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The HsdS subunit from most Type I R-M systems have
been found to be highly insoluble; although the HsdS
subunit of the EcoR124I system was shown to be
moderately soluble fusion protein when fused to
glutathione S-transferase (Kusiak et al., 1992).
It has also been shown that small changes to the
arrangement of the repeated
domains of the HsdS subunit (Janscak et al.,
1998). Therefore, in the bacterial cell HsdM appears
to act as a molecular chaperon by interacting with the
insoluble HsdS subunit in the first step of the subunit
assembly pathway (see below). The resultant M1S1
complex has been identified for the EcoKI R-M
system (Dryden et
al., 1993, but it has never been observed with
the EcoR124I R-M system. This complex can
interact with HsdR to produce an inactive R1M1S1
intermediate, but the correct assembly pathway is through
R1M2S1 complex to the
fully functional R2M2S1
complex (R-M enzyme).
It is though the subunit assembly pathway that EcoR124I
R-M system been shown to produce the
temporal control of the restriction and modification
activities. This has been described as
control through the
quaternary structure of the enzyme.
Alternate subunit assemblies
Early observations with the EcoBI R-M system suggested that
the endonuclease may exist in different stoichiometric forms (e.g.
R1M2S1, R2M2S1
and R4M2S1). We have recently
purified the type IC restriction endonuclease EcoR124I in
milligram amounts and have established a stoichiometry of R1M2S1,
based on gel filtration studies, and scanning of SDS-PAGE
Janscak et al.
(1996). However, the classical Type I enzyme EcoKI has
a stoichiometry of R2M2S1.
Recently, we have observed that addition of excess HsdR to a 1:1
mixture of EcoR124I endonuclease and DNA (which is not
capable of DNA cleavage at this stoichiometry) induces cleavage,
suggesting an alternative
stoichiometry may be capable of more efficient DNA cleavage
than is obtained with the purified endonuclease and this has led
us to understand how this enzyme may
control restriction activity
through subunit assembly.
EcoR124I is controlled through subunit assembly 
Janscak et al (1998)
were able to show that the functional EcoR124I endonuclease is
composed of a R2M2S1 complex and
that the R1-complex is an intermediate in the assembly
pathway, which is restriction-deficient, but
modification-proficient. Studies of the motor activity
(translocation) of the R1-complex showed that the
complex was also able to translocate DNA, but
with a lower level of
processivity (less able to translocate long distances -
Firman &
Szczelkun, 2000;
Seidel et al 2004). Therefore, the key step in the assembly
process - binding of the final HsdR subunit - is able to control
the restriction activity of the restriction enzyme (Firman
et al 2000) providing temporal control of restriction over
modification ensuring that the cell cannot restrict the host DNA
BEFORE methylation affords the normal protection. This is enabled
through the limited concentration of HsdR in vivo, which
guarantees assembly of the MTase in vivo before the
functional ENase is assembled. In fact, this is further
guaranteed y the inherent methylation activity of the R1-complex.
 Recently,
this assembly pathway has been analysed both using bulk
biochemistry and single molecule methods and we have been able to
show a highly dynamic control mechanism based on
association/disassociation of the ENase (Seidel
et al 2005). Where the HsdR subunit concentration controls
motor activity, but the DNA-bound MTase is relatively stable.
This situation is in direct opposition to the situation with EcoKI
where the R2-complex is more stable and temporal
control of restriction versus modification involves a more complex
system known as restriction alleviation (Loennen
et al 1986).
Assembly into an anti-codon nuclease (ACNase)
Recent studies with the type IC R-M system EcoprrI have
identified a novel assembly with another subunit, PrrC, to produce
an enzyme with a new function, namely an anti-codon nuclease (ACNase)
(Amitsur et al.,
1992). This system illustrates again the importance of Type I
R-M systems as a protection system for bacteria, and the ability
of these systems to evolve rapidly into novel restriction systems.
The bacteriophage T4 encodes a small (26 amino acid) polypeptide
called Stp, which can interact with the EcoprrI restriction
endonuclease as an anti restriction determinant. This
interaction destroys the restriction activity of EcoprrI;
although bacteriophage T4 DNA is glucosylated and as a consequence
resistant to cleavage by Type I restriction endonucleases (Penner
et al., 1995).
The EcoprrI restriction endonuclease interacts with another
host protein, PrrC, to produce a latent anti-codon nuclease (ACNase).
The fully functional ACNase is able to cleave tRNALys
at the codon triplet. however, to prevent cleavage of host
(bacterial) tRNA the enzyme is latent and shows no such activity.
Infection of the bacteria by bacteriophage T4 introduces the Stp
polypeptide as an early gene and this polypeptide can activate the
ACNase resulting in cleavage of the bacteriophage tRNALys.
This prevents growth of the bacteriophage; although the phage has
developed a kinase and ligase which is capable of repairing this
damage (Amitsur et al.,
1992).
Recently, it has been demonstrated that
this ACNase, when produced in mammalian cells, is capable of
preventing replication of a retrovirus which utilizes tRNALys
as a primer of replication. This is an important demonstration of
how pure science can lead to major breakthroughs in applied
research. The potential of this enzyme to HIV research is at this
time still under investigation (Shterman
et al., 1995).
We have shown that the Stp polypeptide induces anti-restriction
activity through direct interaction with the type IC
restriction-modification enzyme EcoR124I by disrupting the
subunit assembly pathway. In particular, the R2M2S1,
restriction-proficient form of the enzyme dissociates more readily
into the restriction-deficient R1M2S1
form of the enzyme in the presence of Stp (Figure
1). It seems highly likely that this stp induced dissociation
of the R2-complex allows activation of the latent
ACNase by uncovering an active domain on PrrC, which is normally
blocked by the HsdR subunit of the Type I ENase (Figure
2).
It seems likely that other functions may yet be found for the
alternate assemblies observed with other Type I R-M systems. The
EcoprrI system is simply an extreme example of how these
multimeric enzymes can produce new functions by altering their
subunit assembly.
Figure 1a Assembly of ENase through R1-
and R2-complexes
From left to right MTase is titred with increasing
amounts of HsdR to produce first R1-complex and then R2-complex.
Pure ENase is present in the last lane to allow identification of
the three species.
Figure 1b Assembly of ENase in the presence of Stp
As above but with added stp polypeptide. The
results clearly show that R2-complex formation requires
a higher concentration of HsdR in the presence of Stp.
Figure 2 Interaction between Stp and the latent ACNase
 A
model for activation of the latent ACNase by Stp, and the related
anti-restriction activity of Stp, involves disruption of the
equilibrium between the R2-
and R1-complexes of the Type I R-M system.
The latent ACNase is produced through interaction of the
endonuclease (R2-complex) with PrrC (which would
otherwise kill the cell by cleaving tRNALys). Upon
infection of the bacterium by T4 bacteriophage, Stp is produced
and disrupts the finely balanced equilibrium between R2-
and R1-complexes. As a result of this conformational
change the ACNase is activated by unmasking an active domain of
the PrrC enzyme.
Cleavage of tRNALys will then lead to cell death by
preventing the translation process from occurring. therefore, the
protection afforded by the ACNase is suicidal and can only protect
the bacterial population from T4 infection.
However, the phage has overcome this system by acquiring the means
of ligating the cleavage product!
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Restriction-Modification Systems
