DNA helix destabilization by proline and betaine: possible role in the salinity tolerance process

Transcription

1 FEBS FEBS Letters 410 (1997) DNA helix destabilizatin by prline and betaine: pssible rle in the salinity tlerance prcess Chadalavada S.V. Rajendrakumar, Tangirala Suryanarayana, Arjula R. Reddy* Schl f Life Sciences, University f Hyderabad, Hyderabad , India Received 4 February 1997; revised versin received 14 April 1997 Abstract Evidence is prvided fr the ability f prline, a salinity induced smprtectant, t destabilize the duble helix and lwer the T m f DNA in a cncentratin dependent manner. At the reprted salinity-adaptive bi-accumulatin f 1 M and abve, prline culd cnsiderably decrease the T m and partially cunteract the effect f sdium chlride and spermidine n DNA stability. On the cntrary, several ther amin acids tested did nt shw any such destabilizing effect n DNA helix. Enhanced susceptibility t SI nuclease and insensitivity t DNase I in presence f increasing prline cncentratins have further suggested a clear destabilizatin f the duble helix. Such an effect is smewhat reminiscent f the interactin between betaine, anther salinity induced smlyte, and DNA resulting in decreased T m values. These interactins may be significant in view f the abundance f such smlytes in cells under salinity stress-adapted cnditins, with many a bacterial mutant accumulating them exhibiting imprved tlerance t salinity Federatin f Eurpean Bichemical Scieties. Key wrds: Prlme; Betaine; DNA helix; T m curves; Salinity stress 1. Intrductin Prline and betaine are the tw knwn majr smprtectants which accumulate in plants, bacteria, algae and marine invertebrates in respnse t an array f abitic stresses, mst prminent being the salinity stress [1^1]. Mre ften, this accumulatin is the result f an adaptive de nv synthesis in cells cntributing a majr share amng smlytes [5-8]. Such accumulatins were fund t reach up t 1 M internal cncentratin in certain halphytes and bacteria, accunting fr as much as 10-20% f the dry weight [9-12]. These tw smlytes were als reprted as efficient stabilizers f prteins, lipid membranes, rganelles and cells under severe stress cnditins withut being inhibitry t cellular functins [13-19]. Further, genetically engineered hyper-accumulatin f prline was reprted t cnfer salinity tlerance in tbacc seedlings under labratry cnditins [20]. Many plant and bacterial mutants accumulating prline and betaine have als been fund t exhibit an increased tlerance t salinity stress [11,12,21-24]. We have investigated the interactin f these smlytes with DNA, since their access, even transiently, t DNA in viv under the stress adapted cnditins can nt be ruled ut due t their abundance. In fact, betaine was prved recently t cnsiderably destabilize DNA [25]. We reprt here that prline destabilizes DNA and partially cunteracts the *Crrespnding authr. Fax: (91) arjulsl@uhyd.ernet.in effect f sdium chlride and spermidine n the stability f the duble helix within the adaptive bi-accumulated cncentratins. The present study indicates a pssible rle f these smlytes in salinity tlerance prcess by negating the undesirable effect f NaCl n DNA stability. 2. Materials and methds L-Prline, hydrxy prline, glycine, alanine, valine, leucine, serine; betaine, D-glucse, sarcsine, calf thymus DNA, Tris, EDTA, spermidine, NaCl, agarse and X phage DNA were purchased frm Sigma (St. Luis, MO, USA). E. cli single strand DNA binding prtein (ssb prtein) and puc 18 plasmid were prcured frm Bangalre Genei (Bangalre, India). DNase I was prcured frm Behringer-Mannheim (Mannheim, Germany) and the SI nuclease frm Pharmacia (Uppsala, Sweden). All ther chemicals were f analytical grade purchased lcally DNA melting studies DNA melting studies were cnducted in a buffer (1 ml) cntaining 10 mm Tris-HCl (ph 7.5) and 2 mm EDTA and the indicated cncentratins f NaCl and additives. Calf thymus DNA (1.0 A260) in the abve buffer, with r withut the additives, was taken in a 1 cm path tefln-stppered quartz cell and incubated at the initial assay temperature fr 5 min. The increase in absrbance at 260 nm was mnitred in a Hitachi spectrphtmeter attached t a temperature prgrammer KPC-6 and temperature cntrller SPR-7. Bth the sample and reference cells were heated tgether at a rate f l C/min, and the net absrbance was recrded after every 1 C increase. The T m f DNA was determined graphically frm the transitin mid-pint f the absrbance versus temperature prfile DNase I sensitivity assay The sensitivity f DNA t DNase I digestin was studied spectrphtmetrically (Hitachi) by measuring the increase in absrbance at 260 nm at 37 C in presence f different cncentratins f prline. DNase I (1 ug) was added t duble stranded calf thymus DNA (1.0 A 2m ) in a buffer (1 ml) cntaining 10 mm Tris-HCl (ph 7.8), 50 mm NaCl, 5 mm MgCl 2, 1 mm DTT. The enzyme was diluted t required cncentratin in 10 mm Tris-HCl (ph 7.8) and 50% (v/v) glycerl. DNase I sensitivity f DNA was als analysed by agarse gel electrphresis. Calf thymus DNA, X phage DNA, r puc 18 DNA (1 ug each) in the DNase I assay buffer (30 ul) was incubated at 37 C fr 10 min with 25 ng f DNase I in the presence f different cncentratins f prline and the digestin prducts were separated n a 0.8% agarse gel SI nuclease sensitivity assay The SI nuclease reactin mixture (30 ul) cntained calf thymus DNA (0.5 ug), buffer (5 mm sdium acetate (ph 4.7), 15 mm sdium chlride, 0.1 mm ZnCb) and prline. DNA samples in presence f increasing cncentratins f prline were heated at 65 C fr five minutes and quickly chilled n ice. Reactin was started by adding SI nuclease (1 unit) and incubated at 37 C fr 15 min. The digestin was stpped by adding EDTA and SDS t a final cncentratin f 50 mm and 1%, respectively, and the prducts were separated n a 0.8% agarse gel Single strand binding prtein gel shift assay The X phage DNA (0.5 ug) in 30 ul buffer cntaining 10 mm Tris /97/S Federatin f Eurpean Bichemical Scieties. All rights reserved. P//S (97)

2 202 C.S. V. Rajendrakumar et al.lfebs Letters 410 (1997) C n. i F c ID Csl O a Temperature Fig. 1. Effect f increasing cncentratins f prline n the T m f calf thymus DNA: (a) Cntrl DNA (withut prline), (b) 0.06 M, (c) 0.25 M, (d) 0.5 M, (e) 1.0 M, (f) 2.0 M, (g) 3.0 M, (h) 4.0 M, (i) 4.5 M, (j) 5.0, (k) 5.5 M. ( C) HC1 (ph 8.1), 1 mm EDTA and 20 mm NaCl, was heated at 65 C in the presence r absence f 3.0 M prline fr 5 min and quickly chilled n ice. Increasing cncentratins f ssb prtein was added and after incubatin at rm temperature fr 5 min, the samples were electrphresed n a 0.7% agarse gel Displacement f DNA bund ethidium brmide by prline Ethidium brmide (0.4 iig) in the buffer (10 mm Tris-HCl (ph 7.5) and 50 mm NaCl) was excited at 480 nm and the emissin was recrded between nm in a Hitachi spectrflurimeter. Later, calf thymus DNA (0.5 ug) was added t it t recrd the enhancement in flurescence emissin intensity. Similarly, the emissin spectra were recrded with the additin f increasing cncentratins f prline t the abve mixture after incubating at rm temperature fr 15 min. 3. Results Destabilizatin f DNA duble helix by prline was analysed by varius methds. Prline was fund t significantly lwer the melting temperature f calf thymus DNA in a cncentratin dependent manner. Thugh such an effect fund at Table 1 Effect f prline and ther amin acids n the T m DNA in the presence and absence f additives Cncentratin DNA M prline +2.0 M prline +2.0 M glycine +2.0 M serine M alanine M valine +0.1 M leucine +2.0 M hydrxy prline +0.5 M glycyl glycine +2.0 M sarcsine M glucse M betaine M prline+1.0 M betaine + 10 mm spermidine +0.5 M NaCl + 10 mm spermidine+1.0 M prline +0.5 M NaCl+1.0 M prline +0.5 M NaCl+2.0 M prline +0.5 M NaCl+2.0 M glycine f calf thymus T m f DNA±1.0 C mm was marginal, an appreciable decrease in T m was bserved cnsistently (Fig. 1) at cncentratins ranging frm 250 mm t 1 M, which are widely reprted t be bilgically relevant [9-12]. In rder t knw whether the effects shwn by prline are specific, several ther amin acids were tested as cntrls. The results reveal (Table 1) that nne f the amin acids tested culd induce a similar effect even at high cncentratins. While glycine, glycyl glycine, and serine were fund t significantly stabilize the duble helix and increase the T m, alanine, valine, leucine and sarcsine culd nt greatly alter the T m. Hwever, hydrxy prline at its maximum aqueus slubility pint (2.0 M), culd reduce the T m by 8 C. Prline, unlike its hydrxylated analgue, with a high aqueus slubility (6.0 M) due t the reprted anmalus slutin prperties [18], was fund t destabilize DNA even beynd such a cncentratin (Fig. 1). Hwever, the differential aqueus slubility f tested slutes prevented an ideal cmparisn between them in their interactin with DNA. Prline and betaine (1 M each) were fund t have an 0.35 c u Time (min) Fig. 2. DNase I sensitivity f calf thymus DNA in the presence f increasing cncentratins f prline.

3 C.S. V. Rajendrakumar et allfebs Letters 410 (1997) A ph9p DMA 0 SM Prline 0.75M Prline M.OM Prline tl.sm Prline 2.5 M Prline puc M Prline 2.5M Prline 2,0 M Prl ine 1.25M Prline 1.0 M Pr line 0 M Prline puc 18 prperties f prteins which interact with DNA (see belw). In cntrast, prline at increasing cncentratins was fund t make the duble stranded calf thymus DNA mre susceptible t SI nuclease digestin (Fig. 4). In the gel retardatin assay, binding f increasing amunts f ssb prtein t X DNA in presence f 3.0 M prline was fund t retard the mbility f the DNA-prtein cmplexes which was clearly absent in the cntrl X DNA with the additin f 12 ug f ssb prtein (Fig. 5). These results indicate the nn-interference f prline in interactins between such prteins and DNA. Finally, the ability f prline in replacing the ethidium brmide bund t duble stranded calf thymus DNA was tested and the flurescence emissin data (Fig. 6) revealed a marginal displacement which is expected f cmpunds that destabilize the duble helix. 4 M 3.3 M 2.75M 2 M Prline Prline Prline Prline 1.25 M Prline 1.0 M Prline 0.0 M Prline Cif thymus DNA Fig. 3. DNase I sensitivity f X phage, puc 18 and calf thymus DNA in the presence f different cncentratins f prline. additive effect in the reductin f T m (Table 1). Mrever, prline (1 M) was fund t individually reduce the effect f NaCl (0.5 M) and spermidine (10 mm) n DNA stability as indicated by the decrease in T m by 6 C and 8 C, respectively. On the cntrary, the c-additin f glycine (2 M) with sdium chlride (0.5 M) did nt influence the effect f the latter n DNA indicating the ineffectiveness f glycine in cunteracting the salt effect (Table 1). The helix destabilizatin was further cnfirmed with the DNase I and SI nuclease sensitivity assays. In the spectrphtmetric analysis f DNase I digestin, increased prline cncentratins were fund t prgressively prtect the calf thymus DNA against the digestin, with a near cmplete prtectin bserved at higher than 3.0 M (Fig. 2). This was further demnstrated by gel electrphresis prfile f DNase I digested samples f X phage, plasmid and calf thymus DNA (Fig. 3). Rice and barley DNA did shw a similar pattern f resistance t DNase I activity in the presence f prline (data nt shwn). This effect is either due t a decreased binding f DNase I t DNA r destabilizatin f the duble helix. The frmer is less likely because prline des nt affect the binding 4. Discussin Prline was fund t bring dwn the T m in a cncentratin dependent manner (Fig. 1), smewhat similar t betaine which was reprted t lwer the T m and partly reduce the impact f KC1 n DNA stability [25]. While 1 M prline culd reduce the T m f calf thymus DNA by 6 C (Table 1), betaine at a similar cncentratin culd reduce the T m f ply (dg-dc) by 5 C and the bacterial DNA by 4 C [25]. The results are significant in view f the reprted hyper bi-accumulatin f these smlytes under salinity stress. Such an effect was nt fund with ther tested amin acids, f which, glycine, glycyl glycine and serine were in fact fund t cnsiderably stabilize the DNA. Interestingly, with the additin f methyl grup(s) n the glycine structure, alanine, valine and leucine have crrespndingly lst bth the aqueus slubility and the stabilizing effect n the DNA. Similarly, JV,./V,Af-trimethylglycine (betaine) was fund t be helix destabilizing when cmpared t glycine and sarcsine (Table 1) [25]. In ne such related attempt t test the influence f methyl grups n the ptency f smprtectin, it was demnstrated that, cntrary t glycine and sarcsine, cmpunds f betaine series, with trimethyl grups n the nitrgen were fund t amelirate the effect f high salinity (0.8 M) n the grwth f E. cli [12]. Similarly, the bserved inability f glycine in cunteracting the effect f NaCl n DNA (Table 1) culd prbably be accunted as ne f the reasns fr its failure t prtect E. cli frm high salinity (0.8 M NaCl) stress [12]. Thugh preliminary, these results apparently establish a crrelatin between the reprted capability f these smlytes t prtect the rganism frm salinity stress with their ability t negate the salt effect n DNA stability. 1 M 1.25M 2.0 M 2.75 M 3.0 M 3.3 M Prlin e Prline P r 0I i n * prline prline p r line 4.0 M Prline Calf thymus DNA Fig. 4. SI nuclease sensitivity f calf thymus DNA i: the presence f different cncentratins f prline.

4 204 C.S. V. Rajendrakumar et al.lfebs Letters 410 (1997) Hwever, the destabilizing effect shwn by hydrxy prline is bilgically insignificant as it is nt knwn t accumulate in cells under the stress-adapted cnditins. Prline, n the cntrary, is a widely reprted smprtectant, knwn t stabilize prteins smewhat analgus t chapernes [13] and act as a prtein cmpatible hydrtrpe [26]. Further, the antagnistic effect f prline t that f NaCl n DNA stability in vitr pssibly suggests a similar interactin in viv where prline culd cunteract the effect f high cncentratin f salt and catins accumulating under stress cnditins. Presumably, DNA surrunded by a high cncentratin f salts is bilgically less active than that is surrunded by bth salts and their cunteracting smlytes such as prline and betaine. Mrever, prline and betaine were shwn t have an additive effect n DNA stability (Table 1), and when present tgether culd accunt fr effective cncentratins in viv. Apart frm the suggested effect n DNA, these smlytes are knwn t be highly bi-cmpatible with a prven rle in the stabilizatin f prteins, rganelles and cells [13-19] which can nt be ascertained with ther amin acids. Interestingly, upn increase in salinity f the grwth medium, Lactbacillus plantarum cells were fund t instantaneusly accumulate betaine and prline in preference t alanine as an adaptive measure [27]. Similarly, f the 150 cmpunds tested, nly prline and betaine series were fund t effectively prtect E. cli frm the severe salinity stress suggesting the versatility f these smlytes in cmparisn t ther slutes [12]. DNA destabilizatin by prline in ur study was further cnfirmed by the bserved resistance f DNA t DNase I in the presence f high cncentratins f prline. In fact, it is knwn that the activity f this enzyme n a stable duble helix is 5000 times higher than that n a destabilized helix [28]. Further, this culd nt be due t structural changes in the enzyme induced by prline as there are evidences that prline, even at high cncentratins, des nt substantially affect the structure and functin f prteins [13-18]. On the ther hand, prline was fund t cnfer structural stability t DNase I at higher temperatures (data nt shwn). Increased resistance t DNase I digestin and susceptibility t SI nucle Fig. 5. Gel mbility shift assay f X phage DNA in the absence and presence f 3.0 M prline with increasing cncentratins f ssb prtein. Lane 1, X phage DNA alne. Lane 2, X phage DNA+12 ug f ssbp. Lanes 3-6, X phage DNA in the presence f 3.0 M prline with increasing cncentratins f ssbp as fllws: 3, 3 ug f ssbp; 4, 6 (j.g f ssbp; 5, 9 (ig f ssbp; 6, 12 xg f ssbp. 3O.0 Wavelength (nm) Fig. 6. Flurescence emissin spectra f ethidium brmide in free and DNA-bund frm: effect f prline in displacing the DNAbund ethidium brmide. ase in the presence f increasing prline cncentratins suggest that the destabilized DNA structures culd exist at physilgical temperatures under stress adapted cnditins. Several studies indicate that bth the in vitr binding affinities and rate f binding f certain transcriptinal regulatry prteins t their target sites n DNA are extremely sensitive t the electrlyte cncentratins f the buffers used [29]. Since DNA at physilgical ph exists as a highly charged anin, it is expected t be surrunded by catins which have a natural binding affinity. Mrever, the salts which accumulate during salinity stress may als unduly stabilize the duble helix which culd adversely inhibit the DNA functin in replicatin and transcriptin [3]. Presumably, prline and betaine play an imprtant rle in partially alleviating such an effect. In fact, E. cli cells grwn at very high salinity cnditins (1 M NaCl) were fund t actively cncentrate glycine betaine as much as 10 5 times that f the medium [12]. It was further envisaged that during severe stress cnditins in bacteria, cellular cnstituents may cmpletely be bathed in smprtectants that reach cncentratins abve 1 M and interact with bimacrmlecules [12]. Similarly, the presence f high internal cncentratins f betaine under the stress-adapted cnditins was fund t reverse the effects f salinity mediated smtic stress n DNA replicatin and cell divisin in E. cli which supprts the rle f smprtectants in alleviating the stress effects n DNA functin [30]. Thus, the selective accumulatin f these tw smlytes in a wide range f rganisms under the salt stress appears t be a cnserved adaptive measure rather than a mere cincidence. While such an adaptive value f betaine/ prline-dna interactins can be envisaged in prkarytes where a direct access fr smprtectants t DNA exists, the same can nt yet be ascertained with respect t eukarytes with a distinct nuclear membrane barrier. Hwever, such interactins culd lgically be pssible during certain stages f cell divisin where the nuclear membrane barrier transiently disappears. Thugh a direct interactin in viv f prline r betaine with DNA is yet t be established, these smlytes are the likely bilgical chices t cunteract the effect f accumulated salts n DNA.

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