Genomic DNA of Nostoc commune (Cyanobacteria) becomes covalently modi®ed during long-term (decades) desiccation but is protected from oxidative damage and degradation

Genomic DNA of Nostoc commune (Cyanobacteria) became covalently modi®ed during decades of desiccation. Ampli®cation of gene loci from desiccated cells required pretreatment of DNA with Nphenacylthiazolium bromide, a reagent that cleaves DNAand protein-linked advanced glycosylation end-products. DNA from 13 year desiccated cells did not show any higher levels of the commonly studied oxidatively modi®ed DNA damage biomarkers 8-hydroxyguanine, 8-hydroxyadenine and 5hydroxyuracil, compared to commercially available calf thymus DNA. Different patterns of ampli®cation products were obtained with DNA from desiccated/ rehydrating cells and a liquid culture derived from the dried material, using the same set of primers. In contrast, a reproducible ®ngerprint was obtained, irrespective of time of rehydration of the DNA, using a primer (5¢-GWCWATCGCC-3¢) based upon a highly iterated palindromic repeat sequence present in the genome. In vitro, the desiccation of cccDNA led to loss of supercoiling, aggregation, loss of resolution during agarose gel electrophoresis and loss of transformation and transfection ef®ciency. These changes were minimized when DNA was desiccated and stored in the presence of trehalose, a non-reducing disaccharide present in Nostoc colonies. The response of the N.commune genome to desiccation is different from the response of the genomes of cyanobacteria and Deinococcus radiodurans to ionizing radiation. INTRODUCTION The concept of dormant or `ancient' cells is a controversial topic because of largely untested assumptions about the stability of cellular components within dry cytoplasm (1). DNA in growing cells is thought to exist in the fully hydrated B-form and to have properties similar to those measured for DNA in solution (2). The limited chemical stability of DNA under these conditions is considered to be the major determinant of cell survival as well as a factor that must set limits for the recovery of DNA fragments from old cells and fossils (2,3). Yet it is unclear whether desiccated nucleic acids are subject to the same types, degree and rates of damage or modi®cation as observed (or predicted) for hydrated molecules in growing cells. Obviously some cells do tolerate desiccation and they must do so because of an ability to protect vital components of their cellular machinery from damage and/or repair them quickly upon rehydration. The latter strategy, for example, is employed by the ionizing radiationresistant bacterium Deinococcus radiodurans (4). One potential modi®cation of nucleic acid and protein occurs through Browning reactions (5,6). The modi®cation is initiated by the spontaneous reaction of reducing sugars with the primary amino groups of proteins and nucleic acids (7). The Browning (Maillard) reaction proceeds from reversible Schiff base and Amadori products to a class of irreversibly bound, structurally heterogeneous products referred to as advanced glycosylation end-products (AGEs) (8). Accumulation of AGEs is implicated in many of the pathophysiological alterations associated with normal aging. Such Maillard products occur in plant and animal remains and are a prominent component of ancient DNA extracts (9±11). Other factors that contribute to DNA modi®cation, and ultimately the killing of cells, include metal-catalyzed Haber±Weiss and Fenton reactions (12) and the presence of *To whom correspondence should be addressed at 205 Engel Hall, W. Campus Drive, Virginia Tech, Blacksburg, VA 24061, USA. Tel: +1 540 231 5745; Fax: +1 540 231 9070; Email: [email protected] Nucleic Acids Research, 2003, Vol. 31, No. 12 2995±3005 DOI: 10.1093/nar/gkg404 Nucleic Acids Research, Vol. 31 No. 12 ã Oxford University Press 2003; all rights reserved at Penylvania State U niersity on Feruary 1, 2013 http://narrdjournals.org/ D ow nladed from reactive oxygen species and free radicals (5,13±15). Fivemembered hydantoin rings in DNA originate from oxidative decay of six-membered pyrimidines (3,16,17); their presence is thought to be negatively correlated with the PCR ampli®cation of DNA. Of the free radicals, the highly reactive hydroxyl radical (OH) causes damage to DNA and other biological molecules (18,19). This type of DNA damage is also called `oxidative damage to DNA' and is implicated in mutagenesis, carcinogenesis and aging (20). The occurrence of modi®ed bases is problematic because DNA polymerases are unable to copy these damaged residues. The reagent N-phenacylthiazolium bromide (PTB) disrupts AGE crosslinks and its use made it possible to amplify DNA sequences from ancient samples (11). There are procedural dif®culties when attempting to understand the biochemical properties of cellular components in desiccated cells. Aqueous and organic reagents used in cell extraction and preservation techniques disrupt intracellular microenvironments and the physical properties of dried macromolecules. Non-invasive structural techniques such as Fourier transform infrared spectroscopy and differential scanning calorimetry can provide some information on the physical state of cytoplasmic components (21), but not in suf®cient detail to assess the physiological relevance of any modi®cations. For example, supercoiling of DNA has a profound in ̄uence on the regulation of gene expression in whole genomes yet is very dif®cult to study directly even in fully rehydrated cells (22,23). To understand how ̄uctuating water content may in ̄uence the integrity of nucleic acids (and ultimately gene expression) within cells of Nostoc commune, we designed assays to obtain empirical data on the effects of long-term desiccation on puri®ed supercoiled and linear DNAs in vitro. Nucleic acids were then obtained from cells following different periods of desiccation and rehydration, in the presence and absence of PTB reagent prior to ampli®cation of selected gene loci, and rapid ampli®cation of polymorphic DNA (RAPD)±PCR assays were used to analyze the in vivo dynamics of DNA in the rehydrated cells. MATERIALS AND METHODS Biological materials and growth conditions Desiccated colonies of N.commune of different ages were collected and stored in the dark, until analysis (Table 1). Genotypic analyses indicated these all belonged to `form species' N.commune (24). Most herbarium specimens were obtained in sealed paper envelopes that were unopened since the time of collection. A liquid culture, N.commune strain DRH1, was derived from a desiccated sample and grown as described (25). Human kidney 293H cells were grown and transfected with plasmid DNA as described (26). Drying and rehydration of DNA Escherichia coli strains BL21DE3 (pSpsA), BL21DE3 (pMP005) and DH10B (pEXPcmvgtBgal) were grown overnight in Luria±Bertani (LB) medium containing 200 mg/ml ampicillin. Plasmid DNA was puri®ed using Wizard preparation kits (catalog no. PR-A7510; Promega). Solutions of plasmid and linear DNAs were prepared in 13 TE buffer (1 mM EDTA, 10 mM Tris±HCl, pH 7.5, approximately 1:10, powder:buffer) in the presence or absence of ®lter-sterilized 100 mM trehalose (D[+] Trehalose T-1901; Sigma). Aliquots of the DNA solutions were used to determine the concentration of DNA, for agarose gel electrophoresis, for ®eld Table 1. Treatment of DNA with PTB and ability to amplify gene loci Gene ampli®cation Age (years) Viable tRNALEU introna tRNALEU intronb phr 23S rDNA sodF material +PTB ±PTB +PTB ±PTB +PTB ±PTB +PTB ±PTB +PTB ±PTB WH002 64 Yes + + ± ± + ± + ± ++ ± WH0014 50 Yes ++ ND +++ + + + + + +++ + ALD8122 29 Yes ++ + + + + + + + + + WH010 ~120 ± ± ± ± ± + + + ± ± ± WH012 118 ± ± ± + ± + + + ± ± ± WH001 75 ND + + ± ± + ± + ± ± ± WH004 149 ND + + ± ± + + + + + + WH005 139 ND ± ± ± ± + + + + ± ± WH006 120 ND + ± + ± + ± ++ + ± ± WH011 138 ND ± ± ± ± + ± + ± ± ± WH013 94 ND ± ± + ± + ± + ± ± ± WH016 133 ND ± ± + ± + + + + ± ± MEL1968 30 ND ++ + + + ++ + ++ + +++ ++ SPH1998 26 ND + + + + + + + + ± ± DRH1 NA Yes ND +++ ND + ND + ND + ND + Escherichia coli strains and plasmids Comments Source Escherichia coli BL21DE3 (pSpsA) Ampr, spsA of Synechocystis PCC 6803; sucrose 6-phosphate synthesis (55) Escherichia coli BL21DE3 (pMP005) Ampr; iphP of Nostoc sp. UTEX 584; phosphomonoesterase activity (44) Escherichia coli DH10B (pEXPcmvgtbgal) Ampr; Gateway (Invitrogen) construction; cytomegalovirus promoter; b-galactosidase activity Fred Bloom Escherichia coli DH10B ElectromaxÔ Competent cells Invitrogen Escherichia coli TOP10Ô Competent cells Invitrogen NA, not applicable, liquid culture; ND, not determined; +, ++, +++, relative abundance of a single ampli®cation product; ±, no ampli®cation product. aUsing LEU1 and LEU3 primers (see Table 2). bUsing LEU1 and LEU2 primers (see Table 2). 2996 Nucleic Acids Research, 2003, Vol. 31, No. 12 at Penylvania State U niersity on Feruary 1, 2013 http://narrdjournals.org/ D ow nladed from inversion gel electrophoresis and to transform Electromax competent E.coli DH10B (Invitrogen, CA) as described in the ®gure legends. Aliquots were dried in 1.5 ml microcentrifuge tubes under a stream of sterile air until no liquid remained in the tube (typically overnight). These tubes were then transferred to a glove box and all subsequent manipulations were performed under an atmosphere of nitrogen, under positive pressure. The open tubes were placed on the surface of phosphorus pentoxide powder (P2O5) in 50 ml containers that were later sealed. Storage was continued at room temperature in the light, under an incident photon ̄ux density of 50 mmol photons/m2/s1, or in the dark, for periods from 72 h to 56 days prior to rehydration. In the present experiments the mass ratio of plasmid pMP005 (6.29 kb) DNA to trehalose following drying (50:1711.5 mg) was therefore 1:34.2 (0.0079 3 10±3:0.005 mmol) After a period of storage, replicate tubes were removed and desiccated DNA was rehydrated with sterile distilled water for different periods up to 24 h, in the light, prior to further manipulations. Genomic DNA puri®cation Genomic DNA of N.commune DRH1 was puri®ed as described previously (25). Desiccated materials were lyophilized and ground to a powder under liquid nitrogen. The powder was rehydrated with 13 TE. Extraction buffer (1.4 M NaCl, 20 mM EDTA, 1.5% w/v hexadecyl trimethyl ammonium bromide, 1% v/v b-mercaptoethanol, 100 mM Tris±HCl, pH 7.5, approximately 4 times the volume of 13 TE added to the powder) was added and the mixture was incubated at 65°C, for 30 min. The slurry was homogenized, frozen under liquid nitrogen and thawed at 65°C; the freeze±thaw cycle was repeated six times. The mixture was extracted with chloroform:isoamyl alcohol (24:1) and DNA was recovered from the aqueous phase using isopropanol then stored in 13 TE buffer. In some cases DNA was extracted exhaustively (up to six times) with phenol prior to chloroform:isoamyl alcohol extraction and prior to treatment with PTB (see below). In some cases desiccated colonies were ground to a powder under liquid nitrogen and resuspended in 50 mM EDTA. An equal volume of 1.5% (w/v) molten agarose (type 1-A, low EEO, catalog no. A-0169; Sigma) was added to embed the cell material. The blocks were incubated overnight at 50°C with grade 1 lysozyme (®nal concentration 2 mg/ml) (Sigma) in 50 mM EDTA and then overnight at 50°C in buffer containing proteinase K (®nal concentration 2 mg/ml) (catalog no. P6556; Sigma), 0.1 M EDTA, 1% (w/v) N-lauroylsarcosine, 0.05% (w/v) SDS and 10 mM Tris±HCl (pH 8.0). Finally the blocks were equilibrated in 10 mM sodium phosphate buffer, pH 7.0, prior to the addition of PTB at a ®nal concentration of 10 mM. PTB was synthesized as described (8,11). The blocks were incubated in PTB for 10 h at 27°C, and then equilibrated in TAE buffer (2 mM EDTA, 40 mM Tris±acetate). The blocks were transferred to the wells of a 1% (w/v) agarose (catalog no. A-0169; Sigma) gel and the DNA was resolved at 75 mA for 1 h, excised from the gel and puri®ed further using a QIAEX II gel extraction kit (Qiagen, CA). Highly polymerized calf thymus DNA (ctDNA, type I) was obtained from Sigma Chemical Co. Linear DNA fragments in the size range 75±12 216 bp were obtained from Invitrogen (CA).