Introduction

EcoR124I is a Type I R-M system found in E. coli, working to defend the cells against infection from bacteriophage.  The system recognises a bipartite sequence in the DNA, GAA(N)6RTCG, and flips out the third base (Alanine) on the 5’ strand and the third from last base (Alanine) on the 3’ strand.  These bases are checked for their methylation status, in the absence of methylation on either strand the DNA is recognised as ‘foreign’ and the enzyme switches to a Restriction enzyme.  The enzyme is made up of a central core known as the methyltransferase (MTase) which consists of one HsdS (specificity) subunit and two HsdM (Methylation) subunits (M2S1).  An HsdR subunit binds to this complex (R1 complex; R1M2S1) which allows the enzyme to translocate DNA in one direction and to make a single stranded break (nick) in the DNA.  When a second HsdR subunit binds to form an R2 complex (R2M2S1) the enzyme has been shown to be capable of bi-directional translocation and full double stranded cleavage of DNA.

Figure 1  Restriction assay using plasmid (pCFD30) DNA.

The plasmid contains a single site for EcoR124I combined with an EcoRI site (GAATTC(N)3RTCG). In its native form the plasmid is closed constrained circular DNA (cccDNA). In the presence of an R1 complex the DNA is nicked which releases the supercoiling to produce open circular DNA (ocDNA).  On binding of an R2 complex the DNA is fully cleaved to produce linear DNA. The single EcoRI site allows to linearise the plasmid to run as a control.

 

Fig 2.  Gel shift using a flurocein tagged 30 base pair oligonucleotide containing a single recognition sequence for EcoR124I.

This shows DNA and MTase at equimolar concentrations with increasing concentrations of HsdR. Lanes 1 and 2 are control lanes with DNA alone and DNA with MTase. Lanes 3-8 have ratios of
HsdR to MTase of 0.25, 0.5, 1, 2, 4 and 8 respectively.

 

   

Fig 3.  The affect of the presence and absence of cofactors on the ability to complex and
restriction abilities of EcoR124I.

Restriction assays (Upper) and Gel Shifts (lower) were carried out with varying concentrations
of ATP, Mg²⁺ and SAM respectively.
ATP has been shown to necessary for restriction by the R2 complex but at high concentrations
it does start to affect the ability of the complex to stay together in the R2 conformation.
Mg²⁺ Although magnesium had no effect on complex formation both at R2 complex formation
concentrations and at R1 (data not shown) it is absolutely required for restriction.
SAM has been shown to have absolutely no effect on either restriction ability or complex
formation although there is sometimes a reduction in restriction in the presence of high
concentrations of SAM as the DNA is methylated.

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