DNA looping induced by bacteriophage lambda O protein: implications for formation of higher order structures at the lambda origin of replication

Abstract

A plasmid has been constructed, pOri2, which contains two lambda replication origin sequences separated by 1068 bp; both lambda sequences having the same orientation. When lambda initiation protein O is reacted with linearized pOri2 and examined by electron microscopy it is found to contain a looped area in which two parts of the plasmid are bound together by the O protein complex. Length measurements show that the O protein binds at the expected positions of the lambda origin sequences and that the looped area represents the DNA segment between the two O protein binding domains. Similar looping occurs in reactions with supercoiled pOri2 or if an amino-terminal fragment of O protein is used. When looped molecules are reacted with psoralen, crosslinked by irradiation with uv light, and then denatured, it is found that the looped area is more thermostable than the rest of the molecule. This indicates that the DNA within the looped segment is torsionally constrained while that outside the loop is free to rotate and suggests that simultaneous binding of O to two origins fixes the linkage number of the intervening DNA. The double origin binding ability of O may be diagnostic of the details of the reaction of O with a single origin sequence. A model is presented that rests on the assumption that O can produce microscopic looping between O protein binding sites within a single ori sequence.