Restriction endonucleases and DNA methyltransferases from Haemopilus influenzae and Neisseria gonorrhoeae

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Restriction endonucleases and DNA
methyltransferases from Haemopilus influenzae and
Neisseria gonorrhoeae

• Dr Andrzej Piekarowicz
• Institute of Microbiology
• Warsaw University

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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
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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- m- phenotype
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Phase variation of the type I RM system HindI
from H.influenzae Rd (Glover and Piekarowicz,
1972)

• __________________________________________________
  _________________
• H. influenzae strain
  phenotype
• __________________________________________________
  _________________
• 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)
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Properties of the two forms of restriction
endonuclease HinfIII from H. Influenzae Rf
(Piekarowicz et al.,1978-1982

• _________________________________
• HinfIII with AdoMet bound to it (HinfIII)
• ________________________________
• 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

• ____________________________
• HinfIII free of AdoMet (HinfIII)
• ____________________________
• 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
methyltransfreasesE.coli strain AP1-200-9 helped
to clone unknown genes encoding DNA
Methyltransferases

• Nucleic Acids Res. 1991 Apr 2519(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
  temperature-sensitive 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.

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Restriction endonucleases HaeIV1. The HaeIV gene
contains a homopolimeric tract of 10 guanosine
close to stop codon2. Deletion of one G residue
from poliG tract generate frame shift mutation
extending ORF for 564 bp3. Resulting HaeIV2
variant is 188 aa longer and the enzyme has
second active TRD4. Variant HaeIV 3 was
discovered by genomic analysis in another H.
Influenzae strain
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Restriction
endonucleases HaeIV All three variants show the
same properties1. Single protein has restriction
and methylase activity2. AdoMet is needed for
methylase activity while only Mg ions for
restriction activity3. They differ in
recognition of the specific sites but the
cleavage geometry is identical in relation to the
cognate sequence

• 8 ?nnnnnnnnGAYN5RTCnnnnnnnnnnnnnn? 14 HaeIV
• 13 ?nnnnnnnnnnnnnCTRN5YAGnnnnnnnn? 8
• 8 ?nnnnnnnnGAYN5CTCnnnnnnnnnnnnnn?
  14 HaeIV_S2
• 13 nnnnnnnnnnnnnCTRN5GAGnnnnnnnn? 8
• 8 ?nnnnnnnnGAYN5CTGnnnnnnnnnnnnnn? 14
  
• 
  HaeIV_S3
• 13 ?nnnnnnnnnnnnnCTRN5GACnnnnnnnn? 8

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Restriction and modifications systems of
Neisseria gonorrhoeaeThe combination of the
classical purification method, cloning and
genomic allowed to identified and characterized
most of these enzymes from N. gonorrhoeae
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The genetic structure of some of the analyzed
restriction-modification systems
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• Cloning and characterization of the gene encoding
  a new DNA methyltransferase from Neisseria
  gonorrhoeae.
• Radlinska 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.

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Atypical restriction endonuclease from N.
meningitidis
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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
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Sequence specificity of the Mom-like DNA Mtases

• 5 AB 3 or 5 BA 3
• where B C, G, T
• Poly(A)- tracts are probably not methylated
• Hia5 methylate 61 of adenine residues

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Phase variation of RM systems

• 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)
• 2. Consequences for RM systems
• (a) loss of modification and restriction
  activity
• (b) change of specificity

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Phase variation of the HindI R-M system
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Depending on the number of guanines present in
the poly(G) tract the second TRD is either
expressed or not
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The role of PV

• 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

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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