SPECIALIZED NUCLEOPROTEIN STRUCTURES AT THE ORIGIN OF REPLICATION OF BACTERIOPHAGE-LAMBDA - PROTEIN ASSOCIATION AND DISASSOCIATION REACTIONS RESPONSIBLE FOR LOCALIZED INITIATION OF REPLICATION

Abstract

Binding of the O protein of phage .lambda. to the replication origin (ori.lambda.) results in the formation of an organized nucleoprotein structure termed the O-some. The O-some serves to localize and initiate a six-protein sequential reaction that provides for localized unwinding of the origin region, the critical prepriming step for precise initiation of DNA replication. By the use of electron microscopy of gold-tagged antibody complexes, we have defined four stages of protein association and dissociation reactions that are involved in the prepriming pathway. First, as defined previously, O protein binds to multiple DNA sites and self-associates to form the O-some. Second, .lambda.P and host DnaB proteins add to the O-some to generate an O.cntdot.P.cntdot.DnaB.cntdot.ori.lambda. complex. Addition of the DnaK and DnaJ proteins yields a third stage complex containing DnaK, DnaJ, O, P, and DnaB. With the addition of ATP and single-strand binding protein (SSB), the P protein is largely removed, and the DnaB acts as a helicase to generate locally unwound, SSB-coated single strand DNA. Thus the initiation of .lambda. DNA replication requires ordered assembly and partial disassembly of specialized nucleoprotein structures. The disassembly activity of DnaK and DnaJ may be their general role in the heat shock response.