The assembly of a restriction enzyme that cuts both strands on both sides of its site

1
The assembly of a restriction enzyme that cuts
both strands on both sides of its site
Summer 2009

• Jacqui J.T. Marshall, Alistair J. Jacklin, Frank
  Sobott, and Stephen E. Halford
• The DNA-Protein Interactions Unit, Department of
  Biochemistry, University of Bristol
• Department of Physiology, Anatomy and Genetics,
  Oxford Centre for Gene Function, University of
  Oxford

2
BcgI a Type IIB restriction-modification system
BcgIA Restriction-Modification
BcgIB DNA recognition
N
C
N
C
71.6 kDa
39.2 kDa
TRD1
TRD2
Endonuclease domain (PDExK motif needs Mg2 and
SAM for DNA cleavage)
Methyltransferase domain (transfers methyl group
from S-adenosyl methionine (SAM) to DNA)
DNA recognition subunit (contains two Target
Recognition Domains (TRDs), one for each half of
sequence)
Likely arrangement (Kong, 1998)
2A1B 182.3 kDa
A Targets for methyltransferase, preferentially
on hemimethylated DNA
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Cleavage of DNA by BcgI
(a) Plasmid with one BcgI site
(b) Plasmid with two BcgI sites
(c) Catenane with two BcgI sites
v1
v2

• 3H DNA species separated on 1 agarose,
  quantified by scintillation counting
• BcgI cleaves the two-site plasmid faster than the
  one-site plasmid (v2/v1 6.7), ?
  interacts with two sites.
• Low levels of intermediates. Hence makes
  double-strand breaks. Once bound to two sites,
  cleaves them together.
• Catenane cut at similar rate to the two-site
  plasmid, ? BcgI bridges the two sites through 3-D
  space.

4
Does BcgI have any activity on a single site?

• 61 bp oligoduplex with BcgI recognition site
• Biotinylated at one end
• 32P-labelled at other end
• Immobilisation of this DNA on streptavidin beads
  (at v. low DNA 1 DNA per 100 streptavidins)
  excludes reactions in trans.
• Cleavage can only occur IF BcgI can act at a
  single site.

Streptavidin-coated magnetic bead
14 bp
5
BcgI requires two copies of its site for cleavage
0 nM oligoduplex
5 nM oligoduplex

61mer substrate
Standards
5 nM oligoduplex
Oligoduplex (SDu42) titration (100 nM BcgI)
0 nM oligoduplex
0 1 3 10 30 100 300
0 1 3 10 30 100 300
0 1 2 5 10 20 50
Enzyme (nM)
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How many bonds does BcgI cleave per reaction?
DNA cleavage by BcgI on both sides of its site
releases a small (34 bp) DNA fragment containing
the recognition site. With plasmid substrates,
this is not resolved on agarose. Therefore a
shorter substrate was generated by PCR
e.g.)
20 nM BcgI on 5 nM 235 bp PCR product
The low concentrations of the BC and AB
intermediates show that all 4 target
phosphodiester bonds around each site are cleaved
concertedly.
7
Assembly of the BcgI protomer
The quandry

• Analytical ultracentrifugation (AUC)
  Sedimentation to equilibrium
• Molecular shape independent technique.

Has only two active sites additional active
sites needed to cut 4 bonds
8
AUC High concentrations of BcgI
Can BcgI form multimers with enough nuclease
active sites to cleave (on a two-site substrate)
eight phosphodiester bonds in one event?
7.5 µM BcgI-E53A at 9k rpm after 18 hrs
Fitting to MW for a single ideal species
Fixed 182 kDa MW Kd (M-D) 2.0 µM, Kd (D-T)
2.1 µM (variance 0.98 random residuals)
MW 262 kDa (variance 3.18 non-random
residuals)
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MW of short oligoduplexes bound by BcgI

• Equilibrium AUC, monitoring the absorbance of 42
  bp HEX duplexes at 539 nm, to observe only
  species bound to DNA.
• DNA alone (not shown) MW 27,300 (c.f.
  theoretical MW 26,600)
• DNA BcgI Fitted to single ideal species
  floated MW

Apparent MW (kDa) from fit to single ideal species
0.25
10
BcgI complexes in a QTOF nanospray MS
QTOF nanospray mass spectroscopy a technique for
studying noncovalent complexes Numbers in red
measured MWs (Da)
Theoretical MWs (Da) BcgIA 71,559 BcgIB
39,161 BcgIAB 110,720
BcgIA2 143,118 BcgIA2B 182,279 BcgI(A2B)2
364,558 BcgI(A2B)3 546,837
Rapid desolvation to partially disrupt
noncovalent complexes. 13.3 µM BcgI in 20 mM
Tris-acetate, pH 8.4, 200 mM Ammonium acetate.
Presence of the same MW species at multiple (m/z)
values indicate differently charged states, maybe
one unfolded. Disruption usually removes smaller
subunits first, unless these are inaccessible.
The separate BcgIA (71.5 kDa) and BcgIB (39.2
kDa) polypeptides along with the A2B (180 kDa)
complex can be identified by MS. Additionally,
the presence of an AB but not an A2 complex
supports the model in which the small B subunit
is central within each protomer.
71,566
39,162
71,439
110,733
176,001
Hint of larger aggregates?
Mass spectroscopy performed by F Sobott (Oxford)
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BcgI complexes in a QTOF nanospray MS (2)
Optimisation of conditions leads to maintenance
of noncovalent complexes and clearer
identification of higher MW species.
Slow desolvation to maintain noncovalent
complexes. 23.4 µM BcgI-E53A.
Numbers in red measured MWs (Da)
As in the AUC, multimers of the BcgI protomer can
be seen by mass spectroscopy.
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Conclusions / working model
BcgI cleaves both strands, on both sides of its
recognition site (yielding a 32 bp fragment), at
two separate sites, thus cutting 8 bonds in a
single reaction. The AUC suggest that, at low (lt
1 µM) concs, BcgI exists as a 180 kDa (A2B)
protomer. However at gt 1 µM concs, BcgI forms
multimers. These can also form on DNA larger
multimers on duplexes with the BcgI site. We are
currently pursuing QTOF nanospray mass
spectroscopy as a possible technique for
resolving these complexes.

• The well
• established
• model for the
• action of FokI

• Possible model for BcgI

• BcgI as a double-barrelled FokI
• accounts for observed aggregation

Funding from
Email contact jacqui.marshall_at_bristol.ac.uk