Although restriction enzymes are widely distributed in nature, many bacterial genera are yet to be explored for the presence of this important class of enzymes. We have purified and characterized a new type II restriction endonuclease, OfoI from a nonheterocyst cyanobacterium Oscillatoria foreaui. The recognition sequence has been determined by primer extension analysis. The purified enzyme OfoI recognizes and cleaves the palindromic hexanucleotide 5'-C↓YCGRG-3', generating 5'-protruding ends

Anand, Narayanaswamy

Chandrashekaran, Siddamadappa

Elango, Kathirvel

Nagaraja, Valakunja

Saravanan, Matheshwaran

English

Publications of the IAS Fellows

RESEARCH COMMUNICATIONS 
 
CURRENT SCIENCE, VOL. 85, NO. 2, 25 JULY 2003 188
*For correspondence. (e-mail: vraj@mcbl.iisc.ernet.in) 
§Joint first authors. 
A new type II restriction endonuclease, 
OfoI from nonheterocystous  
cyanobacterium Oscillatoria foreaui  
 
Matheshwaran Saravanan†,§, Kathirvel Elango#,§, 
Siddamadappa Chandrashekaran†,  
Narayanaswamy Anand# and 
Valakunja Nagaraja†,* 
†Department of Microbiology and Cell Biology, Indian Institute of  
Science, Bangalore 560 012, India 
#Centre for Advanced Studies in Botany, University of Madras,  
Guindy Campus, Chennai 600 025, India 
 
Although restriction enzymes are widely distributed 
in nature, many bacterial genera are yet to be explo-
red for the presence of this important class of enzymes. 
We have purified and characterized a new type II  
restriction endonuclease, OfoI from a nonhet rocyst 
cyanobacterium Oscillatoria foreaui. The recognition 
sequence has been determined by primer extension 
analysis. The purified enzyme OfoI recognizes and cle-
aves the palindromic hexanucleotide 5¢-C¯YCGRG -3¢, 
generating 5¢-protruding ends.  
 
THE primary role of restriction–modification systems (R–
M) is to protect the host from invading foreign DNA 
molecules1. Three major types of R–M system are found 
in bacteria, viz. types I, II and III. Type II R–M systems 
comprise a separate restriction endonuclease and a methyl-
transferase that act independent of each other. Type II r-
striction endonucleases generally recognize palindromic 
sequences in DNA, cleave within or near their recogni-
tion sequences and produce DNA fragments of defined 
sizes. These useful properties have led to the screening of 
diverse bacterial species for new restriction enzymes. As 
a result, a large number of type II enzymes have been 
characterized2. Some of these enzymes have been studied 
extensively with respect to their structure, DNA binding 
and reaction characteristics. In spite of their importance 
to the host bacteria, restriction enzymes have not been 
detected in all bacterial species investigated; however, 
some strains have more than one enzyme3. A larg  num-
ber of bacterial species are yet to be subjected to careful 
screening methods to isolate new restriction enzymes.  
 Cyanobacteria, earlier known as blue-green algae, are 
widespread in freshwater, ma ine environment, soil, 
paddy fields and hot springs. The genus Oscillatoria 
comprises many species, which are nonheterocystous, fil-
amentous organisms belonging to the order Oscillatori-
les and family Oscillatoriaceae. So far, no restriction 
enzyme has been identified from this bacter al genus. 
Here, we report the identification and characteriz tion of 
a new restriction enzyme from Oscillatoria foreaui. The 
strain used in the study was isolated from rice fields of 
Kerala and maintained in the culture collection of the 
Centre for Advanced Study in Botany, University of  
Madras, Chennai and registered as O. foreaui A-1340. O. 
foreaui A-1340 appears as trichomes which are sparse, 
elongate, bent, distinctly constricted at the crosswalls, 
and apex not attenuated (Figure 1). O. foreaui cells were 
grown in BG-11 nitrogen-amended medium at 27 ± 1°C 
(ref. 4) under a light intensity of 30–40 mEm–2 s–1 pro-
vided by cool white fluorescent lamp with 12 h alternate 
light/dark regime. The growth was measured in terms of 
chlorophyll-a content5. The cell-free extracts of O. 
foreaui showed a consistent pat ern of substrate DNA 
cleavage into discrete fragments, which is a characteristic 
feature of type II restriction enzymes. 
 The enzyme was purified using two column chroma-
tography steps. Harvested cells were resuspended in 
40 ml of buffer A (10 mM Tris-HCl, pH 7.4, 50 mM 
NaCl, 5 mM b-mercaptoethanol, 0.1 mM EDTA). Cells 
were lysed by sonication and insoluble material from 
crude homogenate was removed by centrifugation at 
92,000 g for 2 h. The supernatant was passed through 
phosphocellulose column, which was pre-equilibrated 
with buffer B (10 mM potassium phosphate, pH 7.4, 
50 mM NaCl, 5 mM b-mercaptoethanol, 0.1 mM EDTA). 
After sample- oading, the column was washed with 400 
ml of buffer B and then eluted with linear gradient of 
0.05–1.6 M NaCl in buffer B. Fractions which showed 
distinct pattern of cleavage were pooled, dialysed against 
buffer A and then loaded onto heparin sepharose column. 
The column was washed with 400 ml of buffer A and 
eluted with linear gradient of 0.05–1.6 M NaCl in buffer 
A. The active fractions were pooled and dialysed against 
buffer A containing 50% glycerol and stored at –20°C. 
 Th  restriction endonuclease assays were perform d by 
incubating the enzyme with pUC18 DNA substrate in a 
40 ml reaction buffer (10 mM Tris-HCl, pH 7.4, 10 mM 
MgCl2, 50 mM NaCl and 5mM b-mercaptoethanol) for 
1h at 37°C. The unit activity of the enzyme was esti- 
 
 
 
Figure 1. Photograph of Oscillatoria foreaui. 
RESEARCH COMMUNICATIONS 
 
CURRENT SCIENCE, VOL. 85, NO. 2, 25 JULY 2003 189
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1      2      3      4 
 
 
Figure 2. Digestion profile of different DNA substrates with OfoI and AvaI. a, Comparative cleavage 
patterns of OfoI and AvaI using different DNA substrates. Lanes 2, 5, 8, Undigested lDNA, pUC18 and 
pBR322 DNA, respectively; lanes 3, 6, 9, lDNA, pUC18 and pBR322 DNA digested with AvaI; lanes 4, 
7, 10, Corresponding OfoI digests; lane 1, DNA marker (0.5–10 kb), Novagen, Madison, USA. b, Double 
digestion of pBR322. Lanes 3, 4, Double digestion of pBR322 using AvaI–EcoRI and OfoI–EcoRI,  
respectively; lane 2, Undigested pBR322; lane 1, DNA marker as indicated. In all the cases, assays were 
terminated by adding 5 ml of stop buffer (20% ficoll, 50 mM EDTA, bromophenol blue 0.1% w/v and  
xylene cyanol 0.1%). The reaction mixture was resolved by electrophoresis for 2 h at 100 V on 1% agarose 
gel in 40 mM Tris-acetate, pH 8.2, 1 mM EDTA. The gel was stained by 0.5 mg/ml ethidium bromide. 
 
 
G     A     T      C     1      2
5¢ 3¢
C  G 
C  G
C  G
G  C
G  C
G  C
3¢ 5¢
 
 
Figure 3. Determination of cleavage site of OfoI. The nucleotide  
position of cleavage of O oI was determined by primer extension 
analysis. The plasmid pUC18, which has a single site for OfoI, was 
used for this analysis. The pUC18 forward primer (5¢-GTT TTC CCA 
GTC ACG AC-3¢) was end-labelled using g-32P-ATP and T4 polynu-
cleotide kinase. The exact cleavage position of the ezyme OfoI was 
mapped by standard dideoxy sequencing reaction using Taq DNA poly-
merase. G, A, T, C represent the sequence ladder. To map the to 
strand cleavage site, pUC18 was digested with OfoI, the resulting 
cleavage products were used for primer extension (lane 1). To map the 
cleavage site in the bottom strand, first the primer was extended and 
then the resulting products were cleaved by OfoI (lane 2).  
 
 
mated by incubating various amounts of the enzyme with 
1 mg of pUC18 DNA under the standard assay conditions 
described above. One unit of enzyme activity was defined 
as the amount of enzyme required to completely digest 
1 mg of lDNA in 1 h at 37°C under the stand rd assay 
conditions. A total of 18,000 units of enzyme was recov-
ered after purification, starting from 7.3 g of O  foreaui. 
The cleavage pattern of the new enzyme was det rmine  
using different DNA substrates such as pUC18, pBR322 
and lDNA in the reaction. Plasmids pUC18 and pBR322 
DNA have a single site, while lDNA contains eight sites. 
The cleavage pattern generated by the new enzyme was 
compared with the pattern of other type II restriction  
enzymes; it was identical to the pattern genrated by 
AvaI. AvaI digestion of lDNA generated nine fragments 
of sizes 14677, 8614, 6888, 4720, 4716, 3730, 1881, 
1674 and 1602 bp. Fragments of the same size were gen-
erated from lDNA with the new enzyme (Figure 2 a). 
Double-digestion pattern of pBR322 obtained using the 
new enzyme and EcoRI was identical to that obtained 
from AvaI and EcoRI (Figure 2 b). The exact cleavage 
site of the new enzyme was determined by primer exten-
sion analysis as described in Balke et al.6. The pUC18 
DNA was digested by the enzyme and a primer (5¢-GTT 
TTC CCA GTC ACG AC-3¢) was annealed to 76 bp 
upstream of the cleavage site and then extended with 
Klenow fragment of DNA polymerase I. To localize the 
bottom strand cleavage site, the primer was first extended 
and then cleaved with the new enzyme. Dideoxy sequenc-
ing reactions were carried out using the same primer, and 
the sequencing products were electrophoresed in parall l 
with the extended products (Figure 3). These data showed 
that the new enzyme recognizes and cleaves the follow-
ing sequence 
 
 5¢-C\CCGGG-3¢ 
 3¢-GGGCC\C-5¢ 
a b 
RESEARCH COMMUNICATIONS 
 
CURRENT SCIENCE, VOL. 85, NO. 2, 25 JULY 2003 190
 
 
Figure 4. Comparison of OfoI and AvaI activities. a, Optimum temperature of the enzymes was determined by estimating the per cent  
activity at different temperatures (30 to 65°C) under standard assay conditions. b, Effect of pH on enzyme activity was evaluated by esti-
mating per cent activity at different pH values such as 6.0, 6.7, 7.0, 7.4 and 8.0. c, Effect of divalent metal ions on AvaI and OfoI activity 
was assessed by estimating the total activity in the presence of different divalent metal ions such as Mg2+, Mn2+, Ca2+, Zn2+, Co2+, Cd2+ and 
Ni2+. d, Optimum ionic strength for activity of the enzymes was determined by adding 0 to 200 mM NaCl to the reaction mixture. 
 
generating four nucleotide-protruding 5¢-ends. The rec-
ognition sequence and cleavage pattern are identical to 
those of AvaI (ref. 7). Since this is the first restriction en-
zyme to be characterized from the O. for aui, it is named 
as OfoI.  
 The properties of OfoI were compared with AvaI, since 
both were isolated from different cyanobacteria (Figure 
4 a–d). The enzyme AvaI is isolated from Anabaena 
variabilis and has been well characterized8. A compari-
son of the activities of isoschizomer OfoI with AvaI is 
important to evaluate its origin and explore its potential 
applications. The temperature optimum for the enzyme 
OfoI was determined by carrying out assays with constant 
amounts of DNA and enzyme over a broad range of tem-
peratures ranging from 30 to 65°C. The enzyme exhibits 
complete activity at 37°C; when temperature was increa-
sed to 45°C, it showed stimulated activity (Figure 4 a). 
OfoI is not inactivated by heating at 65°C for 20 min. 
Similar observations have been made with AvaI. Next, 
optimum pH for the enzyme was determined by estimat-
ing the per cent acivity at different pH values ranging 
from 6.0, 6.7, 7.0, 7.4, 8.0 and 8.5. OfoI exhibits broad 
pH optimum and was found to be most active at pH 7.4 
(Figure 4 b). Type II restriction enzymes require divalent 
m tal ions for their cleavage property. Hence, the 
enzyme OfoI was assayed for its activity in the presence 
of different divalent cations such as Mg2+, Mn2+, Ca2+, 
Zn2+, Co2+, Cd2+ and Ni2+. OfoI showed maximum activ-
ity in the presence of Mg2+ and around 5% activity in 
presence of Mn2+, while no detectable activity was  
obtained with any other divalent cations tested (Figure 
4 c). Another notable property of many Enases is that 
they exhibit star activity in presence of Mn2+ ions9. We 
could not detect any star activity in the presence of Mn2+. 
The effect of ionic strength on enzyme activity was  
determined by estimating per cent activity at different 
salt concentrations. The enzyme is functional between 0 
and 100 mM NaCl concentrations, exhibiting optimum 
activity at 50 mM NaCl concentration (Figure 4 d). Both 
OfoI and AvaI showed nearly identical biochemical prop-
erties (Figure 4 a–d). 
 The enzyme OfoI could be a convenient alternative to 
AvaI because of the following reasons. A. variabilis con-
tains two restriction enzymes AvaI and AvaII, and hence 



A new type II restriction endonuclease, OfoI from nonheterocystous cyanobacterium Oscillatoria foreaui

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A new type II restriction endonuclease, OfoI from nonheterocystous cyanobacterium Oscillatoria foreaui

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