Rad52 depletion in Candida albicans triggers both the DNA-damage checkpoint and filamentation...

Molecular Microbiology (2006)

59

(5), 1452–1472 doi:10.1111/j.1365-2958.2005.05038.x First published online 20 January 2006

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

Blackwell Publishing LtdOxford, UKMMIMolecular Microbiology0950-382X© 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd

? 2006

59

514521472

Original Article

Filamentation of rad52 mutants of C. albicansE. Andaluz

et al.

Accepted 13 December, 2005. *For correspondence. [email protected]; Tel.

+

34 924 289 428; Fax

+

34 924 289 428.

Rad52 depletion in

Candida albicans

triggers both the DNA-damage checkpoint and filamentation accompanied by but independent of expression of hypha-specific genes

Encarnación Andaluz

1

, Toni Ciudad

1

, Jonathan Gómez-Raja

1

, Richard Calderone

2

and Germán Larriba

1

*

1

Departamento de Microbiología, Facultad de Ciencias, Universidad de Extremadura, 06071 Badajoz, Spain.

2

Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, DC 20007, USA.

Summary

We have analysed the effect of

RAD52

deletion inseveral aspects of the cell biology of

Candida albi-cans

. Cultures of

rad52

D

strains exhibited slowgrowth and contained abundant cells with a filamen-tous morphology. Filamentation with polarization ofactin patches was accompanied by the induction ofthe hypha-specific genes (HSG)

ECE1

,

HWP1

and

HGC1

. However, filament formation occurred in theabsence of the transcription factors Efg1 and Cph1,even though disruption of

EFG1

prevented expres-sion of HSG. Therefore, expression of HSG genesaccompanies but is dispensable for

rad52

D

filamenta-tion. However, deletion of adenylate cyclase severelyimpaired filamentation, this effect being largelyreverted by the addition of exogenous cAMP. Fila-ments resembled elongated pseudohyphae, but someof them looked like true hyphae. Following depletionof Rad52, many cells arrested at the G2/M phase ofthe cell cycle with a single nucleus suggesting theearly induction of the DNA-damage checkpoint. Fila-ments formed later, preferentially from G2/M cells.The filamentation process was accompanied by theuncoupling of several landmark events of the cellcycle and was partially dependent on the action of thecell cycle modulator Swe1. Hyphae were still inducedby serum, but a large number of

rad52

cells myceli-ated in G2/M.

Introduction

A key question in biology is to know whether polarizationof growth has a central core where several pathwaysconverge or is the result of a number of independentprocesses. Fungi appear to be excellent models for thistype of study, as changes in morphology are linked tochanges in the polarization of growth. So far, these studieshave been mostly restricted to

Saccharomyces cerevisiae

.However, recent advances in the molecular and cellularbiology of other fungal species make it possible not onlyto test the universality of the currently prevailing models,but also to extend these studies to polymorphic organ-isms, where polarization of growth during hyphal inductionrequires new pathways and can be regulated by a numberof specific environmental conditions. One such organismis

Candida albicans

, an opportunistic human fungalpathogen that causes superficial infections or even moresevere systemic infections of blood and tissues in immu-nocompromised patients (Calderone, 2002).

Both internal stimuli as well as environmental stresscan trigger polarization of growth. In

S. cerevisiae

and

C. albicans

, cell polarization during the budding cycle isrestricted to a short period during the initial steps of bud-ding. When the bud reaches a critical size, polarizedgrowth is shut down and replaced by isotropic growth(Staebell and Soll, 1985; Lew and Reed, 1993). Thesequential combination of polarized/isotropic growthresults in the typical ovoid morphology of the yeast cell.These observations indicate that polarization of growth isintrinsically linked to the cell cycle. As with most of the cellcycle events, polarization of growth in

S. cerevisiae

hasbeen shown to be controlled by cyclic modification ofcyclin-dependent kinases. For instance, G1 cyclinsactivate the Cdk1 cyclin-dependent kinase (Cdc28 in

S. cerevisiae

) to promote apical growth through polariza-tion of actin and polarized secretion to the tip; in laterstages of the cell cycle, B-type cyclins bind to Cdk1 topromote isotropic growth through redistribution of actinand random secretion. A permanent activation of the G1cyclins or abrogation of B-type cyclins resulted in elon-gated cells. Activation of the Cdc28 kinase by B-typecyclins also requires dephosphorylation of Cdc28 by the

Filamentation of

rad52

mutants of

C. albicans 1453

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd,

Molecular Microbiology

,

59

, 1452–1472

tyrosine phosphatase Mih1, as tyrosine inhibitory phos-phorylation of Cdk1 by the kinase Swe1 (the

S. cerevisiae

member of the Wee1 kinase family) prevents both entryinto mitosis and the polar/isotropic shift (Lew and Reed,1995a,b). The Swe1-mediated inhibition of Cdc28 acts inthe morphogenesis checkpoint apparently until the budhas reached a critical size. Deletion of Swe1 results inpremature entry into mitosis and the formation of abnor-mally small cells (Harvey and Kellogg, 2003). Swe1 accu-mulates during the S phase and is hyperphosphorylatedin order to be target for ubiquitin-mediated degradation(Sia

et al

., 1998; Shulewitz

et al

., 1999; Sreenivasan andKellog, 1999; McMillan

et al

., 2002; Sakchaisri

et al

.,2004). Interestingly, a multikinase system, including Clb2-Cdc28 and the polo-like kinase Cdc5, contributes to thishyperphosphorylation (Asano

et al

., 2005; Harvey

et al

.,2005; Lee

et al

., 2005). In contrast, hypophosphorylationof Swe1 results in its stabilization and therefore in a Swe1-dependent inhibition of Clb-Cdc28 (Lew, 2003).

In

S. cerevisiae

, hyperpolarization of growth can be trig-gered by some environmental conditions, such as nitro-gen deprivation or exposure to branched-chain alcohols,which result in the so-called filamentous or invasivegrowth. For instance, under nitrogen limitation, cellsswitch from the typical yeast to the pseudohyphal mor-phology, characterized by the presence of elongated cellsarranged in chains (Gimeno

et al

., 1992). This conditionactivates two partially interconnected morphogeneticpathways, MAPK and cAMP, and results in an alterationof the gene expression pattern (i.e.

FLO11

). However,other parameters related to the cell cycle, including a G2/M cell cycle delay and actin polarization, are also affected.This was explained by the finding that the morphogeneticMAPK and cAMP pathways connect with well-known cellcycle regulators, including Clb2/Cdc28 and its inhibitorSwe1, suggesting that cell cycle components could serveas a link between environmental factors and filamentation(Rua

et al

., 2001). Although

swe1

∆

mutants form pseudo-hyphae in response to nitrogen-limited growth (Ahn

et al

.,1999), Swe1 seems to contribute to pseudohyphal forma-tion under certain conditions (La Valle and Wittenberg,2001), suggesting the existence of at least two pathways.When induced by branched-chain alcohols, filamentationhas been shown unambiguously to be dependent onSwe1 (La Valle and Wittenberg, 2001; Martinez-Anaya

et al

., 2003).

Candida albicans

possesses an even more complicateddifferentiation programme as, in addition to the yeast andpseudohyphal phases, the organism exhibits a hyphalmorphology similar in a number of ways to filamentousfungi (Calderone, 2002). During the hyphal growth of

C. albicans

, deposition of new material leading to theenlargement of the cell wall is restricted to the apical tip.During the

C. albicans

yeast–hyphal transition or morpho-

genesis, polarization of growth is, at least in part, con-trolled by the environment. Several independent circuits,including PKA, MAPK, pH and microaerophilic signallingpathways, promote hyphal development by activating pos-itive effectors or transcription elements, whereas othersfactors, such as a combination of repressors, Nrg1-Tup1and Rfg1-Tup1, repress filamentation (Liu, 2001; Brown,2002; Kadosh and Johnson, 2005). However, there isgrowing evidence that cell cycle components are alsoinvolved in hyphal formation. For instance, the G1 cyclinCln1 is necessary to maintain hyphal growth under someconditions (Loeb

et al

., 1999), and a regulator of B-cyclins(Clb2 and Clb4) gene expression is required for hyperpo-larized bud growth (Bensen

et al

., 2002). More recently, ithas been shown that depletion of a homologue of the polokinase Cdc5, which is involved in the adaptation to theDNA-damage checkpoint in

S. cerevisiae

(Toczyski

et al

.,1997; Sanchez

et al

., 1999), as well as loss of Nimkinases Hsl1 and Gin4 induces hyphal-like growth in

C. albicans

(Bachewich

et al

., 2003; Wightman

et al

.,2004). Compelling evidence for the involvement of cyclinsin hyphal formation has been recently provided by Zheng

et al

. (2004), who discovered a new G1 cyclin-relatedprotein, Hgc1, whose deletion abolishes hyphal growth.In addition, the G1 cyclin Cln3 regulates morphogenesisby negatively regulating the yeast hyphal transition(Bachewich and Whiteway, 2005; Chapa y Lazo

et al

.,2005), and depletion of either mitotic cyclin Clb2 or Clb4results in hyperpolarization of growth (Bensen

et al

.,2005). Finally, cell cycle arrest during S or M phases alsogenerates polarized growth (Bachewich

et al

., 2005).Hyperpolarization of growth in

S. cerevisiae

can also betriggered by internal signals other than regular cyclicevents of the cell cycle as a number of conditions thatslow DNA synthesis and activate checkpoints (includingtreatment with hydroxyurea, MMS, nucleotide-analogues,or defects in DNA polymerase

∈

or DNA ligase Cdc9p)also induce filamentation (Jiang and Kang, 2003). TheDNA damage signal occurs through the checkpoint kinaseRad53 which phosphorylates Swe1p, thus connectingwith the morphogenetic checkpoint transmission pathway.In a recent study, we characterized the Rad52 from

C. albicans

, showing that it plays a role in homologousrecombination (HR), DNA repair and genomic stability in

C. albicans

(Ciudad

et al

., 2004). In addition,

RAD52

isrequired for virulence in a mouse model of disseminatedcandidiasis. Of interest, the

RAD52

gene-reconstitutedstrain repairs a partially deleted chromosome during infec-tion and, in doing so, virulence is restored to the level ofsingle-gene strains (Chauhan

et al

., 2005). In the currentstudy, we further show that null

rad52

mutants arefilamentous and constitutively express ‘hyphal’-specificgenes (HSG). Interestingly, this filamentation was inde-pendent of the presence of transcription factors Efg1 and

1454

E. Andaluz

et al.

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd,

Molecular Microbiology

,

59

, 1452–1472

Cph1, even though disruption of

EFG1

prevented expres-sion of HSG, including the hypha-specific cyclin Hgc1. Alarge number of Rad52-depleted cells arrest in G2/M,suggesting that some intrinsic DNA damage caused bythe absence of Rad52 triggers the DNA replication and/or DNA-damage checkpoints. Filamentation seems tooccur through the subsequent uncoupling of severalevents of the cell cycle.

Results

Effect of Rad52 disruption on growth rate, colony and cell morphology and agar invasiveness

Parental strain CAF2 and several

rad52

Ura

+

mutantderivatives, including heterozygous TCR1, null strainsTCR2.1 and TCR2.2, and two reintegrants, TCR3.1.1 andTCR3.2.1, were subjected to a basic phenotypic analysis.As shown in Table 1 for strains grown in YEPD, null strains(TCR2.1 and TCR2.2) exhibited a generation time signif-icantly longer (140 min) than the heterozygous TCR1(80 min), which grew at the same rate as parental CAF2[or CAI4 supplemented with uridine (Uri)]. ReintegrantsTCR3.2.1 (Table 1) and TCR3.1.1 (not shown) grew at thesame rate as the heterozygous TCR1 (g

=

80 min). Simi-lar conclusions were reached when these strains weregrown in minimal synthetic medium (SC).

Colonies from null strains TCR2.1 and TCR2.2 (notshown) (Ura

+

) were wrinkled with a thorny or spinyappearance and produced radiating filaments (Fig. 1A, cand Fig. 1B, b), whereas those of CAF2 and the heterozy-gote were smooth and lacked peripheral filaments(Fig. 1A, a and b). As shown for reintegrant TCR3.1.1, re-introduction of the gene in either null strain restored thecolonial morphology of the wild type (Fig. 1A, d). Whengrown in liquid YEPD,

rad52

∆

cultures contained, in addi-tion to yeast cells, elongated forms that included filamen-tous cells and pseudohyphal-like elements as well asmalformed cells (Fig. 1A, g and Fig. 1B, a), whereasCAF2, heterozygous and reintegrant grew as a yeast(Fig. 1A, e, f and h). Interestingly, filamentation was sig-nificantly more abundant on agar (Fig. 1B, b) than in liquid

media (Fig. 1B, a), suggesting that a solid surface favoursthe elongation of

rad52

∆

cells. The absence of

URA3

didnot change the phenotypes of these strains (not shown).These differences in morphology decisively influenced thevalue of colony-forming units (cfu) per volume of cell sus-pensions with identical OD

600

(Table 1). For example, thecfu values of the

rad52

null strains were sixfold lower thanthe Rad52

+

counterparts in exponentially growing YEPDcultures. It is likely that, in the mutant, aggregations ofseveral nucleated cells frequently serve as progenitors ofa single colony. A second reason that may account for thisbehaviour is the presence of dead cells in

rad52

∆

cultures,as colonies of this mutant were slightly pink in phloxin Bplates (Fig. 1A, i–l). Finally, the enlargement of

rad52

cellscould also contribute to an increase in the OD value.

The above-mentioned strains were also grown in twodifferent media, M199 and Spider, known to promotehyphal development. As expected, parental and heterozy-gote strains gave rise to colonies with abundant peripheralprojections indicative of hyphal growth, but, unexpectedly,these projections were significantly reduced in the nullmutant (Fig. 1C). This phenotype could be attributed tothe

rad52

mutation, as the revertant TCR3.1.1 partiallyregained the wild-type pattern. These observations sug-gest that most filaments produced by

rad52

∆

cells are nottrue hyphae but result from an incomplete or even differentdevelopmental programme that, like hyphae, includeshyperpolarization of growth.

The filamentous nature of the

rad52

∆

strains was par-alleled by a significant increase in their invasiveness

invitro

. When grown in SC plates (minus Uri),

rad52

∆

Ura

+

strains penetrated the agar whereas wild type or the het-erozygote did not (Fig. 1D), and the same occurred inYEPD plates (not shown). Reintegrants TCR3.1.1 andTCR3.2.1 (Fig. 1D) behaved like the heterozygote, i.e.non-invasive.

Characterization of a conditional RAD52 strain

The fact that two

rad52

strains are filamentous andexhibit a significantly lower growth rate and that re-introduction of

RAD52

consistently restores both pheno-types to those of the wild type strongly suggests thatthose alterations are indeed due to the absence ofRad52 and not to unrelated mutations by cells accumu-lated during growth as a consequence of intrinsic defectsin DNA repair. To further analyse the correlation of geno-type and phenotype, we constructed and characterized aconditional

rad52

strain, EAT4.1, in which a wild-typeallele is regulated by the

MET3

promoter (see

Experi-mental procedures

). Northern analysis indicated that the

RAD52

message was present under non-repressiveconditions but almost disappeared in less than 15 minfollowing the addition of methionine (Met)/cysteine (Cys)

Table 1.

Generation time and relationship of cfu to OD

600

in wild typeand

rad52

mutants grown in YEPD.

Straincfu ml

–1

at OD

600

=

1(exponential phase)

Generation time(min)

SC5314 6

×

10

7

60CAF2 6

×

10

7

80CAI4 (

+

Uri) 6

×

10

7

80

RAD52/rad52

Ura

+

(URA3) 6 × 107 80Reintegrant (TCR3.2.1) 6 × 107 80rad52/rad52 Ura+ 1 × 107 140rad52/rad52 Ura– (+Uri) 1 × 107 140

Filamentation of rad52 mutants of C. albicans 1455

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 59, 1452–1472

and remained at almost undetectable levels during thenext 5 h (Fig. 2A). This indicates that the half life of theRAD52 mRNA is very short and that the MET3 promoterwas effectively repressed by Met/Cys. However, reversetranscription polymerase chain reaction (RT-PCR) exper-iments indicated the presence of small amounts of spe-cific RAD52 mRNA following shift of the conditionalEAT4.1 to repressive conditions for 15 min and 5 h(Fig. 2B) indicating that although repression was effective(compared with the unrepressed control) it was not abso-lute (as seen in the rad52∆ rad52∆ control). In spite ofthis basal expression of RAD52, when incubated underrepressive conditions, strain EAT4.1 showed phenotypesof the rad52∆ null strain suggesting that the levels of

Rad52p are low enough to compromise cell function.Thus, repressed EAT4 was at least as sensitive to MMS(Fig. 2C) and UV light (not shown) as a null strain(TCR2.2). In addition, it was as invasive as the null strain,as shown by its capacity to penetrate agar (Fig. 2E).As expected, under non-repressive conditions, EAT4.1behaved as the heterozygote in the same assays. Whenderepressed EAT4.1 yeast cells were plated on SC orYEPD plates containing Met/Cys, many of them devel-oped into filamentous microcolonies (Fig. 2D, a and b),although the extent of filamentation was lower than withthe null strain, probably due to the presence of lowamounts of Rad52 or to the absence of secondaryeffects in the short-term culture of the depleted strain; the

Fig. 1. Colony and cellular morphology of parental strain CAF2 and rad52 mutants.A. Cells from a 24 h liquid YEPD culture (see Experimental procedures) were streaked on agar plates containing YEPD (a–d), or YEPD plus phloxine B (5 µg ml–1) (i–l) and photographed after 48 h (× 100) or incubated in fresh liquid YEPD (e–h) for 2 h at 30°C in a shaker before being photographed (×400).B. Photomicrographs of rad52∆ filaments in liquid YEPD (a) and the initiation of colony formation on YEPD agar plates (b) at a higher magnification. Cultures were prepared as in (A).C. Colonial morphology of the indicated strains in M199 and Spider agar plates.D. Invasiveness of the indicated strains. Cells of the indicated strains were streaked on a SC agar plate which was incubated for 48 h, and colonies were washed from the plate with sterile water.

CA 2RCT 1.25dar ∆/ 5dar 2∆

CT 1.1.3R/25DAR ra 25d ∆ R

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a b

c d

AR/25DAR 25D

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e f

g h

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hgfe

dcba

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setalpDPEY

EYdiuqiL DP

setalpDPEYpl hloxine BPsu

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1. )1.

mµ02

a

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b

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1456 E. Andaluz et al.


same was true in liquid medium (see Fig. 5D, e). Inaddition, as described for the null strain, filamentation ofrepressed EAT4.1 was much less prominent in liquidmedium than in solid agar. In any case, the early appear-ance of these colonies suggests that the filamentousmorphology yielded by rad52∆ cells is indeed due toRad52 depletion and not to secondary mutations. On theother hand, both unrepressed EAT4.1 (Fig. 2D, c and d)and the heterozygote supplemented with Met/Cys (not

shown) gave rise to smooth colonies formed almostexclusively by yeast cells. A decrease in the number offilaments was observed after long incubation periods (i.e.23 h) in the presence of Met/Cys, when both Northernand RT-PCR analyses indicated the presence of signifi-cant amounts of RAD52 mRNA (Fig. 2A and B). Botheffects are likely due to the consumption of Met/Cys or tothe lysis of filaments. In the absence of Met/Cys, EAT4.1grew as yeast in both liquid SC and YEPD (not shown).

Fig. 2. Characterization of the RAD52 conditional strain EAT4.1.A and B. Northern (A) and RT-PCR analysis (B) of RAD52 expression in EAT4.1 under repressive and non-repressive conditions.C. Sensitivity of EAT4.1 to MMS. About 200 cells were spread on SC plates containing 0.01% MMS supplemented or not with Cys/Met. Plates without MMS were spread in parallel and used as controls. Following a 48 h incubation at 30°C, colonies were counted. Both a heterozygote RAD52/rad52 (TCR1) and a rad52∆ null (TCR2.2) strains were used as controls.D. Colonial and cellular morphology of the indicated strains. EAT4.1 in SC (a and c) or YEPD (b and d) with (a and b) or without (c and d) 2.5 mM Met/Cys. The age of the colonies was 6 h (c and d), 12 h (a and b).E. EAT4.1 behaves invasive in SC plates in the presence but not in the absence of Met/Cys (see legend of Fig. 1).

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51 min h5m0 in

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32 hC/M+DPEY

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b

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M+ e syC/t - syC/teM1.4TAE

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c

a

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Expression of hypha-specific genes in rad52∆ strains

To know whether filamentation of rad52∆ null strains wasaccompanied by the expression of HSG, we performedNorthern hybridization using specific probes of ACT1,ECE1 and HWP1. ACT1 is expressed at similar levels inboth yeast and hyphae, whereas ECE1 and HWP1mRNAs are significantly increased in hyphae (Birse et al.,1993; Staab et al., 1999; Kadosh and Johnson, 2005).Parental CAF2 and the several C. albicans rad52 mutant

strains were first grown under conditions that favour theyeast form (YEPD at 30°C). As shown in Fig. 3A, themRNA levels of both HSG were almost undetectable inthose strains that grew as yeast (CAF2, heterozygoteTCR1, and reintegrant TCR3.2.1). In contrast, expressionof these genes was very high in rad52∆ cells (TCR2.2),which, under the same conditions, produced extensivefilamentation. As expected, the addition of 10% serum (at37°C) to Rad52+ cells resulted in both a massive formationof filaments (not shown) and a significant induction of

Fig. 3. Analysis of morphogenetic programmes involved in rad52∆ filamentation.A and B. Expression of hyphal specific genes in rad52∆ cells. Total mRNA from the indicated strains (heterozygote TCR1, +/–; null rad52∆, –/–; and revertant TCR3.2.1, R) and conditions were electrophoresed and hybridized to internal fragments of ECE1 and HWP1 (A) or HGC1 (B). As loading controls, the C. albicans actin gene (ACT1) (A) or 26S RNA (B) were used.C. Efg1, Cph1 and Hgc1 are not required for filamentation of rad52∆ cells. The indicated mutants were incubated at 30°C for 2 h in liquid (upper row) or 48 h on solid YEPD agar (lower row).D. Efg1 is required for expression of ECE1 in rad52∆. The indicated strains were grown at 30°C in YEPD to mid-log phase and processed for Northern analysis using the ECE1 probe.E. Efg1 is required for expression of HGC1 in rad52∆. The indicated strains were grown at both 30°C or at 37° in the presence of 10% serum, and processed for RT-PCR.

BA

C 2FAC 25dar D 1gfe D 1gfe D 1hpc D 1gfe D 5dar 2D 1gfe D 1hpc D dar 52D gh 1c D ar 25d D

CE 1E

1PWH

TCA

2FAC -/+ - -/ R C 2FA + -/ -/- R

PEY D 03 ˚C PEY D 1+ 0 mures% 37˚C

1CGH ANRm

Cº73,mureSSC 4135 RCT 2 1. RCT 2 2.

25DAR 25dar ∆

ANRr 62 S

1cgh+- + +- --

DE

CE 1E

TCA

25dar D 1gfe D1gfe D1hpc D

1gfe Dar 5d 2 D

1gfe D1hpc D25dar D

1.2 kb

1cgh ∆25dar ∆ 1gfe ∆

1gfe ∆25dar ∆ 25dar ∆

-

+

-

+

+

-

-

+

-

+

+

-

-

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AmureS

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ECE1 and HWP1. However, no significant change in theexpression of these genes was observed when rad52∆cells were supplemented with 10% serum. Figure 3A alsoshows that the level of the ACT1 message remained con-stant in all the strains and was not modified by the additionof serum. These results demonstrate that the constitutivefilamentation of rad52∆ cells is accompanied by theinduction of genes that are usually derepressed duringmyceliation.

Recently, a novel C. albicans G1 cyclin, Hgc1, has beenshown to be specifically expressed during hyphal induc-tion, and a hgc1∆ mutant was defective in hyphal growth(Zheng et al., 2004). In order to know whether filamenta-tion of Rad52-depleted cells was accompanied of theinduction of this cyclin, we performed Northern analysis.As shown in Fig. 3B, parental SC5314 grown in YEPD at30°C did not express HGC1 but did when supplementedwith 10% serum and incubated at 37°C; in contrast,rad52∆ cells from mutant TCR2.1, incubated in YEPD at30°C, showed significant levels of specific HGC1 mRNA,and the amount of this transcript remained at about thesame level upon induction of hyphal growth at 37°C in thepresence of serum. A second rad52∆ strain (TCR2.2) alsoconstitutively expressed HGC1 at 30°C in the absence ofserum (Fig. 3B). As expected HGC1-specific mRNA wasabsent in a hgc1∆ deletant, even in the presence of serum(Fig. 3B). Accordingly, filamentation of rad52∆ cells isaccompanied by expression of several HSG, includingECE1, HWP1 and HGC1.

Filamentation of rad52∆ null cells occurs in the absence of Efg1, Cph1 and Hgc1, and is independent of an operative NHEJ pathway

Various signalling pathways, defined by specific transcrip-tion factors, have been reported to control myceliation inC. albicans. In particular, the transcription factor Efg1, adownstream effector of the Ras1/cAMP pathway, plays animportant role in morphogenetic regulation in response tostarvation, pH and serum stimulation, whereas the tran-scription factor Cph1, an effector of the MAP kinase path-way, only channels responses to a limited set of starvationconditions (Stoldt et al., 1997; Liu, 2001; Brown, 2002).In order to determine the role of these factors in theconstitutive filamentation of rad52∆ cells, we deleted bothcopies of RAD52 in both efg1∆ and efg1∆ cph1∆ geneticbackgrounds (Ciudad et al., 2004). As shown in Fig. 3C(lower row), young colonies of both efg1∆ and efg1∆cph1∆ contained, like parental CAF2, exclusively yeastcells whereas colonies of either efg1∆ rad52∆ or efg1∆cph1∆ rad52∆ developed filaments, like rad52∆. Further-more, old colonies of wild type, efg1∆ and efg1∆ cph1∆were completely smooth whereas those of rad52∆, efg1∆rad52∆ or efg1∆ cph1∆ rad52∆ had abundant peripheral

filaments (not shown). In agreement with this, liquid cul-tures of efg1∆ or efg1∆ cph1∆ contained blastosporesalmost exclusively whereas the introduction of the rad52mutation in any of these backgrounds resulted in a sig-nificant amount of filamentous cells (Fig. 3C, upper row).We conclude that the filamentation caused by theabsence of RAD52 may occur in the absence of the mor-phogenetic pathways defined by Efg1p and Cph1p.

It has been reported that transcription of ECE1 dependson the Efg1 pathway (Sharkey et al., 1999; Braun andJohnson, 2000). In agreement with this result, ECE1 wasexpressed in rad52∆ but not in efg1∆ rad52∆ or efg1∆cph1∆ rad52∆ strains (Fig. 3D), and the same was truefor HWP1 (not shown). Expression of HGC1 has beenreported to be also dependent on the cAMP/PKA signallingpathway and, accordingly, its mRNA was not produced inan efg1∆ null strain even in the presence of serum (Zhenget al., 2004; our own results). Furthermore, as shown inFig. 3E, it was not expressed in the double mutant efg1∆rad52∆. Therefore, the absence of Efg1p prevents expres-sion of ECE1, HWP1 and HGC1 but not filamentation ofrad52∆ strains, an indication that this morphogenetic eventcan occur in the absence of these HSG. In agreementwith this, the double mutant hgc1∆ rad52∆ filamentedconstitutively to the same extent as rad52∆ (Fig. 3C).

We have recently reported that a lig4∆ null mutant,which is defective in the non-homologous end-joining(NHEJ) recombination pathway, was slightly less mycelialthan wild type (Andaluz et al., 2001). Accordingly, wetested the possibility that filamentation of rad52∆ was aconsequence of an imbalance between both recombina-tion pathways caused by deficiencies in HR. However, wecould not find differences in morphology between rad52∆and a double mutant rad52∆ lig4∆ suggesting that fila-mentation in the former is not due to an exacerbation ofthe NHEJ pathway in the absence of HR (data not shown).

Filamentation of rad52∆ null cells is seriously compromised in cells lacking adenylate cyclase

Upstream components of the cAMP pathway, Ras1 andCdc35, are also essential for yeast to hypha morphogen-esis, but the loss of Cdc35 is more restrictive than the lossof Ras1 (Feng et al., 1999; Rocha et al., 2001). Further-more, Bachewich et al. (2003) have shown that Cdc5-depleted and hydroxyurea-induced filaments could alsoform in the absence of Efg1 and Cph1, but required ade-nylyl cyclase signalling, as they were not formed in theabsence of CDC35, the structural gene for the adenylatecyclase. In order to determine whether filamentation ofrad52∆ strains showed a similar requirement, we investi-gated the phenotype of the double mutant cdc35∆ rad52∆.The mutant was constructed by deleting both copies ofRAD52 in a cdc35 null strain, previously shown to lack



both adenylyl cyclase and detectable levels of intracellularcAMP (Rocha et al., 2001). As shown in Fig. 4A (upperrow), filamentation of rad52∆ cells occurred in theabsence of Cdc35, but the number and length of thefilaments were significantly reduced, whereas the numberof yeast-like cells increased in parallel. Addition ofdibutyryl-cAMP (10 mM) to cdc35∆ rad52∆ cells improvedfilamentation to nearly the level of the rad52∆ strain. Thiswas true on YEPD plates, especially in 48–72 h old colo-nies (Fig. 4A, lower row) as well as in liquid SC (Fig. 4B,lower row). As expected, cAMP did not influence thecolony morphology of rad52∆ or cdc35∆ cells (Fig. 4A).Three additional cdc35∆ rad52∆ independent doublemutants behaved similarly (not shown).

rad52∆ cells exhibit a nuclear division defect and constitutive actin polarization

A large number of rad52∆ yeast cells (n = 200) in thestationary phase have small (14%) or large (43%) buds,

suggesting that they are in the late S or G2/M phases ofthe cell cycle, whereas under the same conditions, a largepercentage of wild-type cells (n = 213) are unbudded(86%). More important, DAPI staining of exponentiallygrowing cultures indicated that whereas most wild-type(CAF2) yeast cells carrying medium size or big buds con-tain two nuclei (88%), one in the mother and the other inthe daughter (Fig. 5A, c and d), a significant number ofrad52 cells (from either null rad52∆ strain, TCR2.1 orTCR2.2) with large buds have only one nucleus (97%),either in the mother or in the daughter (54%), or, quitefrequently, as a thread between mother and daughter cells(43%) (Fig. 5A, a and b; Table 2).

In addition to budded yeast and long filaments, a signif-icant number of rad52∆ cells had elongated buds of vari-able sizes and shapes (Fig. 5B) that mimic incipient germtubes. Interestingly, at a high frequency (≥ 70%) the undi-vided nucleus is located in the ‘germ tube’, in such a waythat the rounded basal cell appears empty (Fig. 5B, a).Furthermore, in these cases, the marked constriction

Fig. 4. Effect of the absence of cAMP in the filamentation of rad52∆ cells.A. Cells of the indicated strains were inoculated on YEPD agar plates in the absence (upper row) or in the presence (lower row) of 10 mM dibutyryl-cAMP. They were photographed at the indicated times.B. Cells of the cdc35∆ rad52∆ strains grown in liquid SC media.

A2.2RCT 25dar( ∆/ 25dar ∆)1ARC 53cdc( ∆/ 53cdc ∆ 25dar ∆/ 25dar ∆) 612RC 53cdc( ∆/ 53cdc ∆)

h51

h51

h03

h03

h27 h27

h27 h27 h27

h27

c- AMP

PMAc+

B- c PMA + PMAc

1ARC 53cdc( ∆/ 53cdc ∆ 25dar ∆/ 25dar ∆)



between both cells as well as the calcofluor staining pat-tern suggests that the septum is synthesized beforenuclear division, preventing migration of one of the copiesto the empty cell (Fig. 5B, a). Most (≥ 80%) of the elon-gated evaginations arising in rad52 cells had a taperedappearance suggesting that they were produced by G2/Mcells. In some cases, it is likely that both mother anddaughter cells carry one nucleus, but nuclear division isimpaired in one of them (or later in the progeny) and thatcell polarizes growth (Fig. 5B, b). Pseudohyphal ele-ments, some of them significantly elongated, usuallycoexist with ‘germ tubes’, but again some cells in the chainlack nuclei (Fig. 5B, c).

Fig. 5. Microscopic analysis of rad52∆ mutant cells.A. Exponentially growing yeast cells from rad52∆ (a and b) and CAF2 (c and d). In yeast cells of rad52∆, nuclei are frequently found between mother and daughter cells (arrows).B. Early elongation steps of rad52∆ cells indicating the position of the nucleus in the incipient germ tube. Most cells elongate in G2/M. The stain used is indicated on each frame. For details, see text.C. Detail of elongated cells stained with calcofluor white (CFW) to highlight the shape and the septa showing the typical pseudohyphal morphology (a). Still, some filaments do not show constrictions at the septa and mimic true hyphae (b and c).D. Filaments of rad52∆ (a–d) or repressed EAT4.1 (e) cells showing the position of the nuclei and the basal anucleated cells. For details, see text.E. Rhodamine-phalloidin staining young rad52∆ incipient (b) and longer (d) filaments. DIC pictures are also shown (a and c). Each bar corresponds to 5 µm. Cells were first grown in YEPD broth for 24 h at 28°C and then grown for 2–6 h in fresh liquid YEPD before being fixed.

25dar D

2FAC

CID IPAD

a b

c d

BA

C

E

WFC a

b

c

CID

WFC

D

AD +IP CFW a

AD +IP CFW

c

AD +IP CFW b

db

ac

a AD IPb

c

AD +IP CFW

AD +IP CFW

d

e

AD IP

AD IP

Table 2. Number and position of nuclei in G2/M cells from exponen-tially growing rad52∆ cultures.

1 nucleus 2 nuclei

One nucleusbetween both cells

TCR2.1 (rad52∆) 108 6 86TCR2.2 (rad52∆) 107 10 83SC5314 (wild type) 12 176 12



The presence of more cell bodies than nuclei was alsoa constant feature of longer filaments (Fig. 5D, b and c),although filamentous cells with two nuclei were occasion-ally seen (Fig. 5D, a). Furthermore, not all the nuclei inthe filaments showed the same fluorescence, suggestingthat some of them do not contain a full set of geneticinformation (Fig. 5D, b, arrow). Again, about 70% of thebasal cells were anucleated (Fig. 5D, a–c), and themarked constriction between the basal cell and the fila-ment suggested that, in these cases, a regular septumhad been formed. This was further confirmed by calcofluorwhite staining of this kind of cell (Fig. 5D, c). Long fila-ments formed axial buds, sometimes very elongated, atboth poles, or even in the middle of the filament (Fig. 5C,a), and it was not infrequent to observe nuclei migratinginto the buds (Fig. 5D, d). Although most long filamentsexhibited features of pseudohyphae, some of them wereas narrow as hyphae and, most importantly, did not showconstrictions at the septa, suggesting that some truehyphae may be also present in rad52 cultures (Fig. 5C, band c). In fact, after 24 h, liquid cultures showed somefilaments as long as 100 µm and contained elongatedcells 20 µm long. Also, some elongated cells in thefilaments lyse, suggesting a weak cell wall (Fig. 5D, d).They probably correspond to anucleated filaments. Asexpected, the repressed conditional EAT4.1 showed thecharacteristics described for the null strain with regard tothe distribution of nuclei and cell bodies (Fig. 5D, e).

Actin staining of wild-type yeast cells indicated thetypical pattern (Anderson and Soll, 1986; Hazan et al.,2002), i.e. actin patches clustered at the tip in small ormedium-sized buds, but dispersed over the surface inlarge buds, and forming a double ring at both sites of thesepta (not shown). In elongated rad52∆ cells, a densearea of actin patches locates at the tip (Fig. 5E, a and b),and this was maintained in longer filaments (Fig. 5E, cand d). Still, as actin patches along the entire filamentwere frequently observed, it is feasible that some of themcould be eventually recruited at the site of branching orbudding.

These observations suggest that rad52∆ cells often failto complete nuclear division, possibly as a consequenceof the inefficient repair of spontaneous double-strandbreaks (DSB), which triggers the DNA replication and/orDNA-damage checkpoint. This causes an uncoupling ofother landmark events of the cell cycle, such as the peri-odic rearrangement of the actin cytoskeleton and alterna-tive isotropic/polarized secretion of cell wall material thatultimately leads to filamentation.

Yeast cells present in rad52∆ cultures are still responsive to serum

Addition of 10% serum to rad52∆ cells yielded mycelial

projections which were narrower than the spontaneousfilaments seen in the absence of serum and had theappearance of true hyphae. However, filaments frommutant cells induced by serum exhibited a number ofpeculiarities when compared with those produced by wild-type cells under the same conditions. First, as expectedfrom the cell cycle distribution of rad52∆ cells in liquidcultures, most cells (about 60% of the cells in the yeastform) formed a germ tube when they were in G2/M exhib-iting the characteristic morphology shown in Fig. 6A, c–e.In contrast, in wild type, more than half of the germ tubeswere produced by cells in G1 (Fig. 6A, a and b). In fact,as described before (Hazan et al., 2002), wild-type cellsgerminated regardless of the stage of their cell cycle, butG1 cells were more abundant in wild type than in the nullmutant. Second, even after 2 h in serum, when almost allhyphae developed from wild-type cells had at least oneseptum (Fig. 6A, b), there were almost no septa in theserum-induced rad52 filaments, in spite of the fact thatthey had a similar length (Fig. 6A, d). This could reflectthe longer generation time of rad52∆ cells. However, whena septum was formed it appeared regular (Fig. 6A, e).Third, before mitosis, the nucleus had migrated into thegerm tube in both wild-type and rad52 cells (Fig. 6B).However, whereas in wild-type cells, one of the resultingnuclei from the nuclear division migrated back into themother cell (Fig. 6B, a and b), in rad52 most basal cellswere anucleated (Fig. 6B, c). Again, this is probably dueto the fact that the chitin ring formed before nuclear divi-sion and/or its migration back into the mother cell. In fact,some nuclei seemed to be trapped in the septum (Fig. 6B,d). Other characteristics of the serum-induced rad52∆filaments are similar to those described for wild-type cells(Hazan et al., 2002). For instance, no septum between thebasal cell and the rest of the filament was observed whenthe germinating cell was in G1 (Fig. 6A, b). Also, whenthe basal cell was in G2/M, a chitin ring formed betweenmother and daughter cells (Fig. 6A, c–e), a situation sim-ilar to that described above for the spontaneous filamen-tation of rad52∆. Finally, as expected (Anderson and Soll,1986; Hazan et al., 2002), actin localized almost exclu-sively at the tip of the germ tube in both wild type (notshown) and rad52∆ cells (Fig. 6C, a and b). This situationis different from that observed during the spontaneousfilamentation of rad52∆ cells where additional actinpatches were also distributed along the filaments, sug-gesting the induction of a more specific and finely regu-lated hyperpolarization programme.

Incubation of rad52∆ cells in Lee’s medium alsoinduced germination. Again, many germ tubes arose fromthe buds of cells apparently arrested in G2/M and wereshorter than their counterparts from parental CAF2 andrevertant TCR3.2.1 which usually were formed from G1cells (not shown).



Cell cycle in Rad52-depleted cells

The large number of filaments present in rad52∆ mutantsmakes elutriation difficult. Accordingly, we elutriated cellsof the non-repressed conditional strain EAT4.1. Smallunbudded EAT4.1 cells (G1) were collected and used toinoculate fresh SC medium supplemented with Met/Cys.As the absence of both amino acids increases the gener-ation time of EAT4.1 (not shown), we used as a controlthe heterozygote RAD52/rad52 incubated in the presenceof Met/Cys. The resulting synchronized cultures weremonitored for 195 min (about two cell cycles). As shownin Fig. 7A, downregulation of RAD52 significantly in-creases the length of the cell cycle, initially indicated by adelay in the completion of the first mitosis (transition ofthe G2/M cells to G1). This delay was exacerbated in thesecond cycle, suggesting the progressive induction of theDNA-damage checkpoint. As a consequence of thisbehaviour, the culture partially lost its synchrony and theproportion of G2/M cells never decreased below 25% ofthe total population, whereas in the control it reachedvalues lower than 10%. In addition, during the second

cycle, the transition of small buds to large buds was alsosignificantly retarded, suggesting the additional presenceof a replication block. Fluorescent-activated cell sorting(FACS) analysis of the same samples (DNA content;Fig. 7B) was consistent with these observations. The firstDNA duplication started at about the same time in bothcultures, but in the repressed strain, a delay in the transi-tion from 4N (G2/M) to 2N (G1) occurred. For instance, at105 min about 40% of the tetraploid cells in the controlhad returned to 2N, whereas in the repressed culture 90%still remained as 4N (G2/M). This delay not only subsistedbut was accentuated in the second cycle. Thus, at150 min, nearly 100% of the cells in the control were again4N (G2/M), whereas in the repressed culture only 30%were 4N; it is likely that part of this 30% includes remnantsof the G2/M cells of the first cycle that were arrested atthat phase. Furthermore, as suggested by the buddingpattern, a large percentage of the cells expressing themutation could not finish the second cell cycle, as after150 min transition from 2N to 4N occurred very slowly ifat all in the mutant, whereas Rad52+ cells progressed atthe same rate as in the first cycle (Fig. 7B). Determination

Fig. 6. Response of rad52∆ cells to serum.A. Morphology and septum formation determined by calcofluor white staining of wild-type (a and b) and rad52 cells (c–e). Eighteen-hour-old liquid cultures were supplemented with 10% serum for 2 h. For details, see text.B. Nuclear dynamics during hyphal induction by serum at 37°C (a–d) in wild-type (a and b) and rad52∆ cells (c and d) as determined by DIC and/or DAPI staining.C. Rhodamine-phalloidin staining of rad52∆ (a and b) germ tubes. Each bar corresponds to 5 µm.

A

b

25dar D /D 2D5dar

25dar D /D 2D5dar 25dar D /D 2D5dar

2FAC

d

ea

c

CB 2FAC

a

b

d

c

ab



of the position of the nuclei by DAPI staining in buddedcells was also consistent with the above observations.Figure 7D illustrates the situation at 120 min. Most controlcells had completed nuclear division and some had smallbuds, whereas a large percentage of repressed cellsremained in G2/M with a single nucleus confined to themother cell. As shown in Table 3, after 90 min the propor-tion of budded cells with a single nucleus remained veryhigh in mutant cells whereas it fluctuated in Rad52+ cellsas expected from a regular cell cycle. Concomitantly, theproportion of budded cells with two nuclei remained fairlyconstant in the mutant (likely because of a partial loss ofsynchrony) and yielded the expected pattern in the con-trol, and the same was true for cells with one nucleuslocated in the neck. A similar picture was provided by theanalysis of the cell size (FSC-H) (Fig. 7C). During thesecond cycle, a low percentage of mutant G1 cells shifted

to the size expected for G2/M cells, whereas control cellsbehaved normally. In addition, at 195 min, a significantpercentage of cells exhibited a significantly smaller sizethan the unbudded cells, probably representing anucle-ated cells, as they did not have a DNA signal. Theseresults, together with our morphological observations,confirm that rad52 cells have aberrant DNA replicationand likely induce the DNA-damage checkpoint. However,two cell cycles in the presence of Met/Cys were notenough to induce elongation of rad52 cells.

Filamentation of rad52 mutants is partially dependent on Swe1

The observation that the G2/M arrest was an early phe-notype derived from Rad52 depletion suggests that elon-gation could be a secondary event derived from the action

Fig. 7. Cell cycle analysis of Rad52-depleted cells. Yeast cells of heterozygote TCR1 and the non-repressed conditional EAT4.1 were subjected to elutriation.A–C. G1 cells were inoculated in SC medium supplemented with Met/Cys and the culture followed for 195 min by (A) counting unbudded cells (�), cells with small buds (�) and cells with big buds (�), and FACS analysis determining DNA content (B) and cell size (C).D. DAPI staining of the indicated cells.

0 50 1 00 51 0 0020

05

01 0

51 0

02 0

52 00

05

01 0

51 0

02 0

52 0

)nim( emiT

Num

ber

ofce

lls

aR d + -/M+ syC/te

TAE 4 1.M+ syC/te

lleC H-CSF ,ezis

,tnetnoc AND LF H-2

daR + -/ eM+ t yC/ s TAE 4 1. teM + / yC s

C

A

D IPAD/ygolohprom lleC ts nim 021 ta gninia

B

-/+DAR 1.4TAE

91 m 5 in81 m 0 in

61 m 5 in51 m 0 in

31 m 5 in21 m 0 in

01 m 5 in

09 m in57 m in

06 m in54 m in

03 m in51 m in

m 0 in

91 m 5 in81 m 0 in

61 m 5 in51 m 0 in

31 m 5 in

21 m 0 in

01 m 5 in

09 m in

57 m in

06 m in

54 m in03 m in

51 m in

m 0 in



of the cell cycle regulators involved in that arrest. In addi-tion, as mentioned above, when induced by branched-chain alcohols, filamentation of S. cerevisiae has beenshown unambiguously to be dependent on the cell cycleregulator Swe1, the inhibitory kinase of Clb2-Cdc28 (LaValle and Wittenberg, 2001; Martinez-Anaya et al., 2003).In order to determine whether a similar dependencyoccurred in rad52 mutants of C. albicans, we deleted bothcopies of RAD52 in a swe1 swe1 strain. In C. albicans,swe1∆ cells exhibit a 10% reduction in cell size, suggest-ing a role in the morphogenesis checkpoint, but otherwiseform hyphae and pseudohyphe normally (Wightman et al.,2004). However, the pseudohyphal phenotype of cellsdeleted of the GIN4 kinase (which forms part of the intri-cate signalling network required for proper co-ordinationof cell growth and cell division) was partially dependenton a Swe1-dependent checkpoint (Wightman et al.,2004). As shown in Fig. 8, the double mutant swe1∆rad52∆ was still filamentous in both agar (Fig. 8A) andliquid media (Fig. 8B), but the number of filamentous cellswas significantly reduced as compared with rad52, sug-gesting that elongation of rad52∆ cells is partially depen-dent on the action of Swe1. Similarly, the conditional strainTRS4.1 (MET3-RAD52/rad52∆ swe1∆/swe1∆), in whichthe functional copy of RAD52 is under the MET3 promoter,was significantly less filamentous than EAT4.1 (MET3-RAD52/rad52∆) under repressive conditions in either solidor liquid SC (Fig. 8C). As expected, this strain grew asyeast under non-repressive conditions, and the same wastrue for swe1∆ in liquid or solid YEPD (not shown). Thereduction in filamentation caused by the absence ofSwe1p in rad52 cells was also paralleled by a decreasein the invasiveness of agar medium, whereas the swe1∆strain was non-invasive (Fig. 8D). Finally, the doublemutant swe1 rad52 responded to serum forming truehypha as described for each of the single mutants(Wightman et al., 2004; this work) (not shown).

Discussion

Rad52 depletion triggers the DNA-damage checkpoint in C. albicans

In this study, we have shown that rad52∆ mutants ofC. albicans exhibit defects in growth and a marked fila-mentous morphology. Slow growth seems to be a com-mon phenotype of rad52 mutants in other yeasts, such asS. pombe (Suto et al., 1999; van den Bosch et al., 2001).In S. cerevisiae, 13% of the rad52 cells were unable toform colonies (Toczyski et al., 1997; Melo et al., 2001).In C. albicans, it was not possible to determine this param-eter as the filamentous morphology of rad52∆ complicatesthe determination of cfu. It is likely that the slower growthof rad52 mutants in yeast is due to their inability to repairspontaneous DSB. This results in an arrest in the cellcycle and, eventually, following adaptation, in spontane-ous chromosome loss (Galgoczy and Toczyski, 2001).Under these conditions some cells may lose viability. Wehave shown that a significant percentage of the Rad52-depleted large budded cells of C. albicans do not progressnormally through the next cell cycle but arrest at G2/Mwith a single presumably tetraploid nucleus, suggest-ing the presence of DNA-damage, and perhaps DNA-replication checkpoints. Given the role of Rad52 in DNArepair, these events are likely triggered by the accumula-tion of DSBs.

The absence of Rad52 induces polarization of growth accompanied by but independent of the expression of several HSG

The second relevant phenotype of rad52 strains ofC. albicans is the formation of filaments in media thatusually do not induce hyphal growth, such as YEPD andSC, a behaviour that has not been reported in other yeast.The rad52∆ filaments appears intermediate between

Table 3. Nuclei position in G2/M cells along the cell cycle progression in Rad52+ and Rad52– cells.a

Time (min)

1 nucleus 2 nucleiOne nucleus

between both cells

RAD52/rad52 EAT4.1 RAD52/rad52 EAT4.1 RAD52/rad52 EAT4.1

90 65 109 88 54 47 37105 19 91 166 74 27 35120 9 97 164 53 15 50135 nd 106 nd 63 nd 31150 49 88 84 74 68 38165 15 89 136 71 49 40180 8 77 162 84 30 39195 – 80 – 86 – 34

a. For each sample, 200 budded cells (G2/M) were inspected and classified in the indicated category.nd, not determined (G2/M cells were mostly absent).



pseudohyphae and hyphae, as although they are in gen-eral shorter and wider than true hyphae; some of thembecome thinner upon elongation and are in fact indistin-guishable from hyphal elements. The latter phenotype isone of the several criteria proposed for distinguishinghyphal cells by Sudbery et al. (2004). Interestingly, fila-mentation of rad52∆ cells was also accompanied by dere-pression of HSG, including ECE1, HWP1 and HGC1. Twoof these genes, HWP1 and ECE1, were also significantlyderepressed in cells depleted of the polo-like kinase Cdc5(which also develops highly polarized filaments), at leastduring the later stages of cell elongation (Bachewichet al., 2003; 2005). However, filamentation occurred alsoin both efg1∆ rad52∆ and cph1∆ efg1∆ rad52∆ mutants,indicating that the process was independent of the MAPkinase and cAMP/PKA signalling pathways that includethese transcription factors. As deletion of EFG1 prevents

expression of ECE1, HWP1 and HGC1, we conclude thatexpression of the HSG accompanies but is dispensablefor filamentation of rad52∆ cells. In agreement with this,the double mutant hgc1∆ rad52∆ was also filamentous. Itfollows that the actin polarization signal activated by theRad52 defect is either independent or downstream of theexecution points of Ece1, Hwp1 and Hgc1 and, accord-ingly, bypasses the possible contribution of these factorsto cell elongation. However, the rad52∆-induced signalsomehow activates the cAMP/PKA pathway and, conse-quently, the Efg1-dependent HSGs, accentuating the sim-ilarities between the rad52∆ filamentation and the hyphalprogramme (Fig. 9). A detailed comparison of the filamen-tation patterns yielded by rad52∆ and efg1∆ rad52∆strains will reveal the contribution of the Efg1-controlledHSG to morphogenesis. On the other hand, filamentationwas severely impaired on M199 and Spider agar in the

Fig. 8. A and B. Effect of the rad52∆ disruption in swe1∆ cells. Cells from swe1∆, TCR2.2 (rad52∆) or TRS2 (swe1∆ rad52∆) were grown for the indicated times on minimal SC medium plus uridine on either solid agar (A) or liquid medium (B) (×100).C. Morphology of a conditional strain TRS4.1 (swe1∆/swe1∆ rad52∆/MET3-RAD52) grown for the indicated times in SC liquid (main frame) or agar media (insert) under repressive conditions (×100).D. Cells of the swe1∆ (1), rad52∆ (2) and swe1∆ rad52∆ (3) strains were streaked on a YEPD agar plate which was incubated for 48 h (top), and the cellular mass was washed from the plate with sterile water (bottom).

1ews D 1ews D ra 25d D ra 25d D

ra 25d D

A

B 1ews D r 25da D

h 81 h 42

C/M +1.4TAE

h 42 h 81

C/M + 1.2SRT

1 2

3D

1ews D

h 42 h 42 h 42

h 42 h 42 2 h 4

h 8 8 h 8 h

C



rad52∆ strain, which was still responsive to serum or Lee’smedium at 37°C. These observations emphasize the dif-ficulties inherent to the establishment of cultural, morpho-logical and transcriptional parameters that define thedifferent forms that C. albicans may adopt. In fact, themajority of the HSG seem to be also induced in pseudo-hyphal cells (Kadosh and Johnson, 2005).

Efg1 only controls a limited subset of all the genescontrolled by the cAMP/PKA pathway, in particular thoseactivated in the yeast-to-hyphal transition. Harcus et al.(2004) have shown that loss of upstream components ofthe pathway, in particular the adenylate cyclase, influ-ences expression of, in addition to those controlled byEfg1, a much larger number of genes, including someknown as well as putative transcription factors. The reduc-tion in the number and length of filamentous cells in thecdc35∆ rad52∆ strain indicates that an adequate expres-sion level of some of those genes may facilitate the initi-ation and maintenance of the polarized growth in rad52∆cells.

Finally, the observation that filamentation was signifi-cantly more prominent in solid media than in liquid cul-tures suggests that the agar-induced filamentationpathway may be activated in rad52 strains. It has beenrecently reported that contact activation of a kinase(Mkc1p) signals invasive hyphal growth in C. albicans(Kumamoto, 2005). However, the fact that disruption ofboth copies of either CDC35 or SWE1 reduced signifi-cantly the number and length of rad52∆ filaments, evenon solid agar, suggests that only part of the elongationwould be dependent on the contact activation mechanism,unless both Cdc35 and Swe1 also play some role in this

process. Again, a detailed analysis of the nature of thefilaments formed by the double mutants cdc35∆ rad52∆and swe1∆ rad52∆ appears necessary.

Filamentation of rad52∆ cells is likely derived from the DNA-damage checkpoint rather than from secondary mutations

How is the absence of Rad52 transduced to the growthpolarization machinery in C. albicans? One possibility isthat the latter derives from the genetic instability of rad52Äcells. Many of the phenotypes exhibited by rad52 mutantsof S. cerevisiae, including chromosome loss or anincreased frequency of point mutations (Mortimer et al.,1981; Yoshida et al., 2003), probably are derived from theinability of these mutants to repair some form of intrinsicDNA damage (Paques and Haber, 1999; Galgoczy andToczyski, 2001). In fact, nearly 60% of the S. cerevisiaerad52 cells, versus 8% of the wild-type cells, showedfluorescent Ddc1-GFP foci which are indicative of DNAdamage (Melo et al., 2001). We have evidence that rad52mutants of C. albicans lose chromosomes at an increasedrate (our unpublished results), and this may result in newphenotypes, given the high degree of heterozygosity ofthis organism (Janbon et al., 1998; Forche et al., 2004;Jones et al., 2004). Another cause of genetic alteration isthe introduction of permanent genetic changes as a con-sequence of defective repair using alternative recombina-tion pathways. However, the early filamentation of manycells from repressed cultures of the conditional strain andthe fact that many cells of a rad52 culture are able to formfilaments suggest that the reason for filamentation is not

Fig. 9. A hypothetical model to explain the effects of Rad52 depletion on C. albicans morphogenesis. We assume that the primary effect of Rad52 depletion is accumulation of unrepaired DNA damage and the subsequent activation of the DNA-damage checkpoint. One of the activated molecules triggers polarization of actin. This molecule (x?) could operate also in the Efg1 pathway (convergent pathways) or not (independent pathways). An unknown intermediate of the DNA-damage transduction pathway (marked with an asterisk, *) activates at some point the cAMP/PKA signalling pathway (dotted arrow line). This results in the Efg1-mediated activation of some HSG whose gene products contribute to the hyphal program (thin grey line). Elimination of Efg1 does not substantially affect polarization but prevents expression of the Efg1-dependent HSG. Swe1 has been included as a component of the DNA damage pathway downstream of the putative activator of the cAMP/PKA pathway. The contribution of cAMP and/or PKA to polarization of growth has been indicated with dashed arrow lines. Additional signals induced by environmental factors (lower branch) may activate parallel signalling pathways that contribute to the hyphal program.

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a consequence of secondary mutations, unless a partic-ular open reading frame (ORF) related to that processbehaves as a hot spot.

A second, more attractive possibility is that growthpolarization of rad52∆ cells is derived from the responseof the cells to DNA damage, which, in turn, triggers thecheckpoints that precede filamentation. In S. cerevisiae,cells with an unrepaired DSB arrest in anaphase with asingle nucleus, but, unlike C. albicans, no polarizationof actin has been reported. However, after 8–12 hS. cerevisiae-arrested cells resume progression throughthe cell cycle, a process termed adaptation (Sandell andZakian, 1993; Toczyski et al., 1997; Lee et al., 1998). Anearly landmark event of C. albicans rad52∆ filamentationis the uncoupling between actin polarization and nucleardivision; this uncoupling is maintained for some time, asfilaments with a single nucleus continue to enlarge andeven give rise to new cell compartments in the absenceof nuclear division. We have not investigated the existenceof a phenomenon similar to adaptation in C. albicans, butit is clear that, after some time, at least some nuclei of theelongated, uninucleated cells undergo karyokinesis. How-ever, nuclear division remains uncoupled from otherevents of the cell cycle, such as the rearrangement ofactin cytoskeleton and formation of a new cell, as it ispossible to observe single-celled filaments with two nuclei,empty axial buds and nuclei entering big buds. A likelypossibility is that these uncoordinated events are symp-toms of apoptosis, a phenomenon that can be triggeredby the arrest in cell division that follows a DNA-damageresponse (Kastan and Bartek, 2004). In fact, the fre-quency of dead of filaments supports this possibility.

Is there a pathway connecting DNA damage with filamentation in C. albicans?

Hyperpolarization of C. albicans growth in response toDNA damage has also been observed in the presence ofhydroxyurea (HU), an inhibitor of ribonucleotide reductasethat inhibits DNA replication and arrests (400 mM) ordelays (200 mM) S. cerevisiae cells in S phase (Clarkeet al., 1999; Jiang and Kang, 2003). In the presence of200 mM HU (a concentration that does not arrest com-pletely but slows down DNA replication), each C. albicanscell forms a single uninucleated filament lacking septa orbuds and does not proceed further (Bai et al., 2002;Bachewich et al., 2003; our own observations). rad52 fil-aments also mimic those formed by C. albicans cellsdepleted of the polo-like kinase CDC5, which inC. albicans is required for spindle elongation. Theseobservations suggest that a common mechanism may beresponsible for the polarized growth under several con-ditions. Interestingly, Cdc5 is involved in the hyperphos-phorylation and subsequent degradation of Swe1 in

S. cerevisiae (Asano et al., 2005; Harvey et al., 2005;Lee et al., 2005) (see Introduction). If this role was main-tained in C. albicans, its depletion would result thereforein the stabilization of Swe1 and the maintenance of thesubsequent Swe1-dependent inhibition of Clb-Cdc28 (seealso below). However, once again and in contrast to rad52mutants, filaments induced by the depletion of Cdc5 didnot show buds nor septa (Bachewich et al., 2003), sug-gesting that additional specific mechanisms operateunder each condition.

In different organisms, DNA damage is detected bysensor kinases (Rad17, Rad24, Mec1/Rad3/ATR andTel1/ATM) and then transmitted to effector kinases (Chk1and Rad53/Cds1) (Melo and Toczyski, 2002) that activatetranscription of the DNA repair regulon and connect withcomponents of the cell cycle, halting transiently or delay-ing its progression. On the other hand, recent results havealso shown that filamentous differentiation in S. cerevisiaeoccurs in response to slow DNA synthesis. This processinvolves Mec1 and Rad53 kinases, as well as the cellcycle regulators Cdc28 and its inhibitor Swe1 (Jiang andKang, 2003). Interestingly, Cdc28 and Swe1 are alsoinvolved in the filamentous differentiation of S. cerevisiaetriggered by nitrogen starvation or short-chain alcohols(Liu et al., 1993; Lorenz and Heitman, 1997; Lorenzet al., 2000; Martinez-Anaya et al., 2003). Our finding thatdeletion of Swe1 decreases significantly the ability ofrad52 cells of C. albicans to form filaments suggeststhat the Swe1-mediated inhibition of Cdk1 (Cdc28) thatmediates the morphogenesis checkpoint is also partiallyresponsible for the filamentation process (Fig. 9).Although inactivation of Cdc28 in C. albicans does notseem to play any role in hyphal growth in response toserum (Hazan et al., 2002), Cdc28 could be involved inother kinds of processes requiring cell polarization, suchas rad52 filamentation. For instance, it has been reportedthat Cdc28 functionally interacts with Hgc1 (Zheng et al.,2004). A signal could occur triggered by the Rad52 defectcould relieve the Cdc28 function on growth polarizationfrom the Hgc1 interaction. Residual filamentation of swe1rad52 mutants may result from alternative mechanismsderived from the G2/M arrest. For instance, inS. cerevisiae the DNA damage and replication check-points may also block mitosis independently of the inhib-itory phosphorylation of Cdc28-Y19 by Swe1 (Amon et al.,1992; Sorger and Murray, 1992). In the absence of Swe1,the induction of HSG by a component of the putativepathway (marked with an asterisk in Fig. 9) could promoteand maintain some polarization. As mentioned above, anumber of recent reports have indicated that deletion ofcomponents that regulate cell cycle progression or induc-tion of cell cycle arrest during S or M phases results inpseudohyphal or even hyphal growth (see Introduction) asa consequence of the persistent maintenance of cell



polarity. Our results support this concept and furtherindicate that a disturbance in the DNA-damage repairmachinery may cause a similar effect through the G2/Marrest. Furthermore, they also suggest that cAMP signal-ling facilitates both the induction and the maintenance ofgrowth polarization. The elucidation of the possible con-nection between the Rad52 depletion and the activationof a putative C. albicans counterpart of the central core,shown to be involved in the filamentous differentiation ofS. cerevisiae caused by slow DNA synthesis (Rad53,Swe1, Cdc28), is one of our present objectives.

Experimental procedures

Strains and media

The C. albicans strains used in this study are listed inTable 4. C. albicans SC5314 is a prototrophic strain (Gillumet al., 1984). Therefore, its derivative CAF2-1 (URA3/ura3∆)is used as a control for strains carrying one copy of URA3(Fonzi and Irwin, 1993). C. albicans cells were grown rou-tinely at 30°C in YEPD (2% glucose, 1% yeast extract, 2%Bacto Peptone), or SC medium [0.67% nitrogen base (Difco),2% glucose and a mixture of amino acids] supplemented(Uri–) or not (Uri+) with Uri (25 µg ml–1). Spider and M-199(Gibco-BRL, adjusted to pH 7.5) media were preparedaccording to Gimeno et al. (1992) and Ramon et al. (1999)respectively. Lee’s medium was prepared as described (Leeet al., 1975). To induce germination, cells of C. albicans weretransferred to YEPD pre-warmed at 37°C and supplementedwith 10% serum. To study the cell morphology of the severalstrains, frozen cells (–70°C) were streaked on YEPD agarplates and grown for 48 h at 28°C. The cell mass was thensuspended in water and used to inoculate a liquid YEPDculture that was incubated for 24 h at 28°C. These cultureswere then used to inoculate agar plates or liquid media.

Generation of deletion and conditional mutants

Sequential disruption of both alleles of RAD52 in the indi-cated genetic background (efg1∆/efg1∆, efg1∆/efg1∆ cph1∆/cph1∆, hgc1∆/hgc1∆, swe1∆/swe1∆ and cdc35∆/cdc35∆)was performed as described before (Ciudad et al., 2004)using the URA blaster method (Fonzi and Irwin, 1993). Allthe strains were verified using Southern blot hybridization. Togenerate the CaMET3-RAD52 cassette, a 320 bp fragment,comprising positions –39 to +281 in relation to the first nucle-otide of the CaRAD52 ORF (Ciudad et al., 2004), was ampli-fied by PCR using the appropriate oligonucleotides (R1,5′-CGCGGATCCCCACCCACACTTAAAATACACAG-3′, whichincludes a restriction site for BamH1, and R2, 5′-AACTGCAGCGAGTTCCACCTGGACCATCACG-3′, which includesa restriction site for PstI. The PCR product was cloned inplasmid MET3-URA3 (pCaDis) (Care et al., 1999) followingdigestion both enzymes (BamH1 and Pst1) which are alsopresent in the polylinker of the vector. The construct wasdigested with SacI and used to transform the heterozygousRAD52/rad52 Ura– (TCR1.1). Transformants carrying a con-ditional allele were analysed by PCR using oligonucleotides

MET-R3, 5′-CGACATAGATCCATTAATGCGCC-3′, located inthe MET3 promoter, and RV4, 5′-CCGCTACCACCATGAGTATCT-3′, internal to RAD52 (Ciudad et al., 2004), whosePCR product should correspond to a 0.9 kb fragment. Posi-tive transformants were then analysed for the null allele byPCR, using primers flanking the deleted region of CaRAD52(RV1, 5′-CAACCACGACCACCACAACAA-3′, complemen-tary to positions +25 to +45, and RV2, 5′-TGCGGTATACCCGAAGAAGGA-3′, complementary to positions 1587 to1607 on the complementary strand). As shown before,wild-type and disrupted alleles should yield 1.6 kb and 1.4 kbfragments respectively (Ciudad et al., 2004). Followingthis approach, a conditional strain rad52∆/MET3-RAD52(EAT4.1) yielding the expected fragments was selected andcharacterized. Using the same approach, we constructed anadditional conditional strain in the swe1∆ null background(TSR4.1, swe1∆/swe1∆ rad52∆/MET3-RAD52) (Table 4).Both conditional strains were routinely grown in SC mediumlacking Met, Cys and Uri. To repress RAD52 expression, agarplates were supplemented with 2.5 mM Met and 2.5 mM Cys.Similarly, liquid cultures were supplemented with 2.5 mM Metand 2.5 mM Cys and inoculated with exponentially growingcells (OD600 lower than 2) to reach an initial OD600 of about0.01.

Monitoring growth

Liquid cultures were started by inoculation of a 250 ml flaskcontaining 50 ml of YEPD or SC with a suspension of cellspreviously grown in the same medium to reach a finalOD600 = 0.05. Samples were taken at the indicated times, andthe OD600 was measured in a spectrophotometer. Appropriatedilutions of each sample were plated in duplicate to YEPD orSC plates, respectively, to determine the number of cfu. UVand MMS treatment was carried out as described (Ciudadet al., 2004).

Nucleic acids extraction and analysis

Standard techniques were routinely used for DNA prepara-tion, cell transformation and Southern blot hybridization(Andaluz et al., 2001; Ciudad et al., 2004). To analyse theexpression of filamentation genes, overnight cultures wereused to inoculate YEPD and YEPD supplemented with 10%bovine serum at a final OD600 = 0.5, and these cultureswere incubated for 1.5 h at 30°C and 37°C, respectively,in a rotatory shaker. To analyse the expression of RAD52 inthe conditional strain EAT4.1, cells were inoculated into50 ml of fresh SC with/without Cys/Met at an OD = 0.5.At the indicated times a volume equivalent to 8 × 108

cells was used for RNA preparations. RNA extraction andNorthern analysis have also been described (Ciudad et al.,2004). Probes were generated by PCR from genomicDNA using the following oligonucleotides. For ECE1,F: 5′-TGGCAACATTCCACAAG TAATC-3′ and R: 5′-AGCCGGCATCTCTTTTAACTGG-3′. For HWP, F: 5′-TGCTCCAGGTACTGAATCCGC-3′ and R: 5′-GGCAGATGGTTGCATGAGTGG-3′. For HGC1, F: 5′-CCAACAACAACCCCCAAGCTTCTGGC-3′ and R: 5′-GCCAGTAGAACTAGTTGGTGTAGTAC-3′. For ACT1, F: 5′-GCTGCTTTAGTTATCGAT



AACGG-3′ and R: 5′-TGAACAAAGCTTCTGGAGCTCTG-3′. These primers amplify fragments of 482 bp, 223 bp,1198 bp and 772 bp, respectively, which were cloned in thepGEM-7Zf(+) vector. For determination of RAD52 expres-sion in the conditional strain EAT4.1, we used a 1453 bpSacI–SpeI fragment internal to the RAD52 ORF as a probe(Ciudad et al., 2004). Hybridization bands were visualizedusing a Molecular Imager (Bio-Rad Laboratories). RT-PCRwas performed according to the manufacturer (Promega).

RNA extracts (as above) were incubated with DNase andthen subjected to amplification in the presence of tran-scriptase and polymerase or only polymerase to demon-strate the absence of any contaminating DNA. Foramplification of RAD52 mRNA in the conditional strainEAT4.1, we used the following oligonucleotides: F: 5′-CGTGATGGTCCAGGAACT-3′ and R: 5′-TGAGCAACAATGGTCTTGTCG-3′ that amplify a 365 bp fragment internal to theRAD52 ORF.

Table 4. Strains used in this study.

Strain Genotype Reference

SC5314 Wild type Gillum et al. (1984)CAF2 As SC5314 but ∆ura3::imm434/ΥΡΑ3 Fonzi and Irwin (1993)CAI4 ∆ura3::imm434/∆ura3::imm434 Fonzi and Irwin (1993)TCR1 ∆ura3::imm434/∆ura3::imm434 Ciudad et al. (2004)

RAD52/∆rad52::hisG-URA3-hisGTCR1.1 ∆ura3::imm434/∆ura3::imm434 Ciudad et al. (2004)

RAD52/∆rad52::hisGTCR2.1 ∆ura3::imm434/∆ura3::imm434 Ciudad et al. (2004)

∆rad52::hisG-URA3-hisG/∆rad52::hisGTCR2.2 ∆ura3::imm434/∆ura3::imm434 Ciudad et al. (2004)

∆rad52::hisG-URA3-hisG/∆rad52::hisGTCR3.1.1 ∆ura3::imm434/∆ura3::imm434 Ciudad et al. (2004)

∆rad52::hisG rad52/∆rad52::hisG (RAD52::URA3-hisG)∆ura3::imm434/∆ura3::imm434

TCR3.2.1 ∆rad52::hisG/∆rad52::RAD52-URA3-hisG Ciudad et al. (2004)EAT2 ∆ura3::imm434/∆ura3::imm434 Ciudad et al. (2004)

∆lig4::hisG/∆lig4::hisG∆rad52::hisG-URA3-hisG/∆rad52::hisG

EAT4.1 ∆ura3::imm434/∆ura3::imm434 This work∆rad52::hisG/∆rad52:: MET3-RAD52-URA3

WYZ12 ∆ura3::imm434/∆ura3::imm434 Zheng et al. (2004)∆hgc1::ARG4/∆hgc1::HIS1

HGR1A2 ∆ura3::imm434/∆ura3::imm434 This work∆hgc1::ARG4/∆hgc1::HIS1∆rad52::hisG/∆rad52::hisG-URA3-hisG

HLC52 ∆ura3::imm434/∆ura3::imm434 Lo et al. (1997)∆efg1::hisG/∆efg1::hisG-URA3-hisG

HLC67 ∆ura3::imm434/∆ura3::imm434 Lo et al. (1997)∆efg1::hisG/∆efg1::hisG

HCL54 ∆ura3::imm434/∆ura3::imm434 Lo et al. (1997)∆cph1::hisG/∆cph1::hisG ∆efg1::hisG/∆efg1::hisG-URA3-hisG

HLC69 ∆ura3::imm434/∆ura3::imm434 Lo et al. (1997)∆cph1::hisG/∆cph1::hisG ∆efg1::hisG/∆efg1::hisG

TER2 ∆ura3::imm434/∆ura3::imm434 This work∆efg1::hisG/∆efg1::hisG∆rad52::hisG/∆rad52-hisG-URA3-hisG

CER2 ∆ura3::imm434/∆ura3::imm434 This work∆cph1::hisG/∆cph1::hisG ∆efg1::hisG/∆efg1::hisG∆rad52::hisG/∆rad52::hisG-URA3-hisG

swe1∆ ∆ura3::imm434/∆ura3::imm434 Wightman et al. (2004)swe1∆::ARG4/swe1∆::HIS1

TRS1 ∆ura3::imm434/∆ura3::imm434 This workswe1∆::ARG4/swe1∆::HIS1∆rad52::hisG/∆rad52::hisG-URA3-hisG

TRS4.1 ∆ura3::imm434/∆ura3::imm434 This workswe1∆::ARG4/swe1∆::HIS1∆rad52::hisG/∆rad52:: MET3-RAD52-URA3

CR216 ∆ura3::imm434/∆ura3::imm434 Rocha et al. (2001)∆cdc35::hisG/cdc35::hisG-URA3-hisG

CR276 ∆ura3::imm434/∆ura3::imm434 Rocha et al. (2001)∆cdc35::hisG/cdc35::hisG

CRA1 ∆ura3::imm434/∆ura3::imm434 This work∆cdc35::hisG/cdc35::hisG∆rad52::hisG/∆rad52::hisG-URA3-hisG



Elutriation and cell cycle analysis

A 3 l flask containing 1.5 l of SC medium lacking both Cysand Met was inoculated with the conditional mutant EAT4.1.Following an overnight incubation in a rotatory shaker, cellswere harvested at an OD600 = 1 and subjected to elutriationusing a Beckman centrifuge. About 600 ml of unbudded cellsat a OD600 = 0.2 was collected and split into two cultures of300 ml, one of which was made 2.5 mM Met and Cys. Thesecultures were maintained for 30 min in a rotatory shaker torelieve cells from the elutriation stress and adapt them to thenew conditions. Samples were then taken at the indicatedtimes to determine cell cycle progression (relative proportionof unbudded cells, cells with small buds and cells with bigbuds). Cell size and DNA content were determined by FACSusing a FacsScan (Becton-Dickinson, Franklin Lakes, NJ)flow cytometer.

Cell staining and microscopy

The cell morphology of fresh or fixed cells was inspectedmicroscopically using a Nikon Eclipse 600 fluorescencemicroscope with a 60x DIC objective. A CC-12 digital camerainterfaced with Soft Imaging System software was used forimaging. When cells were fixed (70% ethanol), they were re-hydrated and sonicated to disperse clumps before micros-copy. For fluorescence microscopy 108 cells were fixed in70% of ethanol and supplemented with 5 µl of a DAPI solu-tion (1 mg ml–1). After 5 min, cells were washed several timesin water and resuspended to 106 cells ml–1 for microscopy.Calcofluor staining of the chitin ring and rhodamine-phalloidinwere performed as described (Amberg, 1998; Loeb et al.,1999). Colonial morphology on agar plates was followed in aNikon microscope (× 10).

Acknowledgements

This study was supported by a grant from the Public HeathService, NIH-NIAID 1 R01 AI51949, to R.C. and G.L. Wethank the reviewers for their comments. We also thank W.Fonzi for critical reading of the manuscript, J. Correa for hisadvice in cell elutriation, M.C. López-Cuesta for her help inthe FACS analysis, J. Ernst for strains efg1∆, cph1∆ andefg1∆ cph1∆, Y. Wang, P. Sudbery, and D. Harcus and M.Whiteway for the hgc1∆, swe1∆ and cdc35∆ strains, respec-tively, and Belén Hermosa for her careful technical support.J.G.-R. is the recipient of a fellowship from the NIH-NIAIDgrant and Belén Hermosa is supported by contracts from aJunta de Extremadura grant, 2PR03A044, to E.A. and theNIH-NIAID grant.

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Rad52 depletion in Candida albicans triggers both the DNA-damage checkpoint and filamentation...

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