Unité de Génétique Moléculaire des Levures (URA 2171 CNRS, UFR 927 Université Pierre et Marie Curie), Institut Pasteur, Paris cedex, France
Correspondence: E-mail: gfrichar{at}pasteur.fr.
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Abstract |
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Key Words: hemiascomycete comparative genomics replication repair recombination
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Introduction |
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Materials and Methods |
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Phylogenetic Analyses
Multiple alignments of amino acid sequences were performed using T-coffee (Notredame, Higgins, and Heringa 2000). Gaps and poorly aligned sequences were excluded from alignments using Gblocks (Castresana 2000). Tree reconstruction was performed by the maximum likelihood algorithm as implemented in PHYML (Guindon and Gascuel 2003). The substitution process was modeled by the Jones, Taylor, and Thornton (JTT) model, the heterogeneity of substitution rates among sites was modeled by a gamma distribution, with four categories and a parameter estimated from the data set. Tree topology and support of internal branches were inferred by 500 bootstrap calculations. Calculations of the nonsynonymous/synonymous substitution rate ratio ( = Dn/Ds) were performed with the maximum likelihood method (Goldman and Yang 1994) implemented in the PAML package version 3.14 (Yang 1997).
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Results |
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High Conservation of Genes Involved in S-Phase Replication
It is not surprising that almost all the proteins playing a role in chromosome replication (Burgers et al. 2001) are conserved throughout the hemiascomycete evolution. One notable exception however, is Rfa3p, one of the tripartite components of the yeast single-strand binding protein complex, which is not detected in D. hansenii and Y. lipolytica, the two other members (Rfa1p and Rfa2p) of the same heterotrimeric complex being found. No RFA3 gene relic was found in these two species. In addition, the gene encoding Dna2p, involved in processing Okazaki fragments, contains three in-frame stop codons in Y. lipolytica and thus is most probably not properly translated. Dna2p function is at least partly redundant with Rad27p, suggesting that Rad27p is necessary and sufficient to process Okazaki fragments in Y. lipolytica, or that the translated N-terminal part of the Dna2 protein is sufficient to carry out its essential function.
TOP1 and SGS1 are specifically duplicated in C. glabrata (table 1). The two copies of C. glabrata Top1p (CAGL0E02431g and CAGL0J11660g, supplementary table) are almost perfectly aligned with ScTop1p, except in the N-terminal part of the protein. Synteny shows that CAGL0E02431g is the correct orthologue, the other copy being present in a duplicated chromosomal block present in both S. cerevisiae and C. glabrata (G. Fischer and B. Dujon, unpublished data). The duplicated copy was conserved in C. glabrata but not in S. cerevisiae in which no trace of a pseudogene or a relic could be found in the duplicated block (I. Lafontaine and B. Dujon, unpublished data). Consistent with that, the phylogenetic tree shows that CAGL0E02431g is the closest homologue of ScTop1 (fig. 1A). Calculation of synonymous (Ds) and nonsynonymous (Dn) substitutions show that Ds values are very high (Ds > 5). Because synonymous sites are saturated, Dn/Ds ratios are not a reliable measure of evolutionary rates. Hence, we took in consideration only Dn values. They are low and similar for both paralogues (DnCAGL0E02431g = 0.22; DnCAGL0J11660g = 0.18, as compared to S. cerevisiae). This suggests that both genes have evolved at a similar rate and have both probably retained their catalytic activity. Two copies of Sgs1p were found in C. glabrata (CAGL0L00407g and CAGL0H00759g). According to synteny results, CAGL0L00407g is the correct orthologue, and CAGL0H00759g is found in a duplicated block in S. cerevisiae and C. glabrata only (G. Fischer and B. Dujon, unpublished data). Like previously, the copy in the duplicated block has been erased, and no trace of a pseudogene or relic can be detected in S. cerevisiae. (I. Lafontaine and B. Dujon, unpublished data) The phylogenetic tree shows that the closest homologue of ScSgs1p is CAGL0L00407g, the other copy being more diverged (fig. 1B). Interestingly, the duplicated copy is shorter than the orthologue. It is deleted for the N-terminal part containing the Top3-binding domain and the C-terminal part containing the DNA-binding domain of the Sgs1 protein (fig. 2). Again, synonymous sites are saturated, but Dn values are low. However, the Dn value of the duplicated copy is higher than that of the orthologue (DnCAGL0L00407g = 0.2; DnCAGL0H00759g = 0.46, as compared to S. cerevisiae), meaning that not only the copy lost two important parts of the protein (still retaining the helicase motif) but also the remaining part diverged more rapidly.
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Weak Conservation of Genes Involved in Meiotic Recombination
HR during meiosis is a highly regulated process by which genetic information is reshuffled between homologous chromosomes (for review see Zickler and Kleckner 1998). During this process, DSBs are generated by the Spo11p topoisomerase and then processed by the meiotic recombination machinery involving the Mre11p-Rad50p-Xrs2p complex. Spo11p is homologous to the A subunit of type VI topoisomerases, such as those found in archaebacteria (Bergerat et al. 1997). Homologues to SPO11 are found in all four yeast species, despite extensive sequence divergence. The Tyr135 residue essential for its catalytic activity is conserved, strongly suggesting that the four orthologues are functional in vivo (fig. 3B). The occurrence of crossovers is also regulated during meiosis, although little is known about the precise mechanism by which a recombination intermediate is resolved as a crossover or as a noncrossover, in vivo. It involvesat leasttwo different pathways: the Msh4-Msh5 pathway and the Mus81-Mms4 pathway. Msh4 and Msh5 proteins function as heterodimers in S. cerevisiae, and the corresponding mutants show a reduced frequency of meiotic crossovers as compared to wild-type strains (Pochart, Woltering, and Hollingsworth 1997). The msh5 mutant is profoundly affected at an early stage during meiotic recombination, showing a decreased level of early recombination intermediates leading to crossovers (Börner, Kleckner, and Hunter 2004). Their simultaneous absence in D. hansenii and Y. lipolytica might reflect a different mechanism to control crossovers in these species. The S. cerevisiae Mus81-Mms4 complex is able to process branched structures arising during mitotic or meiotic replication/recombination that are not canonical Holliday junctions (Fricke, Bastin-Shanower, and Brill 2005). Mus81p is conserved in all species, whereas Mms4p was not found in Y. lipolytica. However, although Mus81p is known to be conserved throughout evolution, its partner is poorly conserved (Ögrünç and Sancar 2003). It is therefore possible that a functional homologue of Mms4p is also present in Y. lipolytica but not detected.
The other genes involved in the meiotic recombination pathway are most of the time poorly conserved in D. hansenii and absent in Y. lipolytica, with the exception of MRE2, whose product is involved in the splicing of MER2 and MER3 messenger RNAs in S. cerevisiae. MER2 is predicted to contain an intron only in C. glabrata. Mre2p belongs to the U1 snRNP in S. cerevisiae and therefore splices many transcripts other than those involved in meiosis. It is thus probable that the MRE2 gene does not play a role anymore in meiotic recombination in K. lactis or D. hansenii. In conclusion, the only genes that are found in all five yeast species are genes involved in initiating recombination by making and processing DSBs (SPO11, MRE11, RAD50) in resolving recombination intermediates (MUS81) or the general splicing factor MRE2.
Checkpoint Proteins
Signaling DNA damage during the cell cycle is regulated by a series of proteins that activate the so-called "checkpoints" (for review see Zhou and Elledge 2000). Most of them are conserved except in Y. lipolytica. RAD9 is the only gene that is missing in D. hansenii in addition to Y. lipolytica. Most probably, DDC2, which has a human functional homologue (ATRIP, table 1), is also conserved in Y. lipolytica but is too diverged to be recognized.
Conservation of DNA Maintenance Pathways During Evolution
Given that some of the pathways are very well conserved in the five hemiascomycetous yeasts (e.g., replication or NER proteins) and others are missing several components, we wanted to know if amino acid conservation was the same among the different pathways. We performed pairwise Smith-Waterman alignments between each S. cerevisiae protein and its putative orthologues. Percentages of identity are shown in figure 4 for each species in each pathway. The average identity for each pathway was also calculated. Note that proteins have been classified in a pathway according to one of their functions, although some of them act in several distinct pathways. The best example is the MRE11-RAD50-XRS2 complex, classified in the NHEJ pathway, but which is known to be involved in formation and processing of meiotic DSBs, S-phase checkpoint activation, and HR (for review see Haber 1998). Nevertheless, when amino acid conservations of each pathway are compared, they generally follow the phylogenetic tree, i.e., C. glabrata is the closest to S. cerevisiae and Y. lipolytica is the farthest (fig. 4). The only exception is the meiotic recombination pathway, in which the only three genes to be conserved in Y. lipolytica (MRE2, MUS81, and SPO11) show a higher identity to S. cerevisiae orthologues than the corresponding D. hansenii, K. lactis, and C. glabrata genes. In order to determine if evolutionary rates were similar in the five species for these three genes, we calculated the Dn and Ds rates of nonsynonymous and synonymous substitutions. Because informative sites are saturated (Ds > 5), we took into consideration only Dn values. Using this criterion, we confirmed that SPO11 and MRE2 (but not MUS81) evolved slower in Y. lipolytica (DnSPO11 = 0.76; DnMRE2 = 0.69, as compared to S. cerevisiae) than in D. hansenii (DnSPO11 = 0.98; DnMRE2 = 0.73, as compared to S. cerevisiae). As a control, we also determined the average level of amino acid conservation between all S. cerevisiae proteins and their orthologues in each of the four species and used it as a baseline (see Materials and Methods). As expected, conservation follows the phylogenetic tree, i.e., C. glabrata proteins share a higher percentage of identity with S. cerevisiae proteins (60%) than Y. lipolytica proteins (50%). Hence, the only pathway in which proteins reach the amino acid identity baseline in each species is the replication pathway; most of the others (and all of them in Y. lipolytica) are below the baseline. This suggests that proteins involved in pathways whose average amino acid conservation is under the baseline diverge more rapidly than the average orthologous proteome, perhaps reflecting more flexibility in proteins involved in repair and recombination than in proteins involved in replication.
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Discussion |
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Conservation of Pathways
Pathways have been defined arbitrarily because many proteins belong to several pathways and therefore all pathways are interconnected with each other. However, despite such interconnection, some pathways such as meiotic recombination and checkpoints are less conserved than others, such as replication and NER. There are two independent criteria that may be used to estimate the conservation of pathways. The firstthe presence/absence criterionis used to determine the ratio of genes that are found in each species over the total number of genes in this pathway (table 1). The secondthe conservation criterionis used to calculate the average conservation in amino acid of proteins belonging to a given pathway for each species (fig. 4). Not surprisingly, the replication machinery comes first using both criteria, and almost all genes are present in each species and exhibit a high level of similarity with S. cerevisiae genes (table 1 and fig. 4). The NER pathway is very well conserved (all the genes are found in each species), but amino acid conservation is lower in K. lactis, D. hansenii, and Y. lipolytica than for proteins belonging to the HR pathway, in which many accessory proteins are not found in the more distant species (table 1 and fig. 4). In terms of presence/absence, the meiotic recombination machinery is missing several members, even in species related to S. cerevisiae. Most of the genes that are not found in C. glabrata and K. lactis belong to this pathway (table 1). We found that, in general, proteins interacting with DNA are more conserved than structural proteins, proteins that are part of a scaffold and other cofactors. It is striking that Rad51p and Dmc1p catalyzing strand exchange reactions are the most conserved of their respective pathways. Similarly, the Mre11p-Rad50p complex and Spo11p, necessary to make and process meiotic DSBs, are conserved along with Mus81p, involved in resolving recombination intermediates. All these proteins interact directly with DNA and are much more conserved than proteins involved in making the synaptonemal complex or other structural proteins and cofactors.
Gene Duplications During Evolution
Paralogous sets of genes play a key role in defining functional biological systems. For example, the MutS family of proteins contains six members in S. cerevisiae (MSH1-6, table 1), with distinct functions and specializations. Another example is the replicative helicase, formed by assembly of six distinct subunits, encoded by six different genes (MCM2-7), arising from successive gene duplications during evolution. In the present work, we found that both SGS1 and TOP1 were duplicated in C. glabrata and that SRS2 is tandemly duplicated in K. lactis. SGS1 encodes a DEAD/DEAH helicase of the RecQ/BLM/WRN family and has been shown to interact genetically with Top3p (Gangloff et al. 1994) and Top1p (Tong et al. 2001) and physically with Top2p (Watt et al. 1995). The duplicated Sgs1p and the duplicated Top1p both arose from duplication events prior to the S. cerevisiaeC. glabrata speciation, and both duplicated genes have been conserved in C. glabrata and lost in S. cerevisiae (fig. 1D). Given that Dn values are rather low for both genes, it is probable that both duplicated proteins are under selection pressure in C. glabrata. This could imply being part of an alternative complex involved in replication and/or repair or being part of a Sgs1-containing complex that would be specific to the life cycle of this pathogenic yeast. Interestingly, the duplicated copy of Sgs1p lacks its N-terminal and C-terminal parts (fig. 2) but retains the central helicase domain. It is therefore possible that it lost its DNA-binding activity but is still active as a helicase, maybe as part of a multicomponent complex. In humans, there are five homologues of Sgs1p, and two of them (RecQ5 and RecQL, fig. 2) are shorter versions, lacking either the N-terminal part (RecQL) or both the N- and C-terminal parts (RecQ5) but retaining their helicase domain. It is interesting that in C. glabrata, a short copy of Sgs1p was also found. We performed local and global alignments between the Sgs1p copy and the five human orthologues and concluded that although being a shortened version of Sgs1p, the C. glabrata copy is closer to WRN, BLM, and RecQ4 (RTS) than to the human RecQ5 and RecQL helicases. We therefore concluded that evolution of this protein family in C. glabrata and man was different. The Top1p duplication is interesting because this gene is duplicated in vertebrates but not in S. cerevisiae, Schizosaccharomyces pombe, or plants (Zhang et al. 2004). In vertebrates, one gene product is addressed to the nucleus and the other to mitochondria (Zhang et al. 2001). In C. glabrata, the duplicated copy (CAGL0J11660g) is predicted to encode a nuclear product, but no obvious nuclear nor mitochondrial addressing signal could be found in the original gene (CAGL0E02431g; Y. Pommier, personal communication). However, we know that the S. cerevisiae orthologue functions in the nucleus. Hence, this suggests that both gene products in C. glabrata are nuclear, and therefore that the evolution of this protein family in C. glabrata and in vertebrates was also different. It was previously shown that the very conserved lysine residue (K41) in Srs2p was essential for the adenosine triphosphatase (ATPase) activity (Krejci et al. 2004). This residue is present among the five species in the center of the completely conserved motif 35G36P37G38T39G40K41T42K43. In addition, in the duplicated copy of SRS2 in K. lactis, this motif is also completely conserved, suggesting that all the Srs2p orthologues are functional in the four other hemiascomycetes. The Hmi1p helicase is a paralogue of Srs2p in S. cerevisiae. Hmi1p is a mitochondrial protein and is essential for maintenance of mitochondrial DNA (Sedman et al. 2000). It was found in all species except Y. lipolytica, and the phylogenetic tree shows that the duplication of the SRS2/HMI1 gene ancestor occurred in the common ancestor to S. cerevisiae and D. hansenii (fig. 1C and D). Consistent with this observation, the conserved ATPase motif in Hmi1p only differs by one amino acid (Thr39 Ser39) from Srs2p. This conservative mutation is found in all four species in which a HMI1 orthologue is detected, strengthening the idea that the formation of paralogues occurred before speciation of our yeasts. Finally, it was shown that the C-terminal part of the Hmi1 protein contains the mitochondrial targeting signal (Lee et al. 1999). Alignments of Srs2p and Hmi1p orthologues show that both proteins are very well conserved in the N-terminal part, containing the ATPase motif, but conservation of the C-terminal part is weak. Therefore, Srs2p/Hmi1p is probably a case of gene duplication before speciation, leading after alteration of the C-terminal part of one of the duplicated copies to a specialization of function, with both proteins being DNA helicases but one addressed to the mitochondria and the other to the nucleus. Our results strongly suggest that in each case of gene duplication, both copies are probably functional and have retained their catalytic activity, although they might be active in different cell compartments and/or on different substrates (subfunctionalization) (Lynch and Conery 2000).
Pathway Conservation, Evolution, and Yeast Biological Properties
We showed that genes belonging to the meiotic recombination machinery are poorly conserved in hemiascomycete species (in terms of presence/absence). However, K. lactis, D. hansenii, and Y. lipolytica undergo meiosis (Herman and Roman 1966; Kreger-van Rij and Veenhuis 1975; Casarégola et al. 2000). This means that although most of the genes necessary to go through meiotic recombination in S. cerevisiae are not detected in other yeasts, they must have functional orthologues able to carry out similar functions. Interestingly, the most highly conserved protein of the HR pathway is Rad51p in each species, and the most highly conserved protein of the meiotic recombination pathway is Dmc1p in the three species in which it is detected (fig. 4). Because DMC1 and RAD51 presumably come from the duplication of a common ancestor, our results suggest that this duplication occurred after the divergence between Y. lipolytica and the four other yeast species. It has also been proposed that organisms undergoing meiosis can be classified in two different groups (Stahl et al. 2004). In group I, organisms do not depend on meiotic DSB-repair functions to achieve synapsis (Drosophila melanogaster, Caenorhabditis elegans, Neurospora crassa), whereas in group II organisms, synapsis may only occur if DSB-repair is functional (e.g., S. cerevisiae). In group II organisms, the DMC1, HOP2, and MND1 genes are found, whereas they are apparently absent in group I organisms. This would suggest that group I organisms have lost these three genes or that they have been independently acquired during evolution. Therefore, Y. lipolytica would be classified as a group I organism because none of these three genes is found, whereas the four other species all contain these three genes (table 1 and data not shown for MND1). In addition, the Msh4-Msh5 protein complex involved in crossover control is also missing in this yeast. Taken together, these data suggest that although Y. lipolytica undergoes meiotic recombination (Wickerham, Kurtzman, and Herman 1970; Gaillardin, Charoy, and Heslot 1973), its properties are most probably very different from the four other hemiascomycetous yeasts.
In order to determine if the differences observed among the different yeast species for the NHEJ pathway could reflect a difference in the efficiency of DSB-repair mechanisms, we irradiated haploid cells with a source of -radiation.
-Rays are known to induce single- and double-strand breaks in chromosomes, and resistance to ionizing radiations is a measure of how efficient the DSB-repair systems are in a given organism (Esposito and Wagstaff 1981). At low energy (50 Gys), the four hemiascomycetes are slightly more resistant than a haploid S. cerevisiae strain to ionizing radiations (supplementary figure). At higher doses (300 Gys), all five yeast species show the same sensitivity to
-rays. We concluded that, most probably, no gene dramatically affecting the efficiency of DSB-repair was missing in the four species. This suggests again that the NHEJ pathway is functional, despite the apparent absence of some of its members. The higher resistance at low doses may be hypothesized by the existence of a more efficient pathway, for example HR with the sister chromatid, that would occur more often in those species as compared to S. cerevisiae, perhaps because of a longer S-G2 phase of the cell cycle. Further experimentation will be required to determine if cell cycles are the same in these five hemiascomycetes.
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Supplementary Materials |
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One supplementary figure: comparison of survival to -irradiation between the five hemiascomycetes.
SUPPLEMENTARY FIGURE. Comparison of survival to -irradiation between the five hemiascomycetes. For each haploid strain, approximately 300 cells were plated on YPGlu plates and irradiated at different doses (0, 50, 100, and 300 Gy) using a 137Cs source, at a dose rate of 4 Gy/min. After 3 days of incubation at 30°C, survival was determined as the number of colony forming units (CFU) at each dose divided by the number of CFU at 0 Gy. The average of two independent experiments is shown for each species.
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Acknowledgements |
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Footnotes |
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