MCRB(S), A MODULATOR PEPTIDE FOR MCRBC ACTIVITY

McrBC is a methylation-dependent endonuclease from Escherichia coil K- 12. The enzyme recognizes DNA with modified cytosines preceded by a pu rine. McrBC restricts DNA that contains at least two methylated recogn ition sites separated by 40-80 bp. Two gene products, McrB(L) and McrB (s), are produced from the mcrB gene and one, McrC, from the mcrC gene . DNA cleavage in vitro requires McrB(L), McrC, GTP and Mg2+. We found that DNA cleavage was optimal at a ratio of 3-5 McrB(L) per molecule of McrC, suggesting that formation of a multisubunit complex with seve ral molecules of McrB(L) is required for cleavage. To understand the r ole of McrB(s), we have purified the protein and analyzed its role in vitro. At the optimal ratio of 3-5 McrB(L) per molecule of McrC, McrB( s) acted as an inhibitor of DNA cleavage. Inhibition was due to seques tration of McrC and required the presence of GTP, suggesting that the interaction is GTP dependent. If McrC was in excess, a condition resul ting in suboptimal DNA cleavage, addition of McrB(s) enhanced DNA clea vage, presumably due to sequestration of excess McrC, We suggest that the role of McrB(s) is to modulate McrBC activity by binding to McrC.