Current Biology Vol 15 No 2R50

Correspondence

Ctf7p/Eco1pexhibits acetyl-transferaseactivity — butdoes it matter?

Alex Brands and Robert V.Skibbens*

In eukaryotic cells, faithfulchromosome segregation dependsupon the physical pairing, orcohesion, between sisterchromatids. Budding yeastCTF7/ECO1 (herein termed CTF7)encodes an essential proteinrequired to establish cohesionduring S-phase and associateswith DNA replication factors [1–10].However, the molecularmechanism by which Ctf7pestablishes cohesion remainsunknown. In vitro characterizationof Ctf7p as an acetyltransferase ledto the model that this activityprovides for Ctf7p’s essentialfunction [11]. However, in vivoCtf7p substrates have yet to bedocumented, nor has an in vivoacetyltransferase activity beendemonstrated even when Ctf7p isoverexpressed [11] (A. Brands andR.V. Skibbens, unpublished data).In fact, the effects of acetylation-defective Ctf7p (ctf7ack-) in yeastremain to be rigorously tested,leaving unanswered the criticalquestions of whether Ctf7pacetyltransferase activity isessential for cell viability and towhat extent this activity is requiredfor the establishment of cohesion.Here, we show that yeast strainsharboring acetyltransferase-defective alleles [11] as the solesource of Ctf7p function exhibitrobust growth and high fidelitychromosome transmission.

We first used a plasmid lossassay to test whether ctf7ack-

alleles could support cell viability.CEN TRP1 plasmids containingwild-type CTF7 or ctf7ack- allelesexhibiting abrogated or greatlydiminished acetyltransferaseactivity in vitro [11] weretransformed into an ade2;ade3

yeast assay strain in which the solesource of Ctf7p function wasprovided by a CEN-ADE3-CTF7plasmid. After growth on mediumselective for both plasmids,transformants were placed on richnon-selecting medium to allow forrandom plasmid loss. Cellstransformed with CEN-TRP1-CTF7exhibited CEN-ADE3-CTF7plasmid loss — an event easilydetected by white sectors in anotherwise red colony [12]. Cellstransformed with vector aloneproduced solid red colonies,confirming that the CEN-CTF7-ADE3 plasmid was required inthese cells. Importantly, three ofthe four ctf7ack- alleles producedwhite-sectored colonies identicalto cells transformed with wild-typeCTF7 (Figure S1, Supplementaldata). A fourth allele produced thinand infrequent white sectoredcolonies, but was competent toperform the essential function ofCtf7p. To test whether the resultingctf7ack- strains might exhibitconditional growth phenotypes,serial dilutions were spotted ontonon-selective rich medium platesand incubated at 23°C, 30°C or37°C. Three of the ctf7ack- mutantstrains exhibited wild-type growthat all temperatures tested (FigureS2, Supplemental data). Plasmidrescue and DNA sequencingconfirmed that the ctf7ack- alleles(tested for R222G/K223G) providedthe sole source of Ctf7p function.In summary, these findings revealthat ctf7ack- alleles are competentto provide for the essentialfunction of Ctf7p in vivo. Cellscontaining the G211D ctf7 alleleexhibited conditional growthdefects, suggesting that thismutation may exhibit phenotypesbeyond that associated with lossof acetyltransferase activity.

We next tested whether ctf7ack-

alleles, integrated at single copy,were competent to maintain bothcell viability and high fidelitychromosome transmission. Usingestablished gene replacementmethods, the wild-type CTF7coding sequence was replacedwith single copy integrated ctf7ack-

alleles controlled by theendogenous CTF7 promoter [13].The ability to obtain numeroushealthy ctf7ack- transformantsfurther indicates that theacetyltransferase activity of Ctf7pmay not be necessary for sisterchromatid cohesion – a processthat is essential for viability. Forcontrols, we included wild-typeCTF7 and two alleles (ctf7-108 andctf7-109) obtained from the originalchromosome transmission fidelitycollection [14]. Gene replacementwas performed in a sectoring yeastassay strain in which thephenotypic red colony colorcaused by an ade2 mutation issuppressed by a non-essentialTRP1 SUP11 chromosomefragment [14]. Transformants wereplaced on rich medium to allow forloss of the TRP1-SUP11chromosome fragment, an eventscored as red sectors in a whitecolony background. CTF7 cellsexhibited high fidelity transmissionof the chromosome fragment,evidenced by solid white colonies.As expected, ctf7-108 cells (andctf7-109, data not shown) exhibitedlow fidelity transmission to producehighly red-sectored colonies.Importantly, all three ctf7ack- strainstested produced solid whitecolonies indistinguishable fromthose produced by CTF7 cells(Figure 1). These results reveal thatctf7ack- mutant strains exhibit bothrobust cell viability and high fidelitychromosome transmission.

Figure 1. The acetyltransferase activity of CTF7 is not required for viability.Yeast cells harboring either wild-type CTF7 (WT) or ctf7ack- alleles (R222G/K223G,G225D or D232G) as the sole source of CTF7 function retain the non-essential TRP1SUP11 chromosome fragment and produce non-sectored white colonies. Cells har-boring ctf7-108 (S108) or ctf7-109 (not shown) alleles exhibit dramatic chromosomefragment loss and produce red-sectored colonies.

Magazine R51

ctf7-108 and ctf7-109 mutantstrains both exhibit strongchromosome loss phenotypes[14]. We sequenced these allelesand found that ctf7-108 and ctf7-109 each contain single mutationsin the putative zinc finger domainof Ctf7p [11] — outside of theacetyltransferase domain (Figure2). Biochemical analyses of ctf7-108 expressed in bacteria revealthat this allele exhibitsacetyltransferase activity in vitro(data not shown). Thus, a mutationin the zinc finger domain of Ctf7pis sufficient to producechromosome missegregation invivo.

As the detection of Ctf7pacetyltransferase activity in vivohas failed so far, it formallyremains possible that mutantsdefective for acetyltransferaseactivity in vitro may still retainresidual activity in vivo. Theimportance of addressing thisissue is underscored by the recentidentifications of human (EFO1)and Drosophila (DECO) Ctf7porthologs [15,16]. EFO1 exhibitsacetyltransferase activity in vivo,revealing that this activity isconserved through evolution. Inflies, deco mutants exhibitprecocious sister chromatidseparation. However, theacetyltransferase activity of thesealleles has not been tested. Thus,the role in cell viability for theacetyltransferase activity of Ctf7porthologs remains an openquestion.

Supplemental dataSupplemental data showingdetails of the viability assays areavailable at http://www.current-biology.com/cgi/content/full/15/2/R50/DC1/

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Department of Biological Sciences, 111Research Dr., Lehigh University,Bethlehem, Pennsylvania 18015, USA.*E-mail: rvs3@lehigh.edu

Figure 2. Sequence analyses comparing ctf7 alleles.Comparison of mutations of chromosome loss ctf7 alleles to previously publishedsequence of ctf7ack- alleles [10]. Single mutations in the zinc finger domain (H53Y = ctf7-108; C35Y = ctf7-109) are sufficient to produce massive chromosome missegregation.