence of all other hrr components. it is difficult to imagine this process occurring in the absence of dna resection, strand invasion and gene conversion. therefore, e. cuniculi appears to have drastically reduced both mechanisms for double strand break repair. although e. cuniculi's genome contains very few duplicate genes to use as templates for dna synthesis in hrr, both s. cerevisiae and mammals prefer to use sister chromatids rather than homologous sequences for this process. as e. cuniculi is likely diploid, it is reasonable to assume that this preference would exist in this organism as well. given that such a large number of genes involved in both double strand break repair pathways are absent, it is curious that some of these genes have been retained. when one looks closely at the functions of these genes, it is evident that they all play roles in other critical biological processes. mre and rad both members of the mrx complex, are also involved in telomere maintenance and the generation of meiotic double strand breaks. rad is a nuclease that is implicated in the processing of okazaki fragments during replication. all of the proteins belonging to the hrr pathway that are present in e. cuniculi are also involved in meiosis. although sexual reproduction has not been observed in e. cuniculi, it does contain three of the seven core meiosis-specific genes, as discussed in ramesh et al., and there is evidence that it may possess a mating type locus. sexual reproduction has also been observed in numerous other microsporidia, therefore there is little reason to suspect that e. cuniculi is an exception. as a large number of proteins involved in the hrr pathway are absent in e. cuniculi, the repair functions of the remaining proteins are unknown. it is possible that they have been retained because of their role in meiosis. potential consequences for e. cuniculi reductions within the dna repair pathways have led to two fundamentally different outcomes: reduced complexity by loss of a few proteins and drastic losses of half or more proteins involved in a pathway. although an organism may be able to tolerate a somewhat sloppy repair system, it is difficult to imagine how the organism could exist without any means to mend double-strand dna breaks, especially given their frequency during meiosis and mitosis. e. cuniculi must, therefore, utilize some other form of double strand break repair, or contain such highly divergent copies of most nhej and hrr proteins that they were impossible to identify in this study. along with many of the proteins that carry out the work of repair, e. cuniculi has lost several proteins that participate in cell signaling and cycle control. the 9-1- signaling complex is absent, which has been proposed to play a role in the signaling cascade leading to cell cycle arrest and apoptosis. both the ku and mrx complexes are also involved in cell cycle control, although their roles are not well defined. loss of coordination of cellular activities could result from the absence of these proteins. in addition to their role in repair, the ku proteins protect telomeres from degradation and help to control telomerase activity. as e. cuniculi houses eleven chromosomes that contain telomeres, and encodes the catalytic subunit of the telomerase enzyme, this organism must have developed an alternate method to maintain its telomeres, or it would suffer extreme telomere attrition. like the ku proteins, the dna ligase cdc performs several functions as well. it plays a role in recombination and in the ligation of okazaki fragments during replication, therefore, it is possible that these processes are somewhat impaired. the reduction that is observed within the dna repair pathways is similar to that observed throughout e. cuniculi's genome, as this organism lacks many proteins that participate in diverse biosynthetic pathways. in this way, the genome of e. cuniculi is very similar to those of many endosymbiotic and parasitic bacteria. buchnera aphidicola has also lost many dna repair genes during the process of genome reduction; indeed it has been proposed that it was this lack of dna repair genes that allowed buchnera's genome to become so small in the first place. we cannot rule out the possibility that our bioinformatics tools were unsuccessful in locating highly divergent proteins that act in the dna repair processes. it is also possible that in some cases, other non-homologous proteins carry out essential functions to replace absent proteins. such proteins may still be identified, as roughly half of e. cuniculi's genome consists of hypothetical proteins. another potential explanation for this lack of biosynthetic machinery is that e. cuniculi is able to import many of the products of these pathways from the host's cytoplasm. however, it seems unlikely that this would be the case for dna repair proteins, as protein uptake has not been documented in microsporidia, and these proteins would have to be targeted to the nucleus in order for them to function. for the moment, double strand break repair in e. cuniculi will remain a mystery.  our survey of e. cuniculi's dna repair genes indicates that the process of genome reduction has affected all major dna repair pathways. all of the single strand repair pathways have lost at least one component, indicating that these pathways are less complex than in s. cerevisiae, and could be less efficient. all replicative dna polymerases are present in e. cuniculi, although the specialized repair polymerases are absent. the absence of these enzymes could lead to inefficient dna damage repair and creation of double stranded dna breaks that are not easily repaired. surprisingly, more than half of the proteins participating in both double strand break repair pathways and the sole component involved in methyltransferase repair are absent in e. cuniculi. the proteins that remain are all involved in additional cellular functions. 
a loss of these mechanisms invariably results in accumulation of mutations and/or cell death. 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 from diverse biological pathways, including those that act in dna repair. 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 process of genome reduction has affected the dna repair pathways. e. cuniculi lacks of the dna repair genes sought via blastp and psi-blast searches. six of dna polymerases or polymerase subunits are also absent in e. cuniculi. given the large number of dna repair genes that are absent from the double strand break repair pathways, e. cuniculi is a prime candidate for the study of double strand break repair with minimal machinery. strikingly, all of the double strand break repair genes that have been retained by e. cuniculi participate in other biological pathways. the absence of genes is not distributed equally among the different repair pathways; some pathways lack only one protein, while there is a striking absence of many proteins that are components of both double strand break repair pathways. six major pathways of dna repair in eukaryotes include: non-homologous end joining, homologous recombination repair, mismatch repair, nucleotide excision repair, base excision repair and methyltransferase repair. dna repair is essential to any living cell. dna polymerases are also critical players in dna repair processes. all specialized repair polymerases are also absent.
 encephalitozoon cuniculi is a member of a distinctive group of single-celled parasitic eukaryotes 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 from diverse biological pathways, including those that act in dna repair. dna repair is essential to any living cell. a loss of these mechanisms invariably results in accumulation of mutations and/or cell death. six major pathways of dna repair in eukaryotes include: non-homologous end joining, homologous recombination repair, mismatch repair, nucleotide excision repair, base excision repair and methyltransferase repair. 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 process of genome reduction has affected the dna repair pathways.  e. cuniculi lacks of the dna repair genes sought via blastp and psi-blast searches. six of dna polymerases or polymerase subunits are also absent in e. cuniculi. all of these genes are relatively well conserved within eukaryotes. the absence of genes is not distributed equally among the different repair pathways; some pathways lack only one protein, while there is a striking absence of many proteins that are components of both double strand break repair pathways. all specialized repair polymerases are also absent.  given the large number of dna repair genes that are absent from the double strand break repair pathways, e. cuniculi is a prime candidate for the study of double strand break repair with minimal machinery. strikingly, all of the double strand break repair genes that have been retained by e. cuniculi participate in other biological pathways. 