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The EcoR124I restriction endonucleaseType I enzymes are complex multifunctional proteins which cleave unmodified DNA in the presence of SAM, ATP and Mg2+. If hemimethylated DNA is the substrate then the enzyme is converted into its modifying form and methylates the second strand of the DNA. Fully modified DNA is resistant to DNA cleavage and the enzyme dissociates from it. SAM is thought to act as an allosteric effector, at least for R.EcoKI, which converts the enzyme into an active form which is capable of specific DNA binding. ATP is known to be involved in the determination of the methylation status of the DNA. The enzyme reaction mechanism is complicated and results in DNA cleavage at random sites distant to the recognition site. One model for DNA cleavage has shown how this can result from a cooperative reaction in which two endonuclease molecules, bound to neighbouring recognition sites, meet following DNA translocation and then cleave the DNA. Translocation of the DNA requires ATP and the endonuclease is a powerful ATPase. Interestingly, the endonuclease does not turn over following DNA cleavage and appears to remain bound to the reaction product. This DNA-protein complex appears to be responsible for the massive ATP-hydrolysis continuing for a long period after DNA cleavage. Electron Micrograph of the EcoR124I
endonuclease We have described a two plasmid system which allows overproduction of the R.EcoR124I restriction endonuclease. The endonuclease has been purified to homogeneity in milligram amounts and has been shown to be fully active for both restriction and modification. Unexpectedly, the enzyme was found to require only ATP and Mg2+ for ATPase activity and DNA cleavage; S-adenosyl methionine, (SAM) which has previously been described as a cofactor of Type I restriction enzymes, is NOT required by R.EcoR124I. However, SAM was found to stimulate the rate of ATPase activity and DNA cleavage Janscak (1996). This may occur through an increase in specific DNA binding by R.EcoR124I in the presence of SAM as indicated by our surface plasmon resonance experiments. These functional differences from the well described R.EcoKI restriction endonuclease are reflected in a possible structural difference between the two enzymes; namely that the stoichiometry of R.EcoR124I appears to be R1M2S1 while that of R.EcoKI is R2M2S1 The cleavage of linear DNA substrates is a random process in which the
specifically bound endonuclease translocates DNA until it meets another enzyme.
This collision then promotes cleavage of the substrate
(Studier & Bandyopadhyay, 1988).
This model was suggested following studies of cleavage of T7 DNA by EcoKI,
but does not really explain what happens when only one recognition site is
present on the DNA, it also does not really address the situation with
supercoiled substrates. In the case of a supercoiled substrate
increased degree of supercoiling has been
shown to inhibit restriction (Janscak
1996)which suggests that the process of translocation may be inhibited by
supercoiling. In collaboration with
Prof. Halford's group at
the University of Bristol, we have studied the cleavage of supercoiled DNA with
plasmids carrying one or two sR124I recognition sites. Both
substrates are cleaved by the same mechanism inferring
that the model applied to linear DNA is incorrect for circular DNA
Szczelkun (1996).
Supercoiled DNA, when translocated through the specifically bound enzyme, will
be over-wound on one side of the complex and under-wound on the other side. This
means that the two bound enzymes, on a two-site plasmid, cannot meet and that
some other mechanism must ensure cleavage occurs. We suggest that when all of
the available DNA has been translocated, which may mean when the DNA topology
prevents further translocation, then the translocation process will stop and DNA
cleavage will occur. Therefore, any process which stalls the DNA translocation
will lead to cleavage - on linear DNA this will occur when two enzymes collide -
on circular DNA this will occur when translocation is sufficiently hindered by
the DNA supercoiling that the process stalls. We have also investigated the nature of the attachment of the enzyme to DNA prior to the translocation. In order to determine that the DNA can be bound across a distance (in trans), or whether the adjacent DNA is bound, we have used a catanene carrying a single sR124I site. If the DNA is bound at a second site in trans then both catanene molecules should be cut. However, this was not the case indicating that, following specific binding at sR124I the second binding is non-specific and in cis. Nicked-circle DNA is an intermediate of cleavage reaction. This may be required to allow the initial stage of translocation, a loop will have to be produced following the non-specific binding, and for translocation of this loop to occur the DNA would have to be relaxed. Individual Subunits of the EcoR124I endonuclease
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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.
