genuine heterogeneity in the quaternary structure for two of the four proteins and the dependence on column volume for the ArdBCFT protein further suggested that the apparent switching between the monomer and dimer forms was occurring on the minute timescale of the chro- matography process. In vitro activity of ArdB and KlcA136 proteins against the EcoKI Type IA restriction endonuclease A circular plasmid containing a single copy of the target sequence for the EcoKI type I RM enzyme is eﬃciently cleaved by stoichiometric amounts of EcoKI in the presence of the essential cofactors SAM, ATP and Mg2+ resulting in a linearised plasmid (34). Stoichiometric amounts of the ocr and ArdA anti- restriction proteins inhibit this activity by binding strongly to and blocking the DNA binding site on EcoKI (9,12,13). Figure 3 shows that addition of a 20-fold excess of ocr or ArdA, expected to ensure complete inhibition, did indeed prevent any nuclease activity of the EcoKI restriction enzyme. Addition of the same concentration and degree of excess of any of the examined ArdB or KlcA136 proteins to the endonuclease reaction showed no observable inhibition of the nuclease activity of EcoKI. Thus the anti-restriction activity clearly observed in vivo was not due to an anti-nuclease activity in contrast to what one might expect from the results of the anti-restriction assay. Hence, none of the ArdB or KlcA136 tested appeared able to act as an inhibitor of the Type IA RM enzyme EcoKI in DNA cleavage. The order of addition of the reactants can be important for analysing Type I endonuclease activity. For instance ocr is unable to inhibit EcoKI if it is added after the DNA and ATP (72) so for this reason we added the DNA last to the mixture containing EcoKI, cofactors and anti-restriction protein to avoid this problem. Even with this precaution, ArdB and KlcA136 were unable to inﬂuence the endonuclease activity of EcoKI. KlcA136 does not bind to EcoKI Mtase Biophysical characterization using ITC and ﬂuorescence spectroscopy performed on two other anti-restriction proteins, ocr and ArdA, have shown a strong interaction of the Mtase part of EcoKI with these proteins (13,16). However, ITC experiments performed at 10 and 25 C showed no apparent binding of KlcA136 to Mtase, Figure 4. This result agrees with the in vivo modiﬁcation assays where inhibition of phage DNA modiﬁcation was not observed. Detecting the binding to the EcoKI nuclease was not feasible using ITC due to limited amounts of the nuclease. However, given the result of the nuclease inhibi- tion assay, no interaction is expected with the surface of the nuclease. Do KlcA and ArdB interfere with assembly of EcoKI? Since the KlcA and ArdB proteins appear unable to interact neither with the complete EcoKI RM enzyme nor with the core Mtase in vitro, we also examined whether they could interact with the surface of the Nucleic Acids Research, 2010, Vol. 38, No. 5 1729 Figure 3. Nuclease assay to test for inhibition of EcoKI by ArdB proteins. Lane 1, undigested DNA; lane 2, EcoKI digested DNA; lane 3, plus ocr; lane 4, plus Orf18 ArdA; lane 5, plus ArdBYAF; lane 6, plus ArdBYFJ; lane 7, plus KlcA136; lane 8, plus ArdBCFT; lane M, DNA ladder with sizes in kb. Plasmid DNA used was non-methylated pBRsk1. Ratio of the anti-restriction proteins (monomers) to EcoKI was 20:1. In each case the reaction mixture minus DNA was made up and incubated for 2 min at room tempera- ture. The reaction was then initiated by adding DNA. Digestion was carried out for 8 min at 37 C before quenching at 68 C for 10 min. CC, closed circular plasmid; L, linear plasmid; OC, open circular plasmid. Figure 4. Raw ITC data showing heat changes for the interaction of KlcA136 with the EcoKI Mtase compared with data for the interaction of ArdA with the Mtase as previously published (13). The traces show from top to bottom: Mtase being titrated with ArdA at 25 C, buﬀer being titrated with Mtase at 25 C, Mtase being titrated with KlcA136 at 25 C and Mtase being titrated with KlcA136 at 10 C. It is clear that there is no interaction between the Mtase and KlcA136. HsdR restriction subunit of EcoKI that becomes buried on binding to the Mtase. This would interfere with the assembly of the complete RM enzyme as originally proposed by Belogurov and Delver (26). Addition of ArdB or KlcA prior to assembling the Mtase and HsdR into an intact EcoKI endonuclease failed to inhibit the endonuclease activity when DNA was subsequently added to the mixture, Figure 5. This strongly suggests that ArdB and KlcA do not interact with HsdR to inter- fere with assembly of EcoKI.