Restriction endonucleases and DNA methyltransferases from
Haemopilus influenzae and Neisseria gonorrhoeae
Dr Andrzej Piekarowicz
Institute of Microbiology
Warsaw University
Restriction and modification systems in Haemophilus influenzae strains as
determined on the on the in vivo restriction of phage HP1 (Piekarowicz and Glover
1972)
(1) Each of the strains tested had different specificity except strains Rf and Re
Example of genetic analysis of RM system in H. influenzae strains (Glover Piekarowicz,1974)
(1) Analysis of the restrictions and modification mutants of strain Ra demonstrated existence of
two independent specificity types (strains Re and Rf had also two specificity types while Rd and
Rb only one)
(2) proportion of the r- m+ and r- m- mutants in all strains was the same as for EcoK or EcoP1
(3) Interpretation was that r- m+ mutants arise as a results of mutation in gene hsr and r- m- of
hss gene; three gene model or as two gene model in which mutation in hsm would led r- mphenotype
Phase variation of the type I RM system HindI from H.influenzae Rd (Glover and Piekarowicz, 1972)
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H. influenzae strain
phenotype
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r+ m+
r - m___________________________________________________________________
Rd wild-type
15
85
Rd 123 r + m +
90
10
Rd 200 r - m-
20
80
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Genetic structure of RM system HindI
1 1360627..1363116 HI_1284 translation initiation factor 2 (infB) HindI
1363195..1366362 HI_1285 type I restriction enzyme (hsdR) S.HindI
1366454..1367833 HI_1286 type I restriction/modification specificity M.HindI
1367826..1369157 HI_1287 type I modification enzyme (hsdM) 2
1369672..1370058 HI_1288 ribosome binding factor A (rbfA)
Properties of the two forms of restriction endonuclease HinfIII from H. Influenzae Rf
(Piekarowicz et al.,1978-1982
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HinfIII with AdoMet bound to it (HinfIII*)
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HinfIII free of AdoMet (HinfIII)
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1. Require ATP and Mg++ for restriction
activity, AdoMet stimulates cleavage
activity
2. In the absence and presence of
AdoMet cleaves ColE1 (five sites for
HinfIII) only at one site
3. Does not cleaves DNA in the presence
of imido-ATP
4. Can methylates DNA in the absence
of external AdoMet
5. Preferential cleavage but not
methylation of spercoiled over linear DNA
6. Require minimum in cis two
5’ CGAAT 3’ sites in DNA molecule
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1. Require ATP Mg++ for restriction
activity, AdoMet stimulates cleavage
activity
2. In the presence of AdoMet cleaves
ColE1 (five sites for HinfIII) only at one
site , in the absence cleaves at five sites
3. Does not cleaves DNA in the presence
of imido-ATP
4. Can not methylates DNA in the
absence of external AdoMet
5. Preferential clevage but not
methylation of spercoiled over linear DNA
6. Require minimum in cis two sites in
DNA molecule
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How to clone genes encoding DNA methyltransfreases
E.coli strain AP1-200-9 helped to clone unknown genes encoding DNA
Methyltransferases
Nucleic Acids Res. 1991 Apr 25;19(8):1831-5.
A new method for the rapid identification of genes encoding
restriction and modification enzymes.
Piekarowicz A, Yuan R, Stein DC.
Source
Institute of Microbiology, Warsaw University, Poland.
Abstract
We have constructed derivatives of Escherichia coli that can be used for the rapid
identification of recombinant plasmids encoding DNA restriction enzymes and
methyltransferases. The induction of the DNA-damage inducible SOS response by the Mcr
and Mrr systems, in the presence of methylated DNA, is used to select plasmids encoding
DNA methyltransferases. The strains of E. coli that we have constructed are temperaturesensitive for the Mcr and Mrr systems and have been further modified to include a lacZ gene
fused to the damage-inducible dinD locus of E. coli. The detection of recombinant plasmids
encoding DNA methyltransferases and restriction enzymes is a simple, one step procedure
that is based on the induction at the restrictive temperature of the lacZ gene. Transformants
encoding DNA methyltransferase genes are detected on LB agar plates supplemented with
X-gal as blue colonies. Using this method, we have cloned a variety of DNA
methyltransferase genes from diverse species such as Neisseria, Haemophilus, Treponema,
Pseudomonas, Xanthomonas and Saccharopolyspora.
Restriction endonucleases HaeIV
1. The HaeIV gene contains a homopolimeric tract of 10 guanosine close to stop codon
2. Deletion of one G residue from poliG tract generate frame shift mutation extending ORF for 564 bp
3. Resulting HaeIV2 variant is 188 aa longer and the enzyme has second active TRD
4. Variant HaeIV 3 was discovered by genomic analysis in another H. Influenzae strain
Restriction endonucleases HaeIV
All three variants show the same properties
1. Single protein has restriction and methylase activity
2. AdoMet is needed for methylase activity while only Mg ions for restriction activity
3. They differ in recognition of the specific sites but the cleavage geometry is identical in relation
to the cognate sequence
8 ↓nnnnnnnnGAYN5RTCnnnnnnnnnnnnnn↓ 14
13 ↑nnnnnnnnnnnnnCTRN5YAGnnnnnnnn↑ 8
13
HaeIV
8 ↓nnnnnnnnGAYN5CTCnnnnnnnnnnnnnn↓ 14
nnnnnnnnnnnnnCTRN5GAGnnnnnnnn↑ 8
HaeIV_S2
8 ↓nnnnnnnnGAYN5CTGnnnnnnnnnnnnnn↓ 14
HaeIV_S3
13 ↑nnnnnnnnnnnnnCTRN5GACnnnnnnnn↑ 8
Restriction and modifications systems of Neisseria gonorrhoeae
The combination of the „classical” purification method, cloning and genomic allowed to identified
and characterized most of these enzymes from N. gonorrhoeae
The genetic structure of some of the analyzed restriction-modification systems
Cloning and characterization of the gene encoding a new DNA methyltransferase from
Neisseria gonorrhoeae.
Radlińska M, Piekarowicz A.
Institute of Microbiology, University of Warsaw, Poland.
Abstract
A HindIII fragment of N. gonorrhoeae MS11 DNA coding for DNA methyltransferase (MTase)
activity was cloned and expressed in E. coli AP1-200-9 cells. The sequence of 4681 bp was
determined, and its analysis revealed two open reading frames (ORFs) sharing some similarity with
known DNA MTases. ORF1 encodes an active N4mC MTase (M.NgoMV). The enzyme modifies
only one strand of double stranded DNA and preferentially recognises the sequence GCCHR
although it is able to methylate other sites. The exact recognition sequence cannot be precisely
defined due to a relaxed specificity. The second ORF shows high homology to 5mC Mtases, but we
were unable to demonstrate DNA methylating activity of its product either in vivo or in vitro.
Atypical restriction endonuclease from N. meningitidis
Atypical DNA Mtases encoded by Mu-like phages.
1. mom gene of Mu phage ancode an enzyme that converts adenine to N6-(1-acetoamido)adenine
2. Mu-like prophages (FluMu, Z2491and Hia5) possess a genes located in the same position as mom encoding
DNA methylases with homology to N6 adenine DNA Mtases
Sequence specificity of the Mom-like DNA Mtases
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5’ AB 3’ or 5’ BA 3’
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where B + C, G, T
Poly(A)- tracts are probably not methylated
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Hia5 methylate 61 % of adenine residues
Phase variation of RM systems
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1. Mechanism: slippage of polymerase on
(a) poli G tract
(b) tandem repeat of di-, tri-, four- five- nucleotides
(c) single mutation change both restriction and modification activity (TypeI, Type III,
Type II G)
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2. Consequences for RM systems:
(a) loss of modification and restriction activity
(b) change of specificity
Phase variation of the HindI R-M system
Depending on the number of guanines present in the poly(G) tract the second TRD is either
expressed or not
The role of PV
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1. My studies showed dynamic changes of the restriction and modifications systems
especially Type I and Type III that play a most important role as a defence system
against invasion of cells by phages or plasmids
2. The main mechanism of this dynamic changes lays in the phenomenon of phase
variation
3. What is the role of PV?
a. The change of restriction specificty without its loss may increase the defence
system of bacteria against phage and plasmid invasion and decreasing the level of
possible horizontal gene transfer
b. The loss of restriction activity may allow for increase of the gene transfer by
„window opportunity” for acquiring of new genes increasing the speed of evolution
c. evolution of bacteria and phages
Acknowledgments
I would like to thank to my mentors:
Dr Stuart Glover
Dr Thomas Bickle
Dr Robert Yuan
to my coworkers from other labs
Dr Dan Stein
To my coworkers
Dr Leszek Kauc
Dr Ryszard Brzezinski
Dr Elzbieta Skrzypek
Dr Monika Radlinska
Dr Monika Adamczyk-Poplawska
Dr Agnieszka Kwiatek