PROTEIN-PROTEIN AND PROTEIN-DNA INTERACTIONS IN THE TYPE-I RESTRICTION-ENDONUCLEASE R.ECOR124I

The type I restriction-modification system EcoR124l recognizes and bin ds to the split DNA recognition sequence 5'-GAAN(6)RTCG-3'. The methyl transferase, consisting of HsdM and HsdS subunits with the composition M2S, can interact with one or more subunits of the HsdR subunit to fo rm the endonuclease. The interaction of the methyltransferase with Hsd R has been investigated by surface plasmon resonance, showing that the re are two non-equivalent binding sites for HsdR which differ in bindi ng affinity by at least two orders of magnitude. DNA footprinting expe riments using Exonuclease III suggest that the addition of HsdR to the methyltransferase (at a stoichiometry of either 1:1 or 2:1) increases the stability of the resulting DNA-protein complex but does not incre ase the size of the footprint. More extensive in situ footprinting exp eriments using copper-phenanthroline on the DNA-protein complexes form ed by M2S, R1M2S and R2M2S also show no difference in the detailed cle avage pattern, with similar to 18 nucleotides protected on both strand s in each complex. Thus the HsdR subunit(s) of the endonuclease stabil ise the interaction of the M2S complex with DNA, but do not directly c ontribute to DNA binding. In addition, the thymidine nucleotide in the tetranucleotide recognition sequence GTCG is hyper-reactive to cleava ge in each case, suggesting that the DNA structure in this region is a ltered in these complexes.