Uracil-DNA glycosylase activities in hyperthermophilic micro-organisms

MICROBIOLOGY LETTERS

ELSEVIER

FEMS Microbiology Letters 143 (1996) 267-271

Uracil-DNA glycosylase activities in hyperthermophilic micro-organisms Athanasios

Koulis, Don A. Cowan, Laurence H. Pearl, Renos Savva *

Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WClE 6BT, UK Received 20 June 1996; revised 9 August

1996; accepted

12 August

1996

Abstract Hyperthermophiles exist in conditions which present an increased threat to the informational integrity of their DNA, particularly by hydrolytic damage. As in mesophilic organisms, specific activities must exist to restore and protect this template function of DNA. In this study we have demonstrated the presence of thermally stable uracil-DNA glycosylase activities in seven hyperthermophiles; one bacterial: Thermotoga maritima, and six archaeal: Sulfolobus solfataricus, Sulfolobus shibatae, Sulfolobus acidocaldarius, Thermococcus litoralis, Pyrococcus furiosus and Pyrobaculum islandicum. Uracil-DNA glycosylase inhibitor protein of the Bacillus subtilis bacteriophage PBS1 shows activity against all of these, suggesting a highly conserved tertiary structure between hyperthermophilic and mesophilic uracil-DNA glycosylases. Keywords:

Hyperthermophile;

DNA repair;

Uracil-DNA

glycosylase;

Bacteriophage

PBS1

; Uracil-DNA

glycosylase

inhibitor

-

1. Introduction The informational integrity of the DNA genomes in all organisms is under constant attrition by a variety of chemical and radiological agents [l]. Among these, hydrolytic damage poses a particular threat to hyperthermophiles [l], and one of the most common hydrolytic damage events is the deamination of cytosine bases [l]. Deamination of cytosine results in the formation of the base uracil, normally found only in RNA. As uracil will base pair with adenine, deamination of cytosine will result in a transition mutation from G:C to A:T if the damaged strand is used as a tem-

* Corresponding author. Tel.: +44 (171) 380 7033; Fax: +44 (171) 380 7193. 0378-1097/96/$12.00 Copyright PIISO378-1097(96)00325-4

0 1996 Federation

of European

plate for replication. Estimates of the rate of cytosine deamination vary but figures around 8 X lo-l3 s-l in double-stranded and 2 X lo-lo s-l in single-stranded DNA at 37°C are typical. At 95°C these rates may be 1000 times faster [2] and thus potentially pose more serious problems for hyperthermophilic micro-organisms which grow optimally at such temperatures. Uracil-DNA glycosylases (UDGase; EC 3.2.2.3) are a highly specific and apparently ubiquitous class of repair enzymes which excise the uracil base from DNA and have, to date, been identified in organisms from all kingdoms [3,4,8,9] except the Archaea. Thermophilic examples have been isolated from the thermophilic bacteria Bacillus stearothermophilus [S] (growth

optimum

[6] (growth Microbiological

55’C)

optimum

Societies. Published

and

Thermothrix

75°C). UDGases

thiopara

specifically

by Elsevier Science B.V

cleave the N-glycosidic bond between the uracil base and the deoxyribose sugar. The abasic deoxyribose that results is then repaired by other enzymes [7]. UDGase must be highly specific, as even a low level of thymine-DNA glycosylase activity would have devastating effects on the structure and stability of the chromosomal DNA, while any excision activity against uracil in RNA would completely compromise the function of messenger, ribosomal and transfer RNA molecules. It is therefore important to understand how the exquisite specificity required of uracilDNA glycosylase activity is achieved at extreme temperatures. The crystal structures of the HSV-I [8] and human [9] UDGases show a very high degree of structural conservation and have revealed key conserved residues which provide the exquisite specificity for uracil and play a part in the catalytic mechanism of hydrolytic base excision. The high level of structural identity between the phylogenetically diverse UDGases (39% and 49% homology between HSV-I and the human and E. coli enzymes, respectively) suggests that hyperthermophilic UDGases may be excellent model enzyme systems for studies of more general aspects of molecular recognition at extreme temperatures.

6.5

7.0

7.5

X.0

PH

Fig. 1. pH profiles for UDGase the key box.

reactions

of organisms

shoun

in

2. Materials and methods

lowing the addition of 0.1 volume of 10% w/v streptomycin sulfate, to precipitate nucleic acids and other cell debris which might have interfered with subsequent assays. Clarified supernatants were obtained and stored at 4°C following a 15 min centrifugation at I6 000 X g. Protein concentrations were determined using the Bio-Rad Protein Assay (BioRad Laboratories Ltd.), using bovine serum albumin (fraction V, Sigma Chemical Company) to construct the standard curve.

2. I. Prepurution of’ clur$ied cell supernutunt,s

2.2. Prepurution of’ .suhstrute

Cells of E. coli BL21 (DE3) were harvested by centrifugation at 5000 Xg for 5 min from a IO ml overnight culture shaken at 37°C. Frozen biomass (-70°C) of Suljk~lohus ucidocaldurius, Sulfolohus Sulfolohus so&turicus, Thermococcus shibutue, litorulis, Pyrococcus ji.uYosus, Pyrohuculum islundicum and Thermotogu muritimu were kindly provided by Dr Richard Sharp and Dr Neil Raven of CAMR. Porton Down, UK. Clumps of frozen cells were added to graduated microcentrifuge tubes to approximately 100 yl, resuspended in 400 ul of TrisEDTA (70 mM Tris, 20 mM EDTA, pH 8.3+0.1 mM PMSF), and sonicated on wet ice with IOX 25 s cycles (10 micron power) with 25 s cooling between cycles. The lysates were incubated on ice for 30 min fol-

Radiolabelled (250 pCi, 15.3 Ci mmol-‘) 2’-deoxyuridine 5’-triphosphate (dUTP), tritiated at the 5 position in the pyrimidine ring, was obtained from Amersham International Plc as an aqueous solution in 50% ethanol. The solution was evaporated to dryness in a vacuum rotary evaporator, and 10 1-11 of a 1 in 15.3 dilution of a deoxynucleotide triphosphate (dNTP) solution (25 mM with respect to dATP, dGTP, dCTP, 22.5 mM with respect to dTTP, and 2.5 mM with respect to non-radioactive dUTP) (Pharmacia Biotech.) in deionised water was added. This mixture was then used as the dNTP stock for five PCR reactions, 50 pl each, of bases 10256-10859 of the HSVI genome, part of the UL2 open reading frame. Each PCR was performed using 5 units Taq DNA polymerase, and the 604 base pair product was

A. Koulis et al. IFEMS

Microbiology Letters 143 (1996) 267-271

subsequently isolated from a 1.5% agarose gel following electrophoresis in TAE buffer. The DNA was purified from the gel slices using the Geneclean (Bio- 10 1 Inc.) procedure, recovered in deionised water, 0.22 pm filtered (Millipore Ultrafree MC) and brought to 20 mM Tris-HCYlO mM EDTA pH 8.3. Assuming similar incorporation efficiency for deoxyuridine and thymidine, the distribution of uracil in the PCR generated substrate DNA will be approximately 55% of adenine-containing base pairs, with approximately 50% of adenine-containing base pairs containing tritiated uracil. The number of adenine-containing base pairs in this PCR-generated duplex is 217. 2.3. UDGase assay Assays were performed in 1.5 ml microcentrifuge tubes containing multiples of a standard 50 pl mixture, consisting of 48 ~1 HEPES-EDTA (50 mM HEPES, 1 mM EDTA), 1 ul substrate and 1 ~1 clarified cell supernatant. Reactions were started by adding 1 ~1 of ice cold clarified cell supernatant per 50 pl reaction mixture (pre-incubated for 5 min at the assay temperature). Typically, 50 yl aliquots were removed after 2, 5, 7.5 and 10 min and quenched by adding 200 ul of ice cold ethanol and 50 ml of ice cold stop mixture (0.5 mg ml-’ bovine serum albumin (fraction V), 0.5 mg ml-’ denatured and sonicated DNA from salmon testes (both from Sigma Chemical Company), 0.5 M sodium acetate pH 5.1). The tubes were incubated at -70°C for 30 min and the DNA was pelleted at 16 000 X g for 25 min at 4°C. 200 pl of each supernatant were added to 3 ml aliquots of Ecoscint A (National Diagnostics) scintillation fluid and counted in the tritium Table 1 Specific activities of hyperthermophile Organism

(pH optimum)

S. shibatae (pH 5.8) S. solfataricus (pH 6.0) Th. maritima (pH 6.5) Pb. islandicum (pH 6.5) PC. jiiriosus (pH 7.0)

UDGases

Specific activity protein)

(X

10-s U/mg of

80°C

90°C

1oooc

0.267 1.11

0.290 1.19 1.31 1.20 0.167

0.178 0.905 1.37 1.23 0.133

1.47 1.15 0.082

“A Unit is defined as the quantity 1 nmol of uracil per minute.

of enzyme required

to release

Table 2 Effect of UGI inhibitor UDGases Organism

E. colir S. shibatae S. solfataricus Th. maritima Pb. islandicum

protein

UDGase

269

on E. coli and hyperthermophile

inhibition

by UGI protein (%)

39°C”

80”Cb

100 13 54 22 81

nd. n.d. 35 23 12

“Assayed at pH 7.0. “Assayed at pH 6.5. ‘Assayed 7.5 and 8.0, 37°C. n.d.: not determined.

at pH 6.5, 7.0,

window of a scintillation counter. For the assays carried out with the PBS1 UGI protein, 1 ml of inhibitor (various dilutions from a > 96% pure 30 mg ml-’ stock) was added per 50 ml of reaction mixture. For each assay, positive and negative control reactions were performed using both tritiated uracil and tritiated thymine containing DNA to ensure that only UDGase activity was being measured. A thin layer of paraffin oil was applied to the top of each reaction to prevent evaporation at high temperature.

3. Results and discussion 3.1. UDGase activity in hyperthermophiles UDGase activity was detected in all six archaeal hyperthermophiles as well as in the bacterial hyperthermophile Tt. maritima. Preliminary assays were performed over a pH range spanning 5.5 to 8.0 (Fig. 1). Specific activities of S. shibatae, S solfataricus, Th. maritima, PC. furiosus and Pb. islandicum were determined at 80°C 90°C and 100°C at their optimal pH (Table 1). Clarified supernatants of S. acidocaldarius and Tc. litoralis had low levels of detectable activity which diminished considerably during 2 days storage at 4°C and were not included in any subsequent assays. The hyperthermophile UDGases in this study have pH optima less than 7, and only residual activity around pH 8. However, many mesophilic UDGases have pH optima around 8 [lo]. This might be explained by the fact that the pH for ionisation of histidine decreases with increasing temperature (M. Rossi, personal communica-

tion). The crystal structure of the HSVI enzyme indicated that a conserved histidine may be involved in the catalytic mechanism [S], and it is possible that hyperthermophiles may have a modified active site environment to deal with this temperature dependent pKa shift. The five hyperthermophiles listed in Table I display UDGase specific activities somewhat less than. but of the same order of magnitude as E. cd/ (3.59~ lop2 U mgg’ assayed at 37°C. pH 8.0: see Table 1 footnote for Unit definition). Mesophile UDGases are known to be active against uracil in both single- and double-stranded DNA [3] and the hyperthermophile UDGases appear to be similar in this respect. The 604 base pair substrate used in this assay is likely to be in the single-stranded form at 90°C. and certainly at 100°C. and activities are high at these temperatures. Low levels of activity were detected even at 40°C indicating that these hyperthermophile UDGases are also able to use double-stranded DNA as a substrate. The activity was enhanced with increasing temperature. however this is likely to be due to the hyperthermophilicity of the enzymes rather than a preference for a singlestranded substrate, as the increase is marked at temperatures below those required to melt a 604 base pair duplex (results not shown). 3.2. E#kct of’ PBS1 UDGusr inhibitor protein The B. subtilis bacteriophages PBS1 and PBS2 unusually contain uracil in their DNA genomes rather than thymine [I 11, and they encode several enzymes which maintain their DNA in this condition. These include a UDGase inhibitor protein (UGI), which forms a tight complex with UDGase under mesophilic physiological conditions [ 121. This complex is formed with UDGases from a wide variety of organisms, and the crystal structures for complexes between UGI from PBS1 and PBS2 in complex with HSVI [13] and human [14] UDGases respectively show that the inhibitor interacts with key conserved residues in the DNA binding channel of UDGases. In this respect, the UGI protein should be able to interact with all UDGases of the known type, and provide a simple test of conservation of the DNA binding region of the tertiary structure. Assays were performed in the presence of a signif-

icant molar excess of UGI with respect to the quantity of UDGase present, the UDGases were premixed with UGT for 5 min prior to addition to the assay mixture. E. coli UDGase showed complete inhibition over a broad range of pH (pH 6.5, 7.0, 7.5 and 8.0). The hyperthermophile UDGases were assayed at 80°C pH 6.5 and showed a partial reduction of activity (Table 2). They were also assayed at 39°C pH 7.0 to ensure better conditions for the formation of a UDGase-UGI complex (Table 2). Although inhibition was enhanced at the lower temperature, with the curious exception of Th. maritime, even under these conditions it was only partial. To ensure saturation, assays were performed with increasing concentrations of UGI protein. Even where the UGI concentration exceeded the total protein content of the hyperthermophile clarified cell supernatants used in the assays, inhibition remained at this partial level (results not shown). This result may indicate the presence of more than one uracilDNA glycosylase activity in hyperthermophiles, and that only one of these is of the known type, possessing the correct tertiary structure for inhibition by the UGI protein. Alternatively, some of the uracilDNA glycosylase may not be accessible to UGI, perhaps being part of a multiprotein complex. These effects will need to be re-examined when purer preparations of the hyperthermophile UDGases become available. In any event, these results indicate that there are hyperthermophilic UDGase activities which are likely to have homologous tertiary structure arrangements with known UDGases, which will facilitate structural studies into the mode of specific molecular recognition and modification of DNA at elevated temperatures.

Acknowledgments The authors wish to acknowledge the BBSRC for supporting facilities for the production of hyperthermophile biomass.

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struc-

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