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An evaluation of the role of LIG4 in genomic instability and adaptive mutagenesis in Candida albicans
FEMS Yeast Research 2 (2002) 341^348
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An evaluation of the role of LIG4 in genomic instability and adaptive mutagenesis in Candida albicans Encarnacio¤n Andaluz, Toni Ciudad, Germa¤n Larriba
Departamento de Microbiolog|¤a, Universidad de Extremadura, Avda. de Elvas s/n, 06071 Badajoz, Spain Received 12 October 2001; received in revised form 21 March 2002; accepted 22 March 2002 First published online 30 April 2002
Abstract The non-homologous end-joining (NHEJ) pathway of DNA recombination is important for genomic stability in animal cells, since the absence of Ku70, Ku80, Lig4 or Xrcc4 results in non-reciprocal translocation and chromosome fragmentation. The role of LIG4 in the genomic instability of Candida albicans has been analyzed. We have found that both cell transformation and 5P-fluoroorotic acid selection steps used to obtain several lig4 mutants (LIG4/lig4 Uraþ ; LIG4/lig4 Ura3 ; lig4/lig4 Uraþ ; lig4/lig4 Ura3 ; and revertant lig4/LIG4 Uraþ ) resulted in significant alterations in chromosome R (ChrR). However, this effect is not specific for LIG4, since disruption of SHE9, a gene unrelated to recombination, also caused alterations in the mobility of ChrR. On the other hand, we could not detect reciprocal or nonreciprocal translocations between non-homologous chromosomes in several lig4 mutants. Furthermore, propagation of these mutants in rich medium did not cause other alterations in the mobility of ChrR. Adaptive mutagenesis of C. albicans, determined by the appearance of L-sorbose-utilizing mutants on L-sorbose plates, was also independent of the presence of Lig4 and occurred by monosomy of Chr5. Accordingly, the NHEJ pathway does not appear to be involved in the adaptive mutagenesis mediated by alterations in chromosome copy number. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Genomic instability ; Adaptive mutagenesis; LIG4; Candida albicans
1. Introduction Candida albicans is the most prevalent fungal pathogen of humans. As a commensal of the healthy individual, this fungus lives saprophytically in several niches of the human body, but alterations in the balance between the commensal and the host, as occurs in the immunocompromised patient, may trigger infection [1,2]. A large part of the studies on the biology of C. albicans makes reference to two important properties of the organism. First, it can grow with a variety of morphologies, including the typical yeast form, pseudohyphae, i.e. chains of elongated cells with constrictions between adjacent cells, or true hyphae, which lack constrictions at the septa [3]. Reversibility between the several forms is essential for virulence [1,2]. The second property is that C. albicans behaves as an obligate diploid. On the basis of population genetic studies, it is assumed that C. albicans reproduces primarily by clonal
* Corresponding author. Tel. : +34 (924) 289428; Fax : +34 (924) 289428. E-mail address : [email protected] (G. Larriba).
propagation [4]. However, it has been widely reported that di¡erent isolates of C. albicans exhibit clear di¡erences in heritable phenotypes, including colonial morphology, antigenic pro¢les and electrophoretic karyotypes [5^9]. In addition, the organism has the capacity to adapt to new environments, since during infection of the mucosal epithelia, dissemination via the bloodstream and colonization of internal organs, it copes with new and frequently adverse conditions. As stated above, it is clear that this variability and adaptability do not arise by meiotic recombination of two genomes from cells of di¡erent mating types. The prevailing conclusion is that the organism evolves by accumulation of point mutations, as well as, in a more £exible way, through endogenous recombination events in a heterozygous background, including mitotic recombination, gene ampli¢cation and non-reciprocal chromosomal translocations, as well as gain/loss of speci¢c chromosomes. Recombination events arise from the repair of doublestrand breaks (DSB) in DNA. These DSB can occur as a consequence of the normal cellular metabolism (although the signal that triggers their formation has not been identi¢ed), or as a result of exposure to chemical and physical
1567-1356 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 0 9 4 - 6
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agents. In order to repair DNA damage, cells have evolved a number of mechanisms that can be classi¢ed into two general pathways : homologous recombination (HR) and non-homologous end-joining (NHEJ) [10]. In HR, repair of the damaged DNA requires the presence of an intact homologous partner, and leads to accurate repair. By contrast, in NHEJ, DNA ends are joined with microhomology or no complementarity. Although the repair can be precise, more frequently it produces small insertions or deletions or even large deletions comprising hundreds of base pairs and, accordingly, it is mutagenic [10^13]. It should be noted that, although DNA repair via NHEJ is a minor pathway in Saccharomyces cerevisiae, it is the prevalent pathway for DNA repair in mammals where it is required for precise V(D)J recombination in the generation of immunoglobulins and T-cell receptors [14]. On this basis, we hypothesized that, as an obligate diploid, C. albicans could well carry out recombinational events through the NHEJ pathway whose outcome would be the arrangement of genes or even chromosomes that could help the organism to cope with the hostile conditions found in the human host [15,16]. NHEJ uses a common set of core components in yeast and mammals: these are the DNA end-binding proteins Ku70 and Ku80 (Hdf1p and Hdf2p in yeast), as well as Lig4 and its associated Xrcc4 (Lif1p in yeast) protein [10,17]. Recently, we reported a phenotypic analysis and virulence of lig4 mutants of C. albicans. These studies concluded that the absence of Lig4p results in an impairment in myceliation and an attenuation of virulence. However, LIG4 is neither essential for DNA replication nor for the repair of DNA damage induced by radiomimetics, indicating the presence of a homologous recombination system that operates under most circumstances [15,16,18]. On the other hand, recent studies in mammalian cells have concluded that the NHEJ pathway of DNA repair is a crucial caretaker of the mammalian genome, since null mutants in several components of the pathway, including Ku80, Xrcc4 and Lig4 exhibit genomic instability as well as increased chromosomal fragmentation and non-reciprocal translocations [19^21]. Accordingly, we extended our initial phenotypic analysis of lig4 mutants in order to
know whether Lig4 could also play a role in the genomic stability of C. albicans.
2. Materials and methods 2.1. Strains and growth conditions The C. albicans strains used in this study are listed in Table 1. They were routinely grown in YEPD (2% glucose, 1% yeast extract, 2% Bacto peptone) at 28‡C. The adaptative mutagenesis experiments were performed as described [7,22]. In these experiments cells were plated on L-sorbose plates (2% agar) containing 2% L-sorbose, and 0.67% Bacto yeast nitrogen base without amino acids [23] and incubated at 30‡C. For Ura3 strains, this medium was supplemented with uridine (25 Wg ml31 ). 2.2. DNA extraction and analysis Plasmids containing the disruption cassettes of HST7 and CPH1 were kindly provided by Dr. G. Fink [24]. A set of subtelomeric probes, one for each arm of the eight chromosomes of C. albicans (Table 2), was kindly supplied by Dr. B.B. Magee (University of Minnesota, St. Paul, MN, USA). Standard techniques were routinely used for the preparation and puri¢cation of plasmid DNA [25]. Genomic DNA was prepared from protoplasts. Protoplasts were obtained by incubation of cells with zymolyase in 1 M sorbitol and lysed in 50 mM EDTA and 0.6% SDS. C. albicans cells were transformed by electroporation in a BTX electroporation system. For that purpose, 40 Wl of a cellular suspension were mixed with 5 Wl of transforming DNA (less than 100 Wg). After 5 min in ice, the mixture was transferred to ice-cold electroporation cuvettes (0.2 cm). The voltage applied was 1.5 kV. Disruption of one allele of SHE9 [26] to obtain the heterozygous SHE9/she was carried out using the URAblaster technique and the CAI4 strain as a recipient [27]. One of the several SHE9/she9 Uraþ transformants obtained was then plated on 5P-£uoroorotic acid plates (FOA) to select the Ura3 segregants. Both strains were
Table 1 C. albicans strains used in this study Strain SC5314 CAF2 CAI4 CEA1 CEA15 CEA2 CEA26 CEA3 CGM1.1 CGM1.2
SC5314 CAF2 CAI4 CEA1 CEA15 CEA2 CEA26 CAI4 CAI4
Genotype or description
Source
prototrophic vura3: :imm434/URA3 vura3: :imm434/vura3: :imm434 as CAI4 but LIG4/lig4 : :hisG-URA3-hisG as CAI4 but LIG4/lig4 : :hisG as CAI4 but lig4 : :hisG-URA3-hisG/lig4 : :hisG as CAI4 but lig4 : :hisG/lig4 : :hisG as CEA3 but lig4: :hisG/LIG4 : :URA3-hisG as CAI4 but SHE9/she9: :hisG as CAI4 but SHE9/she9: :hisG
[27] [27] [27] [16] [16] [16] [16] [16] this work this work
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2.4. Studies on adaptive mutagenesis in response to L-sorbose
Chromosome
S¢I fragment
Gene
R
D M L S A U O P N H I M C O C G
INO1 ADE1 ACT1 HGT1 nd SAS2 NPS1 YER132 HIS5 YJR61 CDC1 GCD11 COX12 YMR315 nonea ARG4
1 2 3 4 5 6 7 a
343
Plasmid 321. This probe consists of 4.0 kb HindIII in pUC18.
veri¢ed by Southern blot hybridization and PCR (E. Andaluz and Garc|¤a de la Marta, unpublished results). 2.3. Sample preparation for PFGE 0.1 ml of an exponentially growing culture of C. albicans was used to inoculate 10 ml of YEPD. The culture was maintained in a rotatory shaker at 30‡C for 48 h. Cells were harvested by centrifugation, washed twice with 50 mM EDTA, pH 8.0, and resuspended in 1 ml of CPES (40 mM citric acid, 120 mM sodium phosphate, 20 mM EDTA, pH 8, 1.2 M sorbitol and 5 mM dithiothreitol) supplemented with 0.2 mg of zymolyase 20 000. 1 ml of CPE (as CPES but lacking sorbitol and DTT) containing 1% low melting point agarose at 50‡C was then added and gently mixed. Aliquots of 200 Wl were then transferred into a sample mold and kept at 320‡C. Upon solidi¢cation, plugs were transferred to test tubes, supplemented with 6 ml of CPE and incubated at 30‡C for 4 h. CPE bu¡er was replaced with 5 ml of TESP (1 M Tris^HCl, 0.5 M EDTA, 2% SDS) containing 1 mg ml31 of proteinase K and incubated overnight at 50‡C. Samples were then washed 3U with TE (10 mM Tris^HCl, 1 mM EDTA) at 50‡C and 6U at room temperature. The plugs were stored at 4‡C in 50 mM EDTA, pH 8. The gel containing C. albicans chromosomes was run in 0.6% agarose for 24 h at 80 V with a 120^300 s linear ramp and then for 48 h at 80 V with a 420^900 s linear ramp in a rotating gel electrophoresis apparatus. For Southern analysis, the electrophoresed samples were transferred to TM-nitrocellulose, probed with the indicated probes at high stringency (65‡C in 6USSC, 5UDenhardt’s solution, 0.5% SDS and then washed with 0.1USSC, 0.1% SDS). Xomat X-AR ¢lm (Kodak) was used to expose blots.
The formation of Souþ mutants by parental (LIG4) and mutant (lig4) strains was analyzed as described by Janbon et al. [7,22]. Sorbose plates were prepared using regular agar. In some experiments, scavenger S. cerevisiae cells (strain no. 865) were used to eliminate any contaminant that might be present in agar, as indicated [22], but in our hands, this modi¢cation did not result in any di¡erence in the total number of Souþ colonies. Also, to avoid possible deviations in counts due to di¡erences between individual cells, a cell mass of each C. albicans strain, previously washed twice in sterile water, was used to inoculate the sorbose plates. Two series of approximately 300 and 3000 colony-forming units (CFU) were spread in duplicate on sorbose plates for detection of Souþ colonies, and plates were incubated at 30‡C. A viability experiment was conducted plating the same cell suspension on a number of sorbose plates. Daily for 2 weeks, entire agar discs were transferred to the surface of a YEPD plate to allow viable cells to grow [22]. The ability of the selected Souþ mutants to regrow on L-sorbose plates was investigated as described [22]. Brie£y, Souþ cells from a 370‡C preserved culture were streaked on YEPD plates; smaller colonies were then suspended in distilled water and appropriate dilutions plated again on L-sorbose medium. Plates were incubated at 30‡C and inspected for the appearance of colonies.
3. Results 3.1. Chromosomal alteration of lig4 mutants Fig. 1 shows the karyotypes of wild-type SC5314 and its Ura3 derivative CAI4 as well as those of the several
Fig. 1. Electrophoretical karyotype of the indicated strains. A and B: Ethidium bromide staining. C and D : Southern blot hybridization using the disruption cassettes of HST7 (C) or CPH1 (D) as a probe.
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Fig. 3. Southern blot hybridization of the electrokaryotypes of the indicated strains using subtelomeric probes of Chr2 (A) and Chr3 (B).
Fig. 2. Electrophoretical karyotype of CAI4 and two SHE9/she9 Ura3 strains. Notice the di¡erences in the mobility of ChrR.
strains generated during the disruption of LIG4 (LIG4/lig4 Uraþ ; LIG4/lig4 Ura3 ; lig4/lig4 Uraþ ; and lig4/lig4 Ura3 ) and construction of the revertant (lig4/LIG4 Uraþ ). Ethidium bromide staining indicated signi¢cant alterations in the size of ChrR following each transformation step or selection on 5P-£uoroorotic acid (FOA) plates (Fig. 1A and B). These alterations were further con¢rmed by Southern blot analysis using the deletion cassette of HST7 (that we have located to this chromosome) as a probe. Chr3 is also labelled in wild-type SC5314, but not in its Ura3 derivative CAI4, because the deletion cassette contains the construct hisG-URA3-hisG and the URA3 gene is located in that chromosome in C. albicans. In the same way, the deletion cassette labelled Chr2 in all the lig4 single or double deletants because of the presence of hisGURA3-hisG (LIG4/lig4 Uraþ ), hisG (LIG4/lig4 Ura3 and lig4/lig4 Ura3 ) or both (lig4/lig4 Uraþ ) in the LIG4 locus in these mutants. It should be noted that in the parental strain SC5314 both homologs of ChrR exhibit di¡erent sizes, one migrating slightly faster and the other signi¢cantly slower than Chr1 (Fig. 1C). This observation is not unusual, since ChrR recombines at the level of the rDNA repeats to yield copies that can vary in size from s 4 to s 3 kb. Interestingly, the sizes of the individual R chromosomes are altered in the Ura3 derivative CAI4, where ChrR migrates as a single broad band (Fig. 1C). CAI4 was obtained by sequential deletion of both copies of URA3 present in strain SC5314, the second deletion being followed by selection on 5P-£uoroorotic acid plates [27]. On the basis of the ethidium bromide staining, it appeared that the rest of the chromosomes (1^7) had not undergone gross rearrangements. However, we further analyzed the mobility of chromosome 1 because in some
strains it migrates close to ChrR, and its unambiguous identi¢cation by ethidium bromide staining may be misinterpreted. For this purpose, we performed Southern blot analysis using the deletion cassette of CPH1 (which we have located to this chromosome) as a probe. As shown in Fig. 1D, no signi¢cant di¡erences in the size of this chromosome were seen in the di¡erent lig4 mutants, indicating that the chromosomal rearrangements are speci¢c for ChrR. Because of the involvement of LIG4 in NHEJ, we then asked whether the alterations in the size of ChrR were speci¢c for LIG4 or could be generated also during the disruption of a di¡erent gene. For this purpose, we chose SHE9 which is adjacent to LIG4 in Chr2 [26] and appears unrelated to DNA repair. Disruption of a single copy to yield the heterozygous deletant Uraþ was enough to cause changes in the mobility of the homologs of chromosome R (not shown). Moreover, selection of Ura3 heterozygotes on 5P-FOA plates resulted in a new shift in mobility of ChrR, whereas the rest of the chromosomes remained intact. Interestingly, two di¡erent Ura3 segregants yielded di¡erent patterns for ChrR, indicating that recombination between both homologs does not occur at a precise site, but is random (Fig. 2). We conclude that disruption of LIG4 does not a¡ect the mobility of chromosomes 1^7,
Fig. 4. Southern blot hybridization of the electrokaryotypes of the indicated strains using the HST7 disruption cassette as a probe. Each strain was removed from 370‡C and grown on a YEPD plate. A colony of this plate was considered the ¢rst generation. Then, it was propagated through 15 plates and a colony of the last plate considered as the n generation.
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Fig. 5. All of the Souþ colonies appeared in the ¢rst 12^14 days. Approximately 3000 cells of the indicated strain were spread on each sorbose plate. Plates were photographed after 12 days (row A) and 28 days (row B).
Chr1^7 labelled the correct chromosome (see Fig. 3 for two examples), indicating that no translocations between non-homologous chromosomes had occurred during the generation of the several mutants.
and that there is not a causal relationship between disruption of LIG4 and the changes observed in the size of both homologs of ChrR. Since alterations in the mobility of ChrR are known to be natural events resulting from unequal crossing over between homologs in the rDNA repeats (see below), we believe that the alterations seen here belong to the same category, but their frequency is enhanced by the stress conditions to which cells are subjected during both transformation and FOA selection steps.
3.3. Analysis of chromosomal translocations following propagation of lig4 mutants We next analyzed the possibility that some changes in chromosome size were only seen after several generations in the absence of Lig4. Accordingly, we propagated the parental strain SC5314 as well as the Uraþ derivatives of LIG4/lig4 and lig4/lig4 through 15 YEPD plates. Then, colonies from the ¢rst (¢rst generation) and the 15th (n generation) plates were independently processed and the electrokaryotypes obtained. Ethidium bromide staining failed to indicate gross chromosomal rearrangements and, within every strain, even ChrR seemed to remain unchanged (not shown). This was further con¢rmed by Southern blot analysis using the HST7 disruption cassette as a probe (Fig. 4). We conclude that LIG4 does not behave as a guardian or caretaker of the genome of C. albicans, at least under non-stress conditions, since neither LIG4/lig4 nor lig4 strains exhibit gross chromosomal rearrangements in any of their chromosomes similar to those reported in animal cells.
3.2. Analysis of chromosomal translocations between non-homologous chromosomes in lig4 mutants Still, it was possible that some variations might actually occur due to reciprocal or non-reciprocal translocations involving similar-sized portions of two non-homologous chromosomes, in such a way that our method of analysis was not able to detect them. In order to check this possibility, the electrophoretical karyotypes of the several lig4 mutants were further subjected to Southern blot analysis using a set of subtelomeric probes. This set contains 16 sequences of telomere-proximal genes, one from each arm of each chromosome, in such a way that they allow the detection of translocations between two non-homologous chromosomes. In all the cases, the telomeric probes of Table 3 The number of Souþ colonies appearing each day from the indicated strains Strain
Daily appearance of the colonies 4
5
6
7
8
9
10
11
12
13
CAF2 LIG4/lig4 lig4/lig4 Reintegrant
1 0 1 0
7 5 2 7
237 320 223 217
205 169 235 210
106 65 151 145
29 34 77 71
4 25 34 11
^ 8 4 ^
^ ^ ^ ^
^ ^ ^ ^
Approximately 3000 cells from each strain were spread on duplicated L-sorbose plates. Numbers are means of two plates.
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3.4. Adaptation of lig4 mutants to grow in sorbose The importance of the chromosomal rearrangements in the adaptation of C. albicans under a variety of conditions has been recently reported by Elena Rustchenko and coworkers. One type of genetic alteration resulting in altered chromosome copy number of di¡erent speci¢c chromosomes has been related to the appearance of speci¢c mutants [7,22,28]. These mutants seem to arise by adaptive mutagenesis, i.e. an induced response to environmental stress in which mutation rates rise, producing genetic changes that can adapt cells to stress [22]. Accordingly, we investigated whether LIG4 has any in£uence in adaptive mutagenesis. Parental wild-type strain CAF2 and Uraþ derivatives LIG4/lig4 (CEA1), lig4/lig4 (CEA2), and revertant lig4/ LIG4 (CEA3) were monitored for the appearance of colonies in sorbose plates (Souþ colonies). As shown in Table 3, in all four strains the Souþ colonies accumulated daily over a period of 12^14 days and, on average, no di¡erences in the total number of Souþ mutants was observed between the four strains. These data represent the total number of Souþ mutants, since a further incubation of two additional weeks did not result in an increase in the number of colonies. As expected, all the colonies increased in size with time, due to their ability to utilize L-sorbose as the only carbon source (Fig. 5). Viability of both parental strain and lig4 derivative was determined by the daily transfer of entire agar blocks to YEPD plates, as described in Section 2. As shown in Fig. 6A, a massive cell death occurred with both strains, but their half lives were almost identical. Similarly, both the rate of appearance and the ¢nal number of Souþ colonies were similar for both strains. A reconstruction experiment indicated that, on average, it took about 5 to 6 days for a Souþ cell to yield a visible colony, indicating that most mutant cells were formed 5 or 6 days prior to the detection of the colony. It should be noticed that this time is 2 days longer than that reported for strain 3153A [22]. Mutational rates were then calculated as described before, i.e. mutants per viable cell at the time of mutant formation per day [22]. As shown in Fig. 6B, there was a signi¢cant increase in rates of Souþ mutant formation during the ¢rst 4 days but no di¡erences in mutational rates between LIG4 and lig4 strains were detected. We should also mention that less than 50% of the Souþ colonies initially selected on sorbose plates and then kept at 370‡C yielded colonies when regrown on sorbose plates (reconstruction experiment), indicating that our Souþ strains are very unstable and revert with a high frequency to Sou3 . It has been well documented that in C. albicans the appearance of Souþ strains from their Sou3 counterparts is due to the loss of one copy of Chr5 [7,22], although the molecular mechanism underlying this phenomenon remains unknown. In order to see if lig4 strains become
Fig. 6. A: The survival of CAF2 (open symbols) and lig4 Uraþ (close symbols) cells on L-sorbose medium. About 900 cells were plated on þ L-sorbose. At the indicated times the number of Sou colonies was recorded (inverted triangles) and the entire agar disk was transferred to a YEPD medium and incubated for 48 h in order to determine the total number of viable cells at that time (circles). B: The adjusted rates of Souþ mutants per viable cell per day. The values were calculated from the number of colonies divided by the number of viable cells 6 days prior to the appearance of the colonies (see text).
Souþ by loosing one copy of Chr5, the electrophoretical karyotypes of several independent Souþ strains derived from both wild-type CAI4 and its lig4 derivative were analyzed. Two di¡erent colonies from CAI4, arising at the 5th (large) and 10th (small) days showed one copy of Chr5, but otherwise, their karyotypes were identical to the parental Souþ (not shown). The same was true for the large colony (day 5) derived from the lig4 strain, but a few small colonies arising later (day 10) also exhibited changes in the length of ChrR (not shown), a fact described previously for strain 3153A [22].
4. Discussion In the present work, we have analyzed the e¡ect of the deletion of LIG4 on the genome stability of C. albicans, by comparing the electrokaryotypes of parental LIG4 and lig4 mutant derivatives. This possibility appeared quite
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feasible on the basis of the e¡ect that the deletion of LIG4, as well as other components of the NHEJ pathway, including KU80 and XRCC4, has in mammalian cells [19^ 21]. However, the results indicate that the absence of LIG4 does not signi¢cantly in£uence gross rearrangements of Chr1^7 in C. albicans. Accordingly, although we cannot discard the role of LIG4 in subtle rearrangements that cannot be detected with the technique used in the present report, it is clear that its absence does not cause aneuploidies, non-reciprocal translocation between non-homologous chromosomes, or chromosomal fragmentation. However, drastic changes in the mobility of ChrR homologs were seen during the generation of the lig4 mutants. Di¡erences in the size of ChrR between the di¡erent strains as well as the instability of ChrR within the same strain has been widely reported [29^33]. However, as far as we know, no detailed analysis of the changes in size of ChrR during the several steps required for the widely used gene disruption technique known as the URA-blaster [27] has been reported. Our results clearly indicate that both cell transformation and FOA selection steps result in signi¢cant alterations in the size of both copies of ChrR. It should be noted, however, that this is not peculiar to the disruption of LIG4, since disruption of another gene likely unrelated to recombination (SHE9) caused also the same kind of alterations. In S. cerevisiae, di¡erences in sizes of Chr XII may result from the number of rDNA units, which may vary between homologs due to unequal crossing over or gene conversion [32], and the same could be true for C. albicans. In C. albicans, Rustchenko et al. [34] found that growth conditions are important to de¢ne the size of ChrR; slow-growing populations show few changes whereas rapidly growing cells change the sizes of both ChrR homologs more frequently. It would be reasonable to state that cells adapt the number of rDNA units to each growth condition (E. Rustchenko, personal communication). However, no signi¢cant changes in ChrR occurred during growth of our strains in rich (YEPD) medium, even in the absence of LIG4, indicating that, at least for the strains used in this study (CAF2 and lig4 derivatives), this chromosome is rather stable under rapidly growing conditions. We conclude that spontaneous chromosomal instability, including that of ChrR, occurs with a rather low frequency in strain SC5314 and its derived strains. On the other hand, chromosomal instability in response to a changing environment or stress conditions is related to a phenomenon known as adaptive mutagenesis [35,36]. In adaptive mutagenesis, the mutation rate increases in response to the stress conditions, in such a way that more mutants are produced per generation. Although most of them die, a small fraction of the mutants may acquire the capability to grow under the new circumstances. The appearance of Souþ mutants of C. albicans on sorbose plates follows the basic principles of adaptive mutagenesis and, accordingly, constitutes a good example of this phenomenon, since mutational rates increase signi¢-
347
cantly in the presence of L-sorbose whereas most cells of the initial population die (Fig. 6A) [22]. Our results with CAF2 and lig4 derivatives con¢rm those reported previously for strain 3153A [7,22]. The most signi¢cant di¡erence was that, in our case, the time elapsed between the appearance of the mutant and the detection of the colony was longer (a minimum of 5^6 days for our strains versus 4 days for strain 3153A). This could be due to the fact that we incubated sorbose plates at 30‡C whereas strain 3153A was incubated at 37‡C [22], although we cannot discard additional di¡erences between the strains. Another possibility is that our strains carry a single copy of URA3 whereas strain 3153A carries two copies of this gene. This would slow down their growth in the absence of uridine. It has been demonstrated that there is a causal relationship between the loss of one copy of Chr5 and the ability of the aneuploid cell to utilize sorbose as the only carbon source. Non-disjunction at mitosis [37] has been explicitly proposed to explain chromosomal monosomy associated with sorbose utilization [7,22]. However, no probes supporting this hypothesis have been provided so far. Accordingly, we have analyzed the possibility that LIG4 could regulate in some way the loss of one copy of Chr5. This possibility appeared attractive, since a defect in the repair of DSB in one broken chromosome usually results in the lost of that chromosome. However, our experiments indicate that, if this was the case, the absence of LIG4 does not increase the rate of loss of Chr5, perhaps because the broken chromosome can be repaired by homologous recombination. We conclude that, as described for S. cerevisiae, NHEJ plays a minor role in recombination and genetic instability in C. albicans as compared with the prevalent role that it plays in mammalian cells and that chromosome copy number-dependent adaptive mutagenesis in C. albicans seems to be unrelated to LIG4.
Acknowledgements We are very grateful to Elena Rustchenko and Guilhem Janbon for helpful conversations, suggestions and advice. We also thank M.S. Garc|¤a de la Marta, V. Salguero and Leocadia Franco for technical assistance. This work was supported by grants from FEDER (IDF97-1173) and Junta de Extremadura (IPR98031).
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E. Andaluz et al. / FEMS Yeast Research 2 (2002) 341^348 pseudohyphae show di¡erent patterns of septin ring localization. Mol. Microbiol. 41, 19^31. Pujol, C., Reynes, J., Renaud, F., Raymond, M., Tibayrenc, M., Ayala, F.J., Janbon, F., Mallie¤, M. and Bastide, J. (1993) The yeast Candida albicans has a clonal mode of reproduction in a population of infected human immunode¢ciency virus-positive patients. Proc. Natl. Acad. Sci. USA 90, 9456^9459. Slutsky, B., Bu¡o, J. and Soll, D.R. (1985) High-frequency switching of colony morphology in Candida albicans. Science 230, 666^669. Soll, D.R. (1996) The emerging molecular biology of switching in Candida albicans. ASM News 62, 415^420. Janbon, G., Sherman, F. and Rustchenko, E. (1998) Monosomy of a speci¢c chromosome determines L-sorbose utilization : a novel regulatory mechanism in Candida albicans. Proc. Natl. Acad. Sci. USA 95, 5150^5155. Suzuki, T., Nishibayashi, S., Kuroiwa, T., Kanbe, T. and Tanaka, K. (1982) Variance of ploidy in Candida albicans. J. Bacteriol. 152, 893^ 896. Tsang, P.W.K., Cao, B., Siu, P.Y.L. and Wang, J. (1999) Loss of heterozygosity, by mitotic gene conversion and crossing over, causes strain-speci¢c adenine mutants in constitutive diploid Candida albicans. Microbiology 145, 1623^1629. Haber, J.E. (2000) Partners and pathways : repairing a double-strand break. Trends Genet. 16, 259^264. Boulton, S.J. and Jackson, S.P. (1996) Saccharomyces cerevisiae Ku70 potentiates illegitimate DNA double-strand repair and serves as a barrier to error-prone DNA repair pathways. EMBO J. 15, 5093^5103. Milne, G.T., Jin, S., Shannon, K.B. and Weaver, D. (1996) Mutations in two Ku homologs de¢ne a DNA end-joining repair pathway in Saccharomyces cerevisiae. Mol. Cell. Biol. 16, 4189^4198. Siede, W., Friedl, A.A., Dianova, I., Eckardschupp, F. and Friedlberg, E.C. (1996) The Saccharomyces cerevisiae Ku autoantigen homolog a¡ects radiosensitivity only in the absence of homologous recombination. Genetics 1442, 91^102. Ramsden, D.A., Paull, T.T. and Gellert, M. (1997) Cell-free V(D)J recombination. Nature 388, 488^491. Andaluz, E., Ciudad, A., Calderone, R. and Larriba, G. (1999) Cell cycle regulation of a DNA ligase-encoding gene (CaLIG4) from Candida albicans. Yeast 15, 1119^1210. Andaluz, E., Calderone, R., Reyes, G. and Larriba, G. (2001) Phenotypic analysis and virulence of Candida albicans LIG4 mutants. Infect. Immun. 69, 137^147. Paques, F. and Haber, J.E. (1999) Multiple pathways of recombination induced by double strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63, 349^404. Andaluz, E., Larriba, G. and Calderone, R. (1996) A Candida albicans gene encoding a DNA ligase. Yeast 12, 893^898. Karanjawala, Z.E., Grawunder, U., Hsieh, C.L. and Lieber, M.R. (1999) The non-homologous DNA end joining pathway is important for chromosome stability in primary ¢broblasts. Curr. Biol. 9, 1501^ 1504. DeFilippantonio, M.J., Zhu, J., Chen, H.T., Me¡re, E., Nussenzweig, M.C., Max, E.E., Reid, T. and Nussenzweig, A. (2000) DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature 404, 510^514.
[21] Ferguson, D.O., Sekiguchi, J.M., Chang, S., Frank, K.M., Gao, Y., DePinho, R.A. and Alt, F.W. (2000) The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations. Proc. Natl. Acad. Sci. USA 97, 6630^ 6633. [22] Janbon, G., Sherman, F. and Rustchenko, E. (1999) Appearance and properties of L-sorbose utilizing mutants of Candida albicans obtained on a selective plate. Genetics 153, 653^664. [23] Rustchenko, E.P., Howard, D.H. and Sherman, F. (1994) Chromosomal alterations of Candida albicans are associated with the gain or loss of assimilating functions. J. Bacteriol. 176, 3231^3241. [24] Ko«hler, J.R. and Fink, G.R. (1996) Candida albicans strains heterozygous and homozygous for mutations in mitogen-activated protein kinase signaling components have defects in hyphal development. Proc. Natl. Acad. Sci. USA 93, 13223^13228. [25] Sherman, S., Fink, G.R. and Hicks, J.B. (1982) Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [26] Andaluz, E., Coque, J.J.R., Cueva, R. and Larriba, G. (2001) Sequencing of a 4.3 kbp region of chromosome 2 of Candida albicans reveals the presence of homologues of SHE9 from Saccharomyces cerevisiae and a bacterial phosphatidylinositol-phospholipase C. Yeast 18, 711^721. [27] Fonzi, W.A. and Irwin, M.Y. (1993) Isogenic strain construction and gene mapping in Candida albicans. Genetics 134, 717^728. [28] Perepnikhatka, V., Fisher, F.J., Niimi, M., Baker, R.A., Cannon, R.D., Wang, Y., Sherman, F. and Rustchenko, E. (1999) Speci¢c alterations in £uconazol-resistant mutants of Candida albicans. J. Bacteriol. 181, 4041^4049. [29] Sadhu, C., McEachern, M.J., Rustchenko-Bulgac, E.P., Schmid, J., Soll, D.R. and Hicks, J.B. (1991) Telomeric and dispersed repeat sequences in C. albicans yeast and their use in strain identi¢cation. J. Bacteriol. 173, 842^850. [30] Iwaguchi, S-I., Homma, M. and Tanaka, K. (1992) Clonal variation of chromosome size derived from the rDNA cluster region of Candida albicans. J. Gen. Microbiol. 138, 1177^1184. [31] Iwaguchi, S-I., Sato, M., Magee, B.B., Magee, P.T., Makimura, K. and Suzuki, T. (2001) Extensive chromosome translocation in a clinical isolate showing the distinctive carbohydrate assimilation pro¢le from a candidiasis patient. Yeast 18, 1035^ 1046. [32] Szostak, J.W. and Wu, R. (1980) Unequal crossing over in the ribosomal DNA of Saccharomyces cerevisiae. Nature 284, 426^430. [33] Magee, P.T. (1993) Variation in chromosome size and organization in Candida albicans and Candida stellatoidea. Trends Microbiol. 342, 338^342. [34] Rustchenko, E.P., Curran, T.M. and Sherman, F. (1993) Variations in the number of ribosomal DNA units in morphological mutants and normal strains of Candida albicans and in normal strains of Saccharomyces cerevisiae. J. Bacteriol. 175, 7189^7199. [35] Cairns, J., Overbaugh, J. and Miller, S. (1988) The origin of mutants. Nature 335, 142^145. [36] Foster, P.L. (1998) Adaptive mutation: has the unicorn landed ? Genetics 148, 1453^1459. [37] Fincham, J.R.S. and Day, P.R. (1971) Fungal Genetics. Blackwell Scienti¢c Publications, Oxford.
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An evaluation of the role of LIG4 in genomic instability and adaptive mutagenesis in Candida albicans Encarnacio¤n Andaluz, Toni Ciudad, Germa¤n Larriba
Departamento de Microbiolog|¤a, Universidad de Extremadura, Avda. de Elvas s/n, 06071 Badajoz, Spain Received 12 October 2001; received in revised form 21 March 2002; accepted 22 March 2002 First published online 30 April 2002
Abstract The non-homologous end-joining (NHEJ) pathway of DNA recombination is important for genomic stability in animal cells, since the absence of Ku70, Ku80, Lig4 or Xrcc4 results in non-reciprocal translocation and chromosome fragmentation. The role of LIG4 in the genomic instability of Candida albicans has been analyzed. We have found that both cell transformation and 5P-fluoroorotic acid selection steps used to obtain several lig4 mutants (LIG4/lig4 Uraþ ; LIG4/lig4 Ura3 ; lig4/lig4 Uraþ ; lig4/lig4 Ura3 ; and revertant lig4/LIG4 Uraþ ) resulted in significant alterations in chromosome R (ChrR). However, this effect is not specific for LIG4, since disruption of SHE9, a gene unrelated to recombination, also caused alterations in the mobility of ChrR. On the other hand, we could not detect reciprocal or nonreciprocal translocations between non-homologous chromosomes in several lig4 mutants. Furthermore, propagation of these mutants in rich medium did not cause other alterations in the mobility of ChrR. Adaptive mutagenesis of C. albicans, determined by the appearance of L-sorbose-utilizing mutants on L-sorbose plates, was also independent of the presence of Lig4 and occurred by monosomy of Chr5. Accordingly, the NHEJ pathway does not appear to be involved in the adaptive mutagenesis mediated by alterations in chromosome copy number. : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords : Genomic instability ; Adaptive mutagenesis; LIG4; Candida albicans
1. Introduction Candida albicans is the most prevalent fungal pathogen of humans. As a commensal of the healthy individual, this fungus lives saprophytically in several niches of the human body, but alterations in the balance between the commensal and the host, as occurs in the immunocompromised patient, may trigger infection [1,2]. A large part of the studies on the biology of C. albicans makes reference to two important properties of the organism. First, it can grow with a variety of morphologies, including the typical yeast form, pseudohyphae, i.e. chains of elongated cells with constrictions between adjacent cells, or true hyphae, which lack constrictions at the septa [3]. Reversibility between the several forms is essential for virulence [1,2]. The second property is that C. albicans behaves as an obligate diploid. On the basis of population genetic studies, it is assumed that C. albicans reproduces primarily by clonal
* Corresponding author. Tel. : +34 (924) 289428; Fax : +34 (924) 289428. E-mail address : [email protected] (G. Larriba).
propagation [4]. However, it has been widely reported that di¡erent isolates of C. albicans exhibit clear di¡erences in heritable phenotypes, including colonial morphology, antigenic pro¢les and electrophoretic karyotypes [5^9]. In addition, the organism has the capacity to adapt to new environments, since during infection of the mucosal epithelia, dissemination via the bloodstream and colonization of internal organs, it copes with new and frequently adverse conditions. As stated above, it is clear that this variability and adaptability do not arise by meiotic recombination of two genomes from cells of di¡erent mating types. The prevailing conclusion is that the organism evolves by accumulation of point mutations, as well as, in a more £exible way, through endogenous recombination events in a heterozygous background, including mitotic recombination, gene ampli¢cation and non-reciprocal chromosomal translocations, as well as gain/loss of speci¢c chromosomes. Recombination events arise from the repair of doublestrand breaks (DSB) in DNA. These DSB can occur as a consequence of the normal cellular metabolism (although the signal that triggers their formation has not been identi¢ed), or as a result of exposure to chemical and physical
1567-1356 / 02 / $22.00 : 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 0 9 4 - 6
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agents. In order to repair DNA damage, cells have evolved a number of mechanisms that can be classi¢ed into two general pathways : homologous recombination (HR) and non-homologous end-joining (NHEJ) [10]. In HR, repair of the damaged DNA requires the presence of an intact homologous partner, and leads to accurate repair. By contrast, in NHEJ, DNA ends are joined with microhomology or no complementarity. Although the repair can be precise, more frequently it produces small insertions or deletions or even large deletions comprising hundreds of base pairs and, accordingly, it is mutagenic [10^13]. It should be noted that, although DNA repair via NHEJ is a minor pathway in Saccharomyces cerevisiae, it is the prevalent pathway for DNA repair in mammals where it is required for precise V(D)J recombination in the generation of immunoglobulins and T-cell receptors [14]. On this basis, we hypothesized that, as an obligate diploid, C. albicans could well carry out recombinational events through the NHEJ pathway whose outcome would be the arrangement of genes or even chromosomes that could help the organism to cope with the hostile conditions found in the human host [15,16]. NHEJ uses a common set of core components in yeast and mammals: these are the DNA end-binding proteins Ku70 and Ku80 (Hdf1p and Hdf2p in yeast), as well as Lig4 and its associated Xrcc4 (Lif1p in yeast) protein [10,17]. Recently, we reported a phenotypic analysis and virulence of lig4 mutants of C. albicans. These studies concluded that the absence of Lig4p results in an impairment in myceliation and an attenuation of virulence. However, LIG4 is neither essential for DNA replication nor for the repair of DNA damage induced by radiomimetics, indicating the presence of a homologous recombination system that operates under most circumstances [15,16,18]. On the other hand, recent studies in mammalian cells have concluded that the NHEJ pathway of DNA repair is a crucial caretaker of the mammalian genome, since null mutants in several components of the pathway, including Ku80, Xrcc4 and Lig4 exhibit genomic instability as well as increased chromosomal fragmentation and non-reciprocal translocations [19^21]. Accordingly, we extended our initial phenotypic analysis of lig4 mutants in order to
know whether Lig4 could also play a role in the genomic stability of C. albicans.
2. Materials and methods 2.1. Strains and growth conditions The C. albicans strains used in this study are listed in Table 1. They were routinely grown in YEPD (2% glucose, 1% yeast extract, 2% Bacto peptone) at 28‡C. The adaptative mutagenesis experiments were performed as described [7,22]. In these experiments cells were plated on L-sorbose plates (2% agar) containing 2% L-sorbose, and 0.67% Bacto yeast nitrogen base without amino acids [23] and incubated at 30‡C. For Ura3 strains, this medium was supplemented with uridine (25 Wg ml31 ). 2.2. DNA extraction and analysis Plasmids containing the disruption cassettes of HST7 and CPH1 were kindly provided by Dr. G. Fink [24]. A set of subtelomeric probes, one for each arm of the eight chromosomes of C. albicans (Table 2), was kindly supplied by Dr. B.B. Magee (University of Minnesota, St. Paul, MN, USA). Standard techniques were routinely used for the preparation and puri¢cation of plasmid DNA [25]. Genomic DNA was prepared from protoplasts. Protoplasts were obtained by incubation of cells with zymolyase in 1 M sorbitol and lysed in 50 mM EDTA and 0.6% SDS. C. albicans cells were transformed by electroporation in a BTX electroporation system. For that purpose, 40 Wl of a cellular suspension were mixed with 5 Wl of transforming DNA (less than 100 Wg). After 5 min in ice, the mixture was transferred to ice-cold electroporation cuvettes (0.2 cm). The voltage applied was 1.5 kV. Disruption of one allele of SHE9 [26] to obtain the heterozygous SHE9/she was carried out using the URAblaster technique and the CAI4 strain as a recipient [27]. One of the several SHE9/she9 Uraþ transformants obtained was then plated on 5P-£uoroorotic acid plates (FOA) to select the Ura3 segregants. Both strains were
Table 1 C. albicans strains used in this study Strain SC5314 CAF2 CAI4 CEA1 CEA15 CEA2 CEA26 CEA3 CGM1.1 CGM1.2
SC5314 CAF2 CAI4 CEA1 CEA15 CEA2 CEA26 CAI4 CAI4
Genotype or description
Source
prototrophic vura3: :imm434/URA3 vura3: :imm434/vura3: :imm434 as CAI4 but LIG4/lig4 : :hisG-URA3-hisG as CAI4 but LIG4/lig4 : :hisG as CAI4 but lig4 : :hisG-URA3-hisG/lig4 : :hisG as CAI4 but lig4 : :hisG/lig4 : :hisG as CEA3 but lig4: :hisG/LIG4 : :URA3-hisG as CAI4 but SHE9/she9: :hisG as CAI4 but SHE9/she9: :hisG
[27] [27] [27] [16] [16] [16] [16] [16] this work this work
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2.4. Studies on adaptive mutagenesis in response to L-sorbose
Chromosome
S¢I fragment
Gene
R
D M L S A U O P N H I M C O C G
INO1 ADE1 ACT1 HGT1 nd SAS2 NPS1 YER132 HIS5 YJR61 CDC1 GCD11 COX12 YMR315 nonea ARG4
1 2 3 4 5 6 7 a
343
Plasmid 321. This probe consists of 4.0 kb HindIII in pUC18.
veri¢ed by Southern blot hybridization and PCR (E. Andaluz and Garc|¤a de la Marta, unpublished results). 2.3. Sample preparation for PFGE 0.1 ml of an exponentially growing culture of C. albicans was used to inoculate 10 ml of YEPD. The culture was maintained in a rotatory shaker at 30‡C for 48 h. Cells were harvested by centrifugation, washed twice with 50 mM EDTA, pH 8.0, and resuspended in 1 ml of CPES (40 mM citric acid, 120 mM sodium phosphate, 20 mM EDTA, pH 8, 1.2 M sorbitol and 5 mM dithiothreitol) supplemented with 0.2 mg of zymolyase 20 000. 1 ml of CPE (as CPES but lacking sorbitol and DTT) containing 1% low melting point agarose at 50‡C was then added and gently mixed. Aliquots of 200 Wl were then transferred into a sample mold and kept at 320‡C. Upon solidi¢cation, plugs were transferred to test tubes, supplemented with 6 ml of CPE and incubated at 30‡C for 4 h. CPE bu¡er was replaced with 5 ml of TESP (1 M Tris^HCl, 0.5 M EDTA, 2% SDS) containing 1 mg ml31 of proteinase K and incubated overnight at 50‡C. Samples were then washed 3U with TE (10 mM Tris^HCl, 1 mM EDTA) at 50‡C and 6U at room temperature. The plugs were stored at 4‡C in 50 mM EDTA, pH 8. The gel containing C. albicans chromosomes was run in 0.6% agarose for 24 h at 80 V with a 120^300 s linear ramp and then for 48 h at 80 V with a 420^900 s linear ramp in a rotating gel electrophoresis apparatus. For Southern analysis, the electrophoresed samples were transferred to TM-nitrocellulose, probed with the indicated probes at high stringency (65‡C in 6USSC, 5UDenhardt’s solution, 0.5% SDS and then washed with 0.1USSC, 0.1% SDS). Xomat X-AR ¢lm (Kodak) was used to expose blots.
The formation of Souþ mutants by parental (LIG4) and mutant (lig4) strains was analyzed as described by Janbon et al. [7,22]. Sorbose plates were prepared using regular agar. In some experiments, scavenger S. cerevisiae cells (strain no. 865) were used to eliminate any contaminant that might be present in agar, as indicated [22], but in our hands, this modi¢cation did not result in any di¡erence in the total number of Souþ colonies. Also, to avoid possible deviations in counts due to di¡erences between individual cells, a cell mass of each C. albicans strain, previously washed twice in sterile water, was used to inoculate the sorbose plates. Two series of approximately 300 and 3000 colony-forming units (CFU) were spread in duplicate on sorbose plates for detection of Souþ colonies, and plates were incubated at 30‡C. A viability experiment was conducted plating the same cell suspension on a number of sorbose plates. Daily for 2 weeks, entire agar discs were transferred to the surface of a YEPD plate to allow viable cells to grow [22]. The ability of the selected Souþ mutants to regrow on L-sorbose plates was investigated as described [22]. Brie£y, Souþ cells from a 370‡C preserved culture were streaked on YEPD plates; smaller colonies were then suspended in distilled water and appropriate dilutions plated again on L-sorbose medium. Plates were incubated at 30‡C and inspected for the appearance of colonies.
3. Results 3.1. Chromosomal alteration of lig4 mutants Fig. 1 shows the karyotypes of wild-type SC5314 and its Ura3 derivative CAI4 as well as those of the several
Fig. 1. Electrophoretical karyotype of the indicated strains. A and B: Ethidium bromide staining. C and D : Southern blot hybridization using the disruption cassettes of HST7 (C) or CPH1 (D) as a probe.
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Fig. 3. Southern blot hybridization of the electrokaryotypes of the indicated strains using subtelomeric probes of Chr2 (A) and Chr3 (B).
Fig. 2. Electrophoretical karyotype of CAI4 and two SHE9/she9 Ura3 strains. Notice the di¡erences in the mobility of ChrR.
strains generated during the disruption of LIG4 (LIG4/lig4 Uraþ ; LIG4/lig4 Ura3 ; lig4/lig4 Uraþ ; and lig4/lig4 Ura3 ) and construction of the revertant (lig4/LIG4 Uraþ ). Ethidium bromide staining indicated signi¢cant alterations in the size of ChrR following each transformation step or selection on 5P-£uoroorotic acid (FOA) plates (Fig. 1A and B). These alterations were further con¢rmed by Southern blot analysis using the deletion cassette of HST7 (that we have located to this chromosome) as a probe. Chr3 is also labelled in wild-type SC5314, but not in its Ura3 derivative CAI4, because the deletion cassette contains the construct hisG-URA3-hisG and the URA3 gene is located in that chromosome in C. albicans. In the same way, the deletion cassette labelled Chr2 in all the lig4 single or double deletants because of the presence of hisGURA3-hisG (LIG4/lig4 Uraþ ), hisG (LIG4/lig4 Ura3 and lig4/lig4 Ura3 ) or both (lig4/lig4 Uraþ ) in the LIG4 locus in these mutants. It should be noted that in the parental strain SC5314 both homologs of ChrR exhibit di¡erent sizes, one migrating slightly faster and the other signi¢cantly slower than Chr1 (Fig. 1C). This observation is not unusual, since ChrR recombines at the level of the rDNA repeats to yield copies that can vary in size from s 4 to s 3 kb. Interestingly, the sizes of the individual R chromosomes are altered in the Ura3 derivative CAI4, where ChrR migrates as a single broad band (Fig. 1C). CAI4 was obtained by sequential deletion of both copies of URA3 present in strain SC5314, the second deletion being followed by selection on 5P-£uoroorotic acid plates [27]. On the basis of the ethidium bromide staining, it appeared that the rest of the chromosomes (1^7) had not undergone gross rearrangements. However, we further analyzed the mobility of chromosome 1 because in some
strains it migrates close to ChrR, and its unambiguous identi¢cation by ethidium bromide staining may be misinterpreted. For this purpose, we performed Southern blot analysis using the deletion cassette of CPH1 (which we have located to this chromosome) as a probe. As shown in Fig. 1D, no signi¢cant di¡erences in the size of this chromosome were seen in the di¡erent lig4 mutants, indicating that the chromosomal rearrangements are speci¢c for ChrR. Because of the involvement of LIG4 in NHEJ, we then asked whether the alterations in the size of ChrR were speci¢c for LIG4 or could be generated also during the disruption of a di¡erent gene. For this purpose, we chose SHE9 which is adjacent to LIG4 in Chr2 [26] and appears unrelated to DNA repair. Disruption of a single copy to yield the heterozygous deletant Uraþ was enough to cause changes in the mobility of the homologs of chromosome R (not shown). Moreover, selection of Ura3 heterozygotes on 5P-FOA plates resulted in a new shift in mobility of ChrR, whereas the rest of the chromosomes remained intact. Interestingly, two di¡erent Ura3 segregants yielded di¡erent patterns for ChrR, indicating that recombination between both homologs does not occur at a precise site, but is random (Fig. 2). We conclude that disruption of LIG4 does not a¡ect the mobility of chromosomes 1^7,
Fig. 4. Southern blot hybridization of the electrokaryotypes of the indicated strains using the HST7 disruption cassette as a probe. Each strain was removed from 370‡C and grown on a YEPD plate. A colony of this plate was considered the ¢rst generation. Then, it was propagated through 15 plates and a colony of the last plate considered as the n generation.
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345
Fig. 5. All of the Souþ colonies appeared in the ¢rst 12^14 days. Approximately 3000 cells of the indicated strain were spread on each sorbose plate. Plates were photographed after 12 days (row A) and 28 days (row B).
Chr1^7 labelled the correct chromosome (see Fig. 3 for two examples), indicating that no translocations between non-homologous chromosomes had occurred during the generation of the several mutants.
and that there is not a causal relationship between disruption of LIG4 and the changes observed in the size of both homologs of ChrR. Since alterations in the mobility of ChrR are known to be natural events resulting from unequal crossing over between homologs in the rDNA repeats (see below), we believe that the alterations seen here belong to the same category, but their frequency is enhanced by the stress conditions to which cells are subjected during both transformation and FOA selection steps.
3.3. Analysis of chromosomal translocations following propagation of lig4 mutants We next analyzed the possibility that some changes in chromosome size were only seen after several generations in the absence of Lig4. Accordingly, we propagated the parental strain SC5314 as well as the Uraþ derivatives of LIG4/lig4 and lig4/lig4 through 15 YEPD plates. Then, colonies from the ¢rst (¢rst generation) and the 15th (n generation) plates were independently processed and the electrokaryotypes obtained. Ethidium bromide staining failed to indicate gross chromosomal rearrangements and, within every strain, even ChrR seemed to remain unchanged (not shown). This was further con¢rmed by Southern blot analysis using the HST7 disruption cassette as a probe (Fig. 4). We conclude that LIG4 does not behave as a guardian or caretaker of the genome of C. albicans, at least under non-stress conditions, since neither LIG4/lig4 nor lig4 strains exhibit gross chromosomal rearrangements in any of their chromosomes similar to those reported in animal cells.
3.2. Analysis of chromosomal translocations between non-homologous chromosomes in lig4 mutants Still, it was possible that some variations might actually occur due to reciprocal or non-reciprocal translocations involving similar-sized portions of two non-homologous chromosomes, in such a way that our method of analysis was not able to detect them. In order to check this possibility, the electrophoretical karyotypes of the several lig4 mutants were further subjected to Southern blot analysis using a set of subtelomeric probes. This set contains 16 sequences of telomere-proximal genes, one from each arm of each chromosome, in such a way that they allow the detection of translocations between two non-homologous chromosomes. In all the cases, the telomeric probes of Table 3 The number of Souþ colonies appearing each day from the indicated strains Strain
Daily appearance of the colonies 4
5
6
7
8
9
10
11
12
13
CAF2 LIG4/lig4 lig4/lig4 Reintegrant
1 0 1 0
7 5 2 7
237 320 223 217
205 169 235 210
106 65 151 145
29 34 77 71
4 25 34 11
^ 8 4 ^
^ ^ ^ ^
^ ^ ^ ^
Approximately 3000 cells from each strain were spread on duplicated L-sorbose plates. Numbers are means of two plates.
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3.4. Adaptation of lig4 mutants to grow in sorbose The importance of the chromosomal rearrangements in the adaptation of C. albicans under a variety of conditions has been recently reported by Elena Rustchenko and coworkers. One type of genetic alteration resulting in altered chromosome copy number of di¡erent speci¢c chromosomes has been related to the appearance of speci¢c mutants [7,22,28]. These mutants seem to arise by adaptive mutagenesis, i.e. an induced response to environmental stress in which mutation rates rise, producing genetic changes that can adapt cells to stress [22]. Accordingly, we investigated whether LIG4 has any in£uence in adaptive mutagenesis. Parental wild-type strain CAF2 and Uraþ derivatives LIG4/lig4 (CEA1), lig4/lig4 (CEA2), and revertant lig4/ LIG4 (CEA3) were monitored for the appearance of colonies in sorbose plates (Souþ colonies). As shown in Table 3, in all four strains the Souþ colonies accumulated daily over a period of 12^14 days and, on average, no di¡erences in the total number of Souþ mutants was observed between the four strains. These data represent the total number of Souþ mutants, since a further incubation of two additional weeks did not result in an increase in the number of colonies. As expected, all the colonies increased in size with time, due to their ability to utilize L-sorbose as the only carbon source (Fig. 5). Viability of both parental strain and lig4 derivative was determined by the daily transfer of entire agar blocks to YEPD plates, as described in Section 2. As shown in Fig. 6A, a massive cell death occurred with both strains, but their half lives were almost identical. Similarly, both the rate of appearance and the ¢nal number of Souþ colonies were similar for both strains. A reconstruction experiment indicated that, on average, it took about 5 to 6 days for a Souþ cell to yield a visible colony, indicating that most mutant cells were formed 5 or 6 days prior to the detection of the colony. It should be noticed that this time is 2 days longer than that reported for strain 3153A [22]. Mutational rates were then calculated as described before, i.e. mutants per viable cell at the time of mutant formation per day [22]. As shown in Fig. 6B, there was a signi¢cant increase in rates of Souþ mutant formation during the ¢rst 4 days but no di¡erences in mutational rates between LIG4 and lig4 strains were detected. We should also mention that less than 50% of the Souþ colonies initially selected on sorbose plates and then kept at 370‡C yielded colonies when regrown on sorbose plates (reconstruction experiment), indicating that our Souþ strains are very unstable and revert with a high frequency to Sou3 . It has been well documented that in C. albicans the appearance of Souþ strains from their Sou3 counterparts is due to the loss of one copy of Chr5 [7,22], although the molecular mechanism underlying this phenomenon remains unknown. In order to see if lig4 strains become
Fig. 6. A: The survival of CAF2 (open symbols) and lig4 Uraþ (close symbols) cells on L-sorbose medium. About 900 cells were plated on þ L-sorbose. At the indicated times the number of Sou colonies was recorded (inverted triangles) and the entire agar disk was transferred to a YEPD medium and incubated for 48 h in order to determine the total number of viable cells at that time (circles). B: The adjusted rates of Souþ mutants per viable cell per day. The values were calculated from the number of colonies divided by the number of viable cells 6 days prior to the appearance of the colonies (see text).
Souþ by loosing one copy of Chr5, the electrophoretical karyotypes of several independent Souþ strains derived from both wild-type CAI4 and its lig4 derivative were analyzed. Two di¡erent colonies from CAI4, arising at the 5th (large) and 10th (small) days showed one copy of Chr5, but otherwise, their karyotypes were identical to the parental Souþ (not shown). The same was true for the large colony (day 5) derived from the lig4 strain, but a few small colonies arising later (day 10) also exhibited changes in the length of ChrR (not shown), a fact described previously for strain 3153A [22].
4. Discussion In the present work, we have analyzed the e¡ect of the deletion of LIG4 on the genome stability of C. albicans, by comparing the electrokaryotypes of parental LIG4 and lig4 mutant derivatives. This possibility appeared quite
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feasible on the basis of the e¡ect that the deletion of LIG4, as well as other components of the NHEJ pathway, including KU80 and XRCC4, has in mammalian cells [19^ 21]. However, the results indicate that the absence of LIG4 does not signi¢cantly in£uence gross rearrangements of Chr1^7 in C. albicans. Accordingly, although we cannot discard the role of LIG4 in subtle rearrangements that cannot be detected with the technique used in the present report, it is clear that its absence does not cause aneuploidies, non-reciprocal translocation between non-homologous chromosomes, or chromosomal fragmentation. However, drastic changes in the mobility of ChrR homologs were seen during the generation of the lig4 mutants. Di¡erences in the size of ChrR between the di¡erent strains as well as the instability of ChrR within the same strain has been widely reported [29^33]. However, as far as we know, no detailed analysis of the changes in size of ChrR during the several steps required for the widely used gene disruption technique known as the URA-blaster [27] has been reported. Our results clearly indicate that both cell transformation and FOA selection steps result in signi¢cant alterations in the size of both copies of ChrR. It should be noted, however, that this is not peculiar to the disruption of LIG4, since disruption of another gene likely unrelated to recombination (SHE9) caused also the same kind of alterations. In S. cerevisiae, di¡erences in sizes of Chr XII may result from the number of rDNA units, which may vary between homologs due to unequal crossing over or gene conversion [32], and the same could be true for C. albicans. In C. albicans, Rustchenko et al. [34] found that growth conditions are important to de¢ne the size of ChrR; slow-growing populations show few changes whereas rapidly growing cells change the sizes of both ChrR homologs more frequently. It would be reasonable to state that cells adapt the number of rDNA units to each growth condition (E. Rustchenko, personal communication). However, no signi¢cant changes in ChrR occurred during growth of our strains in rich (YEPD) medium, even in the absence of LIG4, indicating that, at least for the strains used in this study (CAF2 and lig4 derivatives), this chromosome is rather stable under rapidly growing conditions. We conclude that spontaneous chromosomal instability, including that of ChrR, occurs with a rather low frequency in strain SC5314 and its derived strains. On the other hand, chromosomal instability in response to a changing environment or stress conditions is related to a phenomenon known as adaptive mutagenesis [35,36]. In adaptive mutagenesis, the mutation rate increases in response to the stress conditions, in such a way that more mutants are produced per generation. Although most of them die, a small fraction of the mutants may acquire the capability to grow under the new circumstances. The appearance of Souþ mutants of C. albicans on sorbose plates follows the basic principles of adaptive mutagenesis and, accordingly, constitutes a good example of this phenomenon, since mutational rates increase signi¢-
347
cantly in the presence of L-sorbose whereas most cells of the initial population die (Fig. 6A) [22]. Our results with CAF2 and lig4 derivatives con¢rm those reported previously for strain 3153A [7,22]. The most signi¢cant di¡erence was that, in our case, the time elapsed between the appearance of the mutant and the detection of the colony was longer (a minimum of 5^6 days for our strains versus 4 days for strain 3153A). This could be due to the fact that we incubated sorbose plates at 30‡C whereas strain 3153A was incubated at 37‡C [22], although we cannot discard additional di¡erences between the strains. Another possibility is that our strains carry a single copy of URA3 whereas strain 3153A carries two copies of this gene. This would slow down their growth in the absence of uridine. It has been demonstrated that there is a causal relationship between the loss of one copy of Chr5 and the ability of the aneuploid cell to utilize sorbose as the only carbon source. Non-disjunction at mitosis [37] has been explicitly proposed to explain chromosomal monosomy associated with sorbose utilization [7,22]. However, no probes supporting this hypothesis have been provided so far. Accordingly, we have analyzed the possibility that LIG4 could regulate in some way the loss of one copy of Chr5. This possibility appeared attractive, since a defect in the repair of DSB in one broken chromosome usually results in the lost of that chromosome. However, our experiments indicate that, if this was the case, the absence of LIG4 does not increase the rate of loss of Chr5, perhaps because the broken chromosome can be repaired by homologous recombination. We conclude that, as described for S. cerevisiae, NHEJ plays a minor role in recombination and genetic instability in C. albicans as compared with the prevalent role that it plays in mammalian cells and that chromosome copy number-dependent adaptive mutagenesis in C. albicans seems to be unrelated to LIG4.
Acknowledgements We are very grateful to Elena Rustchenko and Guilhem Janbon for helpful conversations, suggestions and advice. We also thank M.S. Garc|¤a de la Marta, V. Salguero and Leocadia Franco for technical assistance. This work was supported by grants from FEDER (IDF97-1173) and Junta de Extremadura (IPR98031).
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