Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: localized unwinding of duplex DNA by a six-protein reaction.

Abstract

The 0 protein ofbacteriophage X localizes the initiation of DNA replication to a unique site on the A genome, oriX. By means of electron microscopy, we infer that the binding of 0 to oriX initiates a series of protein addition and transfer reactions that culminate in localized unwinding of the origin DNA, generating a prepriming structure for the initiation of DNA replication. We can define three stages to this prepriming reaction, the first two of which we have characterized previously. First, dimeric 0 protein binds to multiple DNA binding sites and self-associates to form a nucleoprotein structure, the O-some. Second, A P and host DnaB proteins interact with the O-some to generate a larger complex that includes additional DNA from an A+T-rich region adjacent to the 0 binding sites. Third, the addition ofthe DnaJ, DnaK, and Ssb proteins and ATP results in an origin-specific unwinding reaction, probably catalyzed by the helicase activity of DnaB. The unwinding reaction is unidirectional, proceeding "rightward" from the origin. The minimal DNA sequence competent for unwinding consists of two 0 binding sites and the adjacent A+T-rich region to the right of the binding sites. We conclude that the A 0 protein localizes and initiates a six-protein sequential reaction responsible for but preceding the precise initiation of DNA replication. Specialized nucleoprotein structures similar to the O-some may be a general feature of DNA transactions requiring extraordinary precision in localization and control.Bacteriophage X initiates DNA replication at a single replication origin, oriX (1-3). The DNA sequence of the oriX region has two major characteristics: four direct repeats of 18 base pairs (bp), each of which is an inverted repeat; and an adjacent region to the right of the repeats that is extremely rich in A+T (4-6). Two X proteins, 0 and P, are required for viral DNA replication (7-9). The 0 protein recognizes the origin, and P localizes the essential replication enzymes of Escherichia coli at this site by initiating a series of protein-protein interactions (3). The 0 protein binds to the four repeats (10, 11), probably recognizing each local inverted repeat as a dimer (ref. 12; unpublished work In this study, we show that the third stage ofthe prepriming reaction yields origin-specific unwinding of duplex DNA, presumably catalyzed by the helicase activity of DnaB (26). We infer that a locally unwound DNA structure associated with DnaB becomes the specific substrate for localized priming by DnaG. Thus, the 0 protein builds a nucleoprotein structure at the replication origin of A and initiates a series of protein assembly events culminating in the localized initiation of DNA replication. The DnaA protein of E. coli appears to act in a similar fashion to localize the initiation of bacterial DNA replication to the single site, oriC (27, 28 (18,30,32,33). Restriction enzymes were from New England Biolabs.DNA. The structure and preparation of plasmid pRLM4, which contains the replication origin of phage X, have ...

Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli.

“…The oriC plasmid was pCM960 grown in E. coli strain CM987 (14). The oriX plasmid DNA was RF I (replicative form I; i.e., superhelical circular doublestranded) DNA prepared from E. coli strain JM101 (15) infected with M13 oriX1 (16 (19) by the deletion of the X N gene, was the gift of R. Fuller (Stanford University). E. coli strain SG4148 (Alon), derived from SG4144 (20), was used for the preparation of the host replication factors.…”

Protein-induced DNA linking number change by sequence-specific DNA binding proteins and its biological effects

“…For instance, λ O is the replication initiator of bacterial phage λ (Struble et al 2007;Zahn and Blattner 1985) and specifically binds to the four repeating sequences (iterons) of λ replication origin (Oriλ) (Scherer 1978). Upon O binding, Oriλ wraps around O protein and forms a nucleoprotein complex called BO-some^ (Dodson et al 1986;Schnos et al 1989). As a result, the AT-rich DUE is unwound and ready for subsequent replication initiation (Dodson et al 1989;Schnos et al 1988).…”

Protein-induced DNA linking number change by sequence-specific DNA binding proteins and its biological effects

“…In bacteria, DNA replication usually initiates from a defined single site on the chromosome; the origin of DNA replication (OriC; Leonard and (Struble et al 2007;Zahn and Blattner 1985) and specifically binds to the four repeating sequences (iterons) of λ replication origin (Oriλ) (Scherer 1978). Upon O binding, Oriλ wraps around O protein and forms a nucleoprotein complex called BO-some^ (Dodson et al 1986;Schnos et al 1989). As a result, the AT-rich DUE is unwound and ready for subsequent replication initiation (Dodson et al 1989;Schnos et al 1988).…”