The Subunits of Type I R-M enzymes
In Type I R-M systems, restriction and modification
activities are catalysed by one complex enzyme composed
of three different subunits, which are encoded by the hsdR,
hsdM and hsdS genes (Glover
& Colson 1969). The products of all
three genes are absolutely required for restriction
activity, while the hsdM and hsdS gene products are
sufficient for modification activity.
HsdS
The HsdS subunit is responsible for
DNA-recognition, recognising the 'split' sequences of the Type I
R-M enzymes, which suggests a two domain structure for DNA
specificity (Target Recognition Domains - TRDs). The
non-specific region of the recognition sequence suggests that
these two TRDs are separated by a linker sequence.
The domain structure of HsdS
For the EcoR124I R-M system the recognition
sequence (sR124) is 5'-GAAnnnnnnRTCG-3'.
TRD1 is responsible for recognition of the 5'-end of the
recognition sequence (GAA), while TRD2 recognises the 3'-end
(RTCG).
The conserved regions contain two highly conserved
repeats (shown by the arrows) that were originally
described by Argos (1983)
as the regions of the protein likely to recognise the
target DNA sequence. However, this has now been shown not
to be the case (e.g. Gubler, 1992).
Figure 4
Careful analysis of the arrangement of these repeated
units in all Type I HsdS subunits indicates that all the
subunits consist of a similar set of sub-repeats and, as shown in Figure
4, this
has led to a prediction of a
circular structure for the
HsdS subunit (Kneale
1994). To test this model, Janscak & Bickle (1998)
circularly permuted the HsdS subunit of the Type IB R-M enzyme EcoAI, at
the DNA level, by direct linkage of codons for the original termini and
introduction of new termini elsewhere along the N-terminal and central
conserved regions. By analysing the activity of these mutant enzymes,
two circularly permuted variants of HsdS, which had termini located at
equivalent positions in the N-terminal and central repeats,
respectively, were found to fold into a functional DNA recognition
subunit with wild-type specificity. This result suggests that the
N- and C-termini of the native protein are in close proximity.
The central conserved region has been subjected
to intensive mutagenesis using deletion mutagenesis,
site-directed mutagenesis and PCR-misincorporation mutagenesis.
This has allowed us to show that this region is critical for
protein-protein interactions with the other subunits of the
endonuclease (Abadjieva
et al., 1994).
TRD1 and the central conserved region are sufficient to
assemble an active enzyme (Abadjieva
et al., 1993;
Macwilliams & Bickle, 1996). this reflects the circular structure of the
HsdS subunit and the deletion mutants assemble through dimerisation of a M1S½
subunit. Since the recognition sequence of this deletion mutant is GAAnnnnnnnTTC,
and TRD1 recognises the GAA component of the sR124 sequence, it is
apparent that each half subunit recognises the opposite strand on the DNA
strand.
The recent crystallisation of HsdS from two putative Type I
R-M systems has allowed Obarska
et al (2006) to model the EcoR124I HsdS subunit in silico. This
structure has confirmed the circular structure detailed above and shown that the
central conserved region consists of two a-helices
(see right - D).
The bent DNA structure is based on observations from AFM images of
the MTase bound to DNA (HsdS is insoluble and has not been
isolated bound to DNA).
HsdM
The HsdM subunit is responsible for DNA methylation at the
appropriate adenine within the DNA recognition sequence.
Recently a crystal structure for the EcoKI HsdM subunit (in
which HsdM appears as a dimer - see figure on left), which clearly identifies
two major domains and a short tail within the protein. This fits with
proteolysis data, where limited proteolysis quickly releases a short
polypeptide, followed by production of two larger polypeptides.
The two domains may be required to 'clamp' to the DNA during
binding by the MTase.
HsdR
The HsdR subunit is required for DNA cleavage and, therefore,
restriction activity. The active site for this endonuclease
site was first identified for EcoKI and coined "Motif X" (see
below). It is also the motor component of the enzyme
responsible for movement of DNA (translocation),
through the bound complex, until cleavage occurs. This
activity is ATP dependent and the enzyme has the well described
DEAD-box motifs (Gorbalenya
& Koonin, 1991) associated with DNA helicases.
Mutations within each of the seven DEAD-box motifs identified
within EcoKI resulted in loss of translocation activity and
severely impaired ATPase activity (Davies
et al., 1998;
1999a).
Motif X - the endonuclease motif:
 There is no published structure for
this subunit, but we have initiated an attempt to model the structure
in silico (see right). However, progress toward a structure
for HsdR seems imminent as a crystal isolation for HsdR(EcoR124I) has been
described (Lapkouski et al 2007).
This analysis, using site-directed mutagenesis of the DEAD-box motifs, lead to
the identification of specific domains (confirmed by limited proteolysis
experiments - Davies et al., 1999b),
which enabled a more detailed structural model for the endonuclease to be
produced:
HsdR(EcoR124I)
We have cloned the hsdR gene of the EcoR124I
restriction endonuclease, confirming the presence of an independent promoter for
this gene by means of a complementation assay with MTase
(Zinkevich
1997). In addition, we have also cloned the hsdR gene into the
expression vector pTrc99A. This has allowed us to purify the subunit in
milligram quantities and to study the enzymatic properties of the individual
subunit. The presence of a Walker Type I ATP-binding site within the HsdR
subunit suggested that the subunit might be capable of independent enzymatic
activity. The purified HsdR subunit was found to be a soluble monomeric protein
capable of a DNA and Mg2+-dependent ATP hydrolysis. The subunit was
found to have a weak nuclease activity both in vivo and in vitro,
and to bind plasmid DNA; although was not capable of binding a DNA oligoduplex.
We were also able to reconstitute the fully active endonuclease from purified M.EcoR124I
and HsdR. This is the first clear demonstration that the HsdR subunit of a Type
I restriction endonuclease is capable of independent enzyme activity, and
suggests a mechanism for the evolution of the endonuclease from the independent
methylase by acquisition of an additional subunit capable of nuclease activity.
The DNA binding was found to be unusual in that while it was weak (Kd = 0.2uM)
the binding was not non-specific. The subunit cannot bind an oligonucleotide
(30-mer) but was able to bind two HinfI fragments of the plasmid pDRM-1R
(a derivative of pTZ19R carrying a sR124 recognition site). Analysis
of these two DNA fragments
 failed
to reveal a common sequence greater than 4-bp in length suggesting the
specificity of the binding may involve DNA curvature rather than DNA sequence.
The length of the fragment is not the sole determinant for DNA binding since the
digest used in the study had fragments present of intermediate size to those
retarded in the gel. The level of ATPase activity is similar to that observed
with Type III restriction endonucleases. This suggests a "looping" model may be
involved in the DNA translocation carried out by both enzymes, perhaps with the
DNA being wrapped around the HsdR subunit of the Type I restriction endonuclease
prior to the main translocation mechanism.
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