Báo cáo Y học: The DNA-polymerase inhibiting activity of poly(b-L-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum pot

6 260 0
  • Loading ...
1/6 trang

Thông tin tài liệu

Ngày đăng: 31/03/2014, 21:21

The DNA-polymerase inhibiting activity of poly(b-L-malic acid)in nuclear extract during the cell cycle ofPhysarum polycephalumSabine Doerhoefer1, Christina Windisch1, Bernhard Angerer1, Olga I. Lavrik2, Bong-Seop Lee1and Eggehard Holler11Institut fu¨r Biophysik und physikalische Biochemie, Universita¨t, Regensburg, Germany;2Novosibirsk Institute of Biorganic Chemistry,Siberian Division of the Russian Academy of Sciences, Novosibirsk, RussiaThe naturally synchronous plasmodia of myxomycetessynthesize poly(b-L-malic acid), which carries out cell-spe-cific functions. In Physarum polycephalum,poly(b-L-malate)[the salt form of poly(b-L-malic acid)] is highly concentratedin the nuclei, repressing DNA synthetic activity of DNApolymerases by the formation of reversible complexes. Totest whether this inhibitory ac tivity is cell-cycle-dependent,purified DNA polymerase a of P. polycephalum was addedto the nuclear extract and the activity was measured by theincorporation of [3H]thymidine 5¢-monophosphate into acidprecipitable nick-activated salmon testis DNA. MaximumDNA synthesis by the reporter was measured in S-phase,equivalent to a minimum of inhibitory activity. To t est forthe activity of endogenous DNA polymerases, DNA syn-thesis was followed by the highly sensitive photoaffinitylabeling technique. Labeling was observed i n S-phase inagreement with the minimum of the inhibitory activity. Theactivity was constant throughout the c ell cycle when theinhibition was neutralized by the addition of spermidinehydrochloride. Also, the concentration of poly( b-L-malate)did not vary with the phase of the cell cycle [Schmidt, A.,Windisch, C. & Holler, E. (1996) Nuclear accumulation andhomeostasis of the unusual polymer poly(b-L-malate) inplasmodia of Physarum polycephalum. Eur. J. Cell Biol. 70,373–380]. To explain the variation i n the cell cycle, a p eriodiccompetition for poly( b-L-malate) between DN A polym-erases and most likely certain histones was assumed.These effectors are synthesized in S-phase. By c ompeti-tion they displace DNA polymerase from the complex ofpoly(b-L-malate). The free polymerases, which are no longerinhibited, engage in DNA synthesis. It is speculated thatpoly(b-L-malate) is active in maintaining mitotic synchronyof plasmodia b y playing the mediator between the periodicsynthesis of certain proteins and the catalytic competence ofDNA po lymerases.Keywords: poly(malic acid); cell cycle; S-phase; DNA syn-thesis; h istones.Poly(b-L-malic acid) consists ofL-malic acid units, which arecovalently linked by ester bonds between the hydroxylgroup and the carboxyl group in the b position, while thecarboxyl group in a position points away from the polyesterchain [1]. The ionized form of the polymer, poly(b-L-malate)(PMLA), amounts to high concentrations comparable toDNA in the naturally synchronous nuclei of the plasmo-dium, the giant polynuclear cell form of the slime mouldPhysarum polycephalum [1–3]. This organism differentiatesinto several cell forms during its life cycle (e.g. spores andamoebae) [4], but only the plasmodium produces poly(b-L-malic acid). In contrast to the giant cell dimensions, thebillions of nuclei display cyclic events, such as mitosis andDNA replication, with a high degree o f natural synchrony.Because of these featu res, the plasmodium is suited forstudying molecular biology of the cell cycle. One particularquestion is the organization of the catalytic competence ofDNA polymerases in the context of synchrony.Poly(b-L-malate) was discovered by its activity to bindand reversibly inactivate the endogeneous DNA polymerasea [1,5]. The other replicatively active DNA polymerases(types d and e)havealsobeenshowntobindandbecomeinactivated, whereas the putative repair enzyme, DNApolymerase b-like, was not inhibited [6,7]. Binding experi-ments with synthetic polyanions, which differed fromPMLA in the distance between the negative charges,demonstrated that specific ity of binding is a ttributed tothe particular distance b etween the n egative charges inPMLA [5]. This distance is similar to that betweenphosphate groups in the nucleic acid backbone, in agree-ment with the competitive binding of PMLA and DNA tothe polymerases. The molecular mimicry suggested thatPMLA could bind to histones and to other DNA interact-ing proteins. Indeed, large complexes of PMLA not onlywith DNA polymerases but also with histones and otherproteins have been found under conditions close to in vivo[2,7]. The binding to histones has been further investigatedby in vitro experiments [5].If histones and DNA polymerases are together, they areprone to compete for the binding to PMLA. The periodicCorrespondence to E. Holler, Institut fu¨r Biophysik und physikalischeBiochemie der Universita¨t Regensburg, D-93040 Regensburg,Germany. Fax: + 49 941943 2813, Tel.: + 49 941943 3030,E-mail: eggehard.holler@biologie.uni-regensburg.deAbbreviations: AFBdCTP, exo-N-{[[((4-azido-2,3,5,6-tetra-fluorobenzylidene)hydrazino)carbonyl]butyl]carbonyl}deoxycytidine-5¢-triphosphate; PMLA, poly(b-L-malate).Enzymes: D NA polymerase (E.C.; benzonase (E.C. a website is available at h ttp://www.biologie.uni-regensburg.de/Biophysik(Received 8 O ctober 2001, revised 27 December 2001, accepted7 January 2002)Eur. J. Biochem. 269, 1253–1258 (2002) Ó FEBS 2002production of histones (or of any other PMLA-bindingmolecule) in S-phase could evoke a cycling of free DNApolymerases and DNA synthetic a ctivity, although theindividual levels of PMLA and DNA polymerases need notvary. For a p roper understanding of the role of PMLA itwas thus interesting to know its inhibitory activity over thecell cycle. Because the inhibitory activity could not be testedunder in vivo conditions, experiments were carried out withnuclear extracts. The results were consistent with theassumption that PMLA was a mediator between increasedconcentrations of certain nuclear constituents and thecompetence of DNA polymerases in DNA synthesis duringS-phase.MATERIALS AND METHODSMaterialsMicroplasmodia of P. polycephalum,strainM3CVIII(ATCC 96951), were grown in shaken cultures at 27 °C, asdescribed p reviously [8]. Macroplasmodia were obtained assurface cultures on fi lter paper by the fusion of mi cro-plasmodia, as described previously [9]. The mitotic stageswere identified b y phase-co ntrast m icroscopy [10]. O ne gramof wet plasmodia corresponded to % 2 · 108nuclei. DNApolymerase a (110 UÆmL)1) was purified from plasmodia asdescribed previously [11]. DNase-I-activated salmon testisDNA for the s tandard DNA polymerase a ssay and forphotoaffinity labeling was prepared as described previously[12]. Rabbit antiserum against DNA polymerases type aand type e, was prepare d with a m ixture of the purifiedP. polycephalum DNA polymerases [13], a nd rabbit anti-serum against DNA polymerase d by immunization withsynthetic peptides of the enzyme [6]. Peroxidase-coupledanti-(rabbit IgG) Ig was purchased from Pierce. Proteinaseinhibitors were used in a cocktail of the followingconcentrations after dilution with the e xtracts: 5 mMsodiumbisulfite, 0.2 mMphenylmethanesulfonyl fluoride, 1 mMbenzamidine (Sigma), 1 lMpepstatin A (Merck), 10 lMleu-peptin (Sigma), 1 mgÆmL)1aprotinin (Merck), 10 lMtosyl-L-lysine chloromethyl ketone (Calbiochem), 100 lMpefablock S C (Merck), and 2 lgÆmL)1E 64 (BoehringerMannheim). For photocrosslinking, the dCTP analogueexo-N-{[[((4-azido-2,3,5,6-tetrafluorobenzylidene)hydrazi-no)carbonyl]butyl]carbonyl}deoxycytidine-5¢-triphosphate(AFBdCTP) was prepared as described previously [14], andwas a gift from Safronov (Novosibirsk). Benzonase grade II(25 0 00 UÆmL)1) was purchased from Merck. Nonradio-active dNTPs and standard proteins for SDS/PAGEwere obtained from P harmacia (Sweden). [3H]dTTP(60 CiÆmmol)1,1Ci¼37 GBq) and [a-32P]dATP(3000 CiÆmmol)1) were purchased from Amersham.Preparation of nuclear extractNuclei were prepared either from macroplasmodia follow-ing their third mitosis, or from microplasmodia harvestedafter 2 days of inoculation. The plasmodia were washed bycentrifugation (500 g,10min,4°C) in cold water, suspen-ded in disruption buffer (2 g of solvent per 1 g o f wetplasmodia; 15 mMTris/HCl pH 7.5, 5 mMEGTA, 0.5 mMCaCl2,15 mMMgCl2, 500 mMhexylenglycol, 10% dextran,14 mM2-mercaptoethanol, and p rotease inhibitor c ocktail)and disrupted in a Dounce homogenizer (10–12 strokes).Nuclei were pellete d over a 25% Percoll g radient in t heabove buffer, as described previously [2]. The pelletcontained 2 ± 1 · 108nuclei per g of wet m icroplasmodia.Nuclear extracts were prepared by incubating for 10 min onice in an equal volume of extraction buffer (final concen-trations 50 mMTris/HCl pH 7.5, 0.3MKCl, 20 mMMgCl2, 0 .5% T riton X-100, 20% glycerol, 1 mM2-merca-ptoethanol, protease inhibitor cocktail) and centrifugationat 700 g. The nuclear extract contained > 85% o f the totalnuclear PMLA and > 75% o f t he total nuclear DNApolymerase activity in the standard assay. Results by SDS/PAGE and Western blotting with specific antisera againstDNA polymerases a and e [13], and DNA polymerase d [14]were consistent with the recovery of > 95% of DNApolymerase a and > 75% of the other DNA polymerases inthe extract.Standard DNA polymerase activity and inhibition assaysTotal DNA polymerase activity was assayed as describedpreviously [12]. The standard assay contained in 150 lL50 mM3-(N-morpholino)propanesulfonic acid/potassiumsalt (pH 7.5), 50 mMKCl, 10 mMMgCl2,3mMEDTA,3mM2-mercaptoethanol, 3 3 lMeach of dATP, dCTP,dGTP, 3 lM[3H]dTTP (1 CiÆmmol)1), 20 lg DNase-Iactivated salmon testis DNA, 80 lg bovine serum albumin,and DNA polymerase. After a 30-min lcubation at 37 °C,10% (v/v) saturated cold trichlo roacetic acid in water w asadded and the precipitate collected on Whatman GF/Cfilters, which were washed with trichloroacetic acid, thenwith 70% (v/v) ethanol/H2O dried, and counted with 20%efficiency. If r equired, either 0 .4 mMspermineÆ4HCl or2mMspermidineÆ3HCl were included to suppress bindingand inhibition of polymerases by PMLA [1]. One unit ofpolymerase activity is equivalent to the amount of enzymethat catalyses the incorporation of 1 nmol nucleotidesduring 1 h.The same conditions were used in the inhibition experi-ments, except that the biogenic amines were omitted. Tomeasure the inhibitory activity of n uclear extracts during t hecell cycle, 0.4 U of purified DNA polymerase a was presentin the assay under the above standard conditions comparingthe reaction rates in the presence (vi) and the absence(reference, vo) of nuclear extract equivalent to 1.5 · 105nuclei. To account for effects of particular ingredients in theextract buffer, appropriate amounts of these reagents wereadded to t he reference reaction mixture. The low poly-merase activity contained in t he extract due to DNApolymerase b-like (which was not inhibited by PMLA)was measured in parallel and subtracted from the crudevalue for vi. The inhibitory activity is defined in terms ofthe reciprocal value of the inhibition constant KÀ 1i¼[E–PMLA]/[E]Æ[PMLA]. The concentration of the poly-merase–inhibitor complex, [E–PMLA], can be expressed i nterms of [E]o) [E]. The concentration of free polymerase,[E], and of the total polymerase [E]ois proportional to viandvo. The expression for the relative inhibitory activity is then(vo) vi)/vi¼ [PMLA]/Ki. Therefore, the higher the con-centration of free PMLA, and the lower the value of theinhibition constant (Ki), the higher the relative inhibitoryactivity (vo) vi)/vi. In the presence of ligands that bind com-petitively to PMLA, the value of Kiincreases as a function1254 S. Doerhoefer et al. (Eur. J. Biochem. 269) Ó FEBS 2002of rising concentrations and a ffinities of these ligands. Forexample, if spermine hydrochloride, which binds to PMLA,is present, the inhibition may b e totally neutralized. Thereciprocal of the direct value s of the relative inh ibitoryactivity will be shown in the results, as they correlate directlywith the degree of residual DNA polymerase activity.DNA replication of single macroplasmodia w as followedunder in vivo conditions at various phases in the cell cycle.The plasmodia were grown on filter paper for a particularperiod of time in the cell cycle. One fourth of theplasmodium, usually 80 lg, was transferred to freshmedium containing 5 lCiÆmL)1[methyl-3H]thymidine(25 CiÆmmol)1) and grown for 15 min at 27 °C. Theremainder was allowed to grow and used as the source offurther samples. The incubation was terminated by fixationin a 10-mL solution containing ice-cold 5% saturatedtrichoroacetic acid and 50% acetone. The samples weredisrupted in a Dounce homogenizer and filtered on GF/C.After washing with trichloroacetic acid/acetone and etha-nol, the filters were dried, and the radioactivity was countedin a scintillation cocktail.Photoaffinity labeling of DNA polymerasesLabeling o f DNA polymerases was carried out in a 20-lLsolution containing 5 lL nuclear extract, 3–5 lM[32P]dATP(3000 CiÆmmol)1), 125 lMAFBdCTP, 33 lMof eachdGTP and dTTP, 50 mM3-(N-morpholino)propanesulfonicacid buffer (pH 7.5), 50 mMKCl, 10 mMMgCl2,3mMEDTA and 5 lg activated salmon testis DNA [7]. Escheri-chia coli DNA polymerase I served as a positive control in aparallel, but oth erwise identical, reaction mixture. In othercontrol reactions, to exclude staining due to DNA poly-merase adenylation or phosphorylation, the photoreactivenucleotide was omitted. The polymerization reaction wascarried out in the dark for 10 min at 37 °C. An aliquot wasthen irradiated for 2 min. Free DNA and DNA protrudingfrom crosslinked complexes with proteins were digestedwith benzonase (25 U per sample) for 10–15 min a t 37 °C.The sample was heated for 3 min with Laemmli buffer [15]and e xamined b y denaturating SDS/PAGE (10% poly-acrylamide gel). Proteins were electroblotted onto MilliporeImmobilon membranes [16] and visualized by autoradio-graphy (Kodak X-OMAT LS) at )70 ° (5–7 days). Theidentities of blotted proteins were verified by immunostain-ing of the same membranes. Intensities of bands werequantified with a Boehringer Mannheim Lumi ImagerTM.The intensity of labeled E. coli DNA polymerase I in thesame gel served as a reference.RESULTSFinding optimal assay conditions to measurethe inhibitory activity in nuclear extractsIn previous analytical and preparative experiments, it hasbeen shown that PMLA was the constituent that s pecific-ally inhibited replicative DNA polymerases in nuclearextracts [1,2,7]. In the present study, we measured theinhibitory activity of PMLA using purified DNA poly-merase a of P. polycephalum as an added reporter. To findthe optimal assay conditions, t he extracts were preparedfrom the nuclei of microplasmodia that naturally includedall phases o f the c ell cycle. As considered in Materials andmethods, t he degree of inhibition depends on both theconcentration of free PMLA and the inhibition constant.This parameter reflected the affinity of the polymerase andboth the affinity and concentration of competing ligandsfor binding to the polyanion. In the (added) nuclear extract,such ligands were histones, and probably other DNA-binding proteins [2]. T o obtain an optimal response by thereporter polymerase to a varying i nhibitory activity in thenuclear extract, the amounts o f the added polymerase a ndextract had to be optimized. To this end, the titration of afixed amount of nuclear extract, c orresponding to 1.5 · 105nuclei, was performed in the first experiment (Fig. 1). In thebeginning of the titration, the polymerase activity remain edsuppressed until the inhibitory activity was neutralized byan amount of 0.38 ± 0.03 U of the reporter DNApolymerase (the arrow in Fig. 1). During continued addi-tion of the polymerase, the enzyme a ctivity increased inparallel with the activity of the control experiment in theabsence of extract. An a mount of 0.4 U of the r eporterDNA polymerase, close to the neutralization point, waschosen for the measurement o f the inhibitory activityduring t he cell cycle.We have previously shown that the inhibitory activity ofpurified PMLA is neutralized in the presence 0.4 mMspermine hydrochloride [1]. To confirm t hat PMLA wasthe only inhibitor of DNA polymerases in the extract above,we measured the polymerase activity in the presence ofadded spermine hydrochloride. A value of 1.2 ± 0.03 U(five measurements) was ob se rved and r eferred t o theendogenous DNA polymerases. The experiment was repea-ted with the extract containing in addition 0.4 U reporterDNA polymerase a. A n amount of 1.6 ± 0.03 U wasmeasured in this case (five measurements). The difference of0.4 ± 0.04 U was in agreement with the added 0.4 U. Thesame results were obtained when nuclear extracts in theS-phase and in G2-phase were compared. The agreementwas consistent with the assumption that the inhibitoryactivity was a property of PMLA in the extract.Fig. 1. The inhibition of purified DNA polymerase a by po ly(b-L-malate) in the nuclear extract. (d) The activity was measured as afunction of added a mounts of DNA polymerase a in the standardDNA polymerase assay that contained the extract of 2 · 106nucleiisolated from microplasmodia. (j) The control in the absence ofthe nuclear extract containing an equivalent of the ingredients in thenuclear e xtraction buffer. The arrow refers to the equivalence ofthe added DNA p olymerase activity and t he inhibitory activity.Ó FEBS 2002 Poly(b-L-malate) mediated DNA polymerase activity (Eur. J. Biochem. 269) 1255The inhibitory activity during the cell cycleThe inhibitory activity during the cell cycle was measured inthe p resence of 0.4 U of (added) purified DNA polymerasea and the extract of 1.5 · 105nuclei. The dependence isshown in Fig. 2A in terms of the reciprocal values,corresponding to the residual DNA polymerase activity.The m aximum at 1 h after m itosis corresponded t o aminimum in inhibitory activity and a maximum i n t heresidual polymerase a ctivity (63% of the reference activity).After 2 h following mitosis and during the remainder of t hecell cycle, the polymerase activity approached a basal levelof 10–20% of the reference activity.The activity of DNA polymerases measuredby photo affinity labelingAccording to Fig. 2A, the inhibitory activity in the nuclearextract s howed a minimum between 0 and 2 h after mitosis(Fig. 2 A). It was of interest whether this interval coincidedwith some endogenous residual activity of the DNApolymerases in the n uclear extract (DNA polymerase anot added). Because t he (residual) activity of the e ndo-genous DNA polymerases was too low to be detected withthe standard assay, we introduced a highly sensitivetechnique of affinity photo crosslinking [7]. Briefly, theenzymatically active DNA polymerase c atalysed the primerelongation w ith r adioactively labeled nucleotides o f h ighspecific radioactivity. Then , the e longated primers w erephoto crosslinked to the active polymerases within theelongation complex. The amount of radioactivity covalentlyattached to DNA polymerases was an i ndicator of thepolymerase activity and was measured after SDS/PAGE byautoradiography. S eparate results are shown in Fig. 2C forDNA polymerase e and for DNA polymerases of type a,type-b-like, and type d in Fig. 2B, which were not resolvedfrom each other. DNA polymerase e showed the highestactivity during the first hour after mitosis. Then the activityFig. 2. DNA polymerase activitiy during the cell cycle of macroplasmodia. All graphs are drawn to the same scale to facilitate comparison. In thisscale, the measured value at 0.7 h in the nuclear division c ycle is arbitrarily set equal to one u nit in each panel. M denotes m itosis. (A) The reciprocalinhibitory activity vi(vo) vi))1(see text) calculated from values of the r esidual polymerase activity (vi) and the reference activity (vo)ofadded0.4 Uof purified DNA po lymerase a in the standard DNA polymerase assay with (vi) and without (vo) nuclear extract. The extract of 2 · 106nuclei wasprepared from macroplasmodia at various times during the cell cycle. Bars refer to standard deviations (three determinations). One unit on scalerefers to 1.64 U of th e recip rocal in hibitory activity. (B) Activity o f endo geno us D NA polymerases a, d,andb-like in the nuclea r e xtract, measuredin arbitrary units (staining intensity) by the highly sensitive technique o f photoaffinity labeling. Single types of DNA polymerases could n ot beresolved. Bars refer to standard deviations (three determinations). (C) Activity of endogenous DNA polymerase e, measured in parallel with t heDNA polymerases in panel B. One u nit o n scale compares to half a unit in (B). (D) DNA synthesizing activity o f plasmodia at various times in thecell cycle. The incorporation of radioactivity into acid precipitable material has been measured during a brief exposure to [methyl-3H]thymidine.One u nit on scale refers to 6 Bq [3H]TMP incorporated. (E) The activity of endogenous D NA polymerases in the extract o f 2 · 108nuclei at varioustimes during the cell cycle. One unit on scale refers to 90 U of DNA po lymerase activity (see Materials an d metho ds). T he activity was measured i nthe presence of added 2 mMspermidineÆ3HCl (?) to neutralize t he inhibitory activity o f PMLA. I n the absence o f spermidineÆ3HCl, activities ofDNA polymerase, except o f DNA polymerase b-like, are inhibited by PMLA contained in the extracts.1256 S. Doerhoefer et al. (Eur. J. Biochem. 269) Ó FEBS 2002decreased and fell to a basal level 2 h after mitosis. Thedependence was sim ilar f or the unresolved DNA poly-merases. The high basal level was explained by thecontribution of DNA polymerase b-like, which was notinhibited by PMLA. The results show that the minimum inthe inhibitory activity corresponded with a maximum ofDNA polymerase activity in the cell cycle.Thein vivoactivity of DNA polymerasesThe cell cycle depen dence of DNA polymerase activity w asfollowed under in vivo conditions. The incorporation ofradioactivity into DNA was determined following a briefexposure to [methyl-3H]thymidine. The results in Fig. 2Dshow a maximum in activity of DNA synthesis a t 1 h aftermitosis, followed by a decrease approaching a basal level at2 h after mitosis. Thus, the in vivo and in vitro activities ofDNA synthesis c orresponded with the minimum of theinhibitory activity in the cell cycle.The activity of DNA polymerases in the nuclear extractsafter neutralization of the inhibitory activityby spermidine hydrochlorideAlthough the appearance of an activity peak of DNApolymerases was consistent with the minimum in theinhibitory activity of PMLA, an additional periodicvariation in the intrinsic activities of the endogenousDNA polymerases was not excluded. It has b een shownthat biogenic polyamines bind to PMLA and n eutralize itsinhibitory activity against DNA polymerases [1]. Thisfinding allowed us to examine whether the intrinsic DNApolymerase activity of the extract varied during the cell cycleor whether it w as totally modulated by the degree ofcomplex formation of DNA polymerases with PMLA. Thecircles in Fig. 2E show the DNA polymerase activity ofnuclear extract in the standard assay in the case whenspermidine hydrochloride was not present. This a ctivityreferred to DNA polymerase b-like, which was not inhibitedby PMLA [1] and thus did not show a cell cycle dependence.The other DNA polymerases displayed no m easurableactivity in the standard assay due to the strong inhibition byPMLA (see also above). The squares i n Fig. 2E refer to theaddition of spermidine hydrochloride. The activities of theDNA polymerases were derepressed, because the inhibitoryactivity was neutralized. The data show the superpositionsfor DNA polymerases a–e. Importantly, a cell cycledependence was not indicated. The variations observed inFig. 2A–C were explained by the change in the inhibitoryactivity of PMLA during the cell cycle.DISCUSSIONMyxomycetes comprise a large family of organisms thattypically gen erate a plasmodium, a polynucleated giant cellamong other cell forms in the life c ycle [4]. Interestingly, thebillions of nuclei in these syncytia participate with highdegree of synchrony in the division cycle. All of themyxomycetes species so far examined contained PMLA inthe plamodia. The PMLA level in the nuclei is high andcomparable to that of chromosomal DNA [1,3]. It remainsconstant during the cell cycle, and PMLA synthesized inexcess amounts is secreted into the culture medium. Incontrast to varying degrees of PMLA synthesis, the contentin the nuclei is conserved among different species (Karl, M.,Anderson, R. W. & Holler, E., unpublished data). Thevarious observations suggest that PMLA may play a role inmany biological functions. One function has been attributedto the induction of sporulation of P. polycephalum [17] andanother to the carriage and s torage of DNA polymerases,histones, and other nuclear proteins in the plasmodium[2,18].In connection with the role as a carrier and storagefunction, the inhibitory activity of PMLA towards thereplicative DNA polymerases was of interest [ 1,6,7]. Thecoupling of the carrier/storage function and the inhibition ofDNA synthesis suggested an effect on the availability ofDNA polymerase activity during t he cell cycle. Our r esultsshowed an inverse relation of the inhibitory activity with theactivity of the endogenous DNA polymerases in the extractas well as with the DNA synthetic activity (S-phase) in livingplasmodia. While the DNA synthetic a ctivity in t he nuclearextract was periodic with a maximum in S-phase, t heactivities of the DNA polymerases in the standard assaywere constant after n eutralization of PMLA. The cell cycle-independent biosynthesis and activity of DNA polymerase ahas been described by Western blotting and a ctivity gelanalysis [19].The findings extend t he storage/carrier role of PMLA to acontroller function of the catalytic competence of DNApolymerases. The results in Fig. 2 E revealed that theactivities of DNA polymerases on their own did not varyand that they were inhibited by complex formation withPMLA. The inhibition would be permanent unless thecomplexes dissociated in S-phase. The dissociation could beprincipally controlled by a periodical decrease in the level ofPMLA. However, the nu clear content of PMLA has beenshown to be constant over the cell cycle [3]. As a carrier,PMLA binds also histones and other nuclear proteins [2].Core histones, for example, are heavily synthesized inS-phase [20], and are likely to compete with DNA poly-merases for the binding of PMLA. Once free, DNApolymerase is c ompetent for DNA synthesis. However,the i dentity of these effectors i s still unclear. We f avorhistones, because they are depleted from the PMLAcomplexes by forming nucleosomes, when their synthesisceases at the end of S-phase. This would explain the e nd ofthe activity period of DNA polymerases. Instead ofassuming a neutralization of the inhibitory a ctivity, theDNA polymerases could bind effectors, which induce therelease of polymalate from t he complex. While this mech-anism is principally possible, it is not supported byexperimental evidence. PMLA binds to DNA polymerasescompetitively with DNA [1], and such factors would alsoinhibit the binding of template-primer DNA and thus thepolymerase activity. Although t he inhibitory activity ofPMLA and its cycling has been established i n the nuclearextract, it is speculated th at a s imilar mechanism exists in t henuclei of plasmodia. A major reason for this assumption isthe finding that PMLA forms complexes with DNApolymerases, histones a nd other nuclear proteins underconditions close to in vivo [2,7].The PMLA-dependent cycling of DNA polymerases doesnot account for the timing of DNA r eplication. It also doesnot take into account the periodic synthesis of factors suchas the proliferating-cell nuclear antigen (PCNA) andÓ FEBS 2002 Poly(b-L-malate) mediated DNA polymerase activity (Eur. J. Biochem. 269) 1257replication factor C ( RF-C) [7], but merely links the catalyticcompetence of DNA synthesizing polypeptides to the S-phase. These factors are recognized only with specializedtemplate-primers but not with activated salmon testis DNA,as used here. Because PMLA is only found in themultinucleated plasmodia and not in the mononucleatedamoebae, it is speculated that the periodic change of theinhibitory activity is involved in the maintenance of theplasmodial synchrony by coordinating the catalyticcompetence o f DNA polymerases throughout the giantcell. We are currently investigating the transfer of PMLAbetween the nuclei, and the competitive exchange of DNApolymerases and effector proteins.REFERENCES1. Fischer, H., Erdmann, S. & Holler, E. (1989) An unusual p oly-anion from Physarum. polycephalum that inhibits homologousDNA polymerase a in vitro. Biochemistry 28, 5219–5226.2. Angerer, B. & Holler, E. (1995) Large complexes of b-poly(L-malate) with DNA polymerase a, histones, and other proteinsin nuclei of growing plasmodia of Physarum polycephalum. Bio-chemistry 34, 14741–14751.3. Schmidt, A., Windisch, C. & Holler, E. (1996) Nuclear accumu-lation and homeostasis of the u nusual polymer b-p oly (L-malate)in plasmodia o f Physarum polycephalum. Eur. J. Cell. Biol. 70,373–380.4. Burland, T.G., Solnica, K.L., Bailey, J., Cunningham, D.B. &Dove, W .F. (1993) Pattern s of inheritance, development an d themitotic cycle in t he protist Physarum polycephalum. Adv. Microb.Physiol. 35, 1–69.5. Holler, E., Achhammer, G., Angerer, B., Gantz, B., Hambach, C.,Reisner, H., Seidel, B., Weber, C., Windisch, C., Braud, C.,Guerin, P. & Vert, M. (1992) Specific inhibition of Physarumpolycephal um DNA-polymerase-a-primase by poly (L-malate) andrelated polyanions. Eur. J. Biochem. 206, 1–6.6. Achhammer, G., W inkler, A., Angerer, B.& Holler, E. (1995) DNApolymerase d ofPhysarum polycephalum. Curr. Genet. 28, 534–545.7. Doerhoefer, S ., Khodyreva, S., Safronov, I .V., Wlassoff, W.A.,Anarbaev, R., Lavrik, O.I. & Holler, E. (1998) Molecularconsituents of the replication apparatus in the plasmodium ofPhysarum polycephalum: identification by photoaffinity labelling.Microbiology 144, 3181–3193.8. Daniel, J.W. & Baldwin, H.H. (1964) Methods of culture forplasmodial myxomycetes. Methods Cell Physiol. 1, 9 –14.9. Nygaard, O.P. & Guttes, S.R. H.P. (1960) Nucleic acid metabolismin a slime mold with synchronous mitosis. Biochim. Biophys. Acta38, 298–306.10. Mohberg, J. (1982) Recognition of mitosis. In Cell Biology ofPhysarum and Didymium (Aldrich, H.C. & Daniel, J.W., eds),pp. 273–276. Academic Press, New York, NY.11. Weber, C., Fischer, H. & Holler, E. (1988) Purification andcharacterization of DNA polymerase a from plasm odia ofPhysarum polycephalum. Eur. J. Biochem. 176, 199–206.12. Holler, E., Fischer, H., Weber, C., Stopper, H., Steger, H. &Simek, H. (1987) A DNA polymerase with unusual propertiesfrom the slime mold Physarum polycephalum. Eur. J. Biochem.163, 397–405.13. Achhammer, G., Angerer, B., Windisch, C., Uhl, A. & Holler, E.(1992) DNA Polymerase a-primase complexes of Physarumpolycephal um. Cell. Biol. Int. Reports 16 , 1047–1053.14. Safronov, I.V., Sherbick, N.V., Khodyreva, S.N., Wlassoff, W.A.,Dobrikov, M.I., Shishkin, G.V. & Lavrik, O .I. (1997) N ewphotoreactive N4-substituted dCTP analogues: preparation,photochemical characteristics, and substrate properties in HIV-1reverse transcriptase-catalyzed DNA synthesis. Russ.J.Bioorg.Chem. 23, 576–585.15. Laemmli. U.K. (1970) Cleavage of structural proteins duringtheassemblyoftheheadofbacteriophageT4.Nature 227,680–685.16. Towbin, H., Staehlin, T. & Gordon, J. (1979) Electrophoretictransfer of proteins from polyacrylamide gels to nitrocellulosesheets: procedure and some applications. Proc. Natl Acad. Sci.USA 76, 4350–4354.17. Renzel, S., Esselborn, S., Sauer, H.W. & Hildebrandt, A. (2000)Calcium and Malate are sporulation-promoting factors ofPhysarum polycephalum. J. Bacteriol. 182, 6900–6905.18. Rathberger, K., Reisner, H.W.B., Molitoris, H P. & Holler, E.(1999) Comparative synthesis and hydrolytic degradation of poly(L-malate) by myxomycetes and fungi. Mycol. Res. 103, 513–520.19. MacNicol, A.M., Banks, G.R. & Cox, R.A. (1987) Biosynthesisand activity of DNA polymerase throughout the mitotic cycle ofPhysarum polycephalum. FEBS Lett. 221, 48–54.20. Loidl, P. & Gro¨bner, P. (1987) Histone synthesis during the cellcycle of Physarum polycephalum. Synthesis of different histonespecies is not under a common regulatory control. J. Biol. Chem.262, 10195–10199.1258 S. Doerhoefer et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . orresponded with the minimum of the inhibitory activity in the cell cycle. The activity of DNA polymerases in the nuclear extracts after neutralization of the inhibitory activity by spermidine hydrochloride Although. 1255 The inhibitory activity during the cell cycle The inhibitory activity during the cell cycle was measured in the p resence of 0.4 U of (added) purified DNA polymerase a and the extract of 1.5. the minimum in the inhibitory activity corresponded with a maximum of DNA polymerase activity in the cell cycle. The in vivo activity of DNA polymerases The cell cycle depen dence of DNA polymerase
- Xem thêm -

Xem thêm: Báo cáo Y học: The DNA-polymerase inhibiting activity of poly(b-L-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum pot, Báo cáo Y học: The DNA-polymerase inhibiting activity of poly(b-L-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum pot, Báo cáo Y học: The DNA-polymerase inhibiting activity of poly(b-L-malic acid) in nuclear extract during the cell cycle of Physarum polycephalum pot

Gợi ý tài liệu liên quan cho bạn