Specialized nucleoprotein structures at the origin of replication of bacteriophage lambda: complexes with lambda O protein and with lambda O, lambda P, and Escherichia coli DnaB proteins.

Abstract

The 0 protein of bacteriophage A is required for initiation of DNA replication at the A replicative origin designated onik. The binding sites for 0 protein are four direct repeats, each of which is an inverted repeat. By means of electron microscopy, we have found that phage A 0 protein utilizes these multiple binding sites to form a specific nucleoprotein structure in which the origin DNA is inferred to be folded or wound. The phage K 0 and P proteins and host DnaB protein interact at onA to generate a larger structure than that formed by 0 protein alone; P and DnaB proteins fail to form any observable complex when 0 protein is excluded from the reaction mixture. We conclude that the specialized nucleoprotein structure formed by phage K 0 protein and oniK provides for localized initiation of DNA replication by serving as the foundation for the assembly of the initial priming structure. Specialized nucleoprotein structures may be a general means to confer exceptional accuracy on DNA transactions requiring extraordinary precision.Replication by bacteriophage X initiates at a unique site designated oriX and proceeds bidirectionally (1-3). The DNA sequence in the ori region has two notable features: (i) four direct repeats of 18 base pairs (bp), each of which is an inverted repeat; and (ih) adjacent A+T-rich regions, one of which has partial homology to the DnaG priming site ofphage G4 (4-7). Two phage X proteins, 0 and P, are required for viral DNA replication (8)(9)(10). These proteins serve to direct the host replicative machinery to phage X through a sequence of interactions inferred from a variety of genetic and biochemical experiments (3). The 0 protein associates with the ori region (11-13), binding to the four direct repeats (14). 0 protein interacts with P protein (15-17), and P, with Escherichia coli DnaB protein (18-21). The addition of DnaB directs other host proteins, including DnaG, DnaJ, and DnaK, to the initiation of DNA replication at oriX (3,(21)(22)(23)(24)(25). Thus, the binding of 0 protein to oriA is the initiating event for a complex series of reactions, culminating in originspecific initiation of DNA replication.In this study, we used electron microscopy to examine the interactions that occur between the phage X origin of replication and X 0, X P, and host DnaB proteins. We find that 0 protein interacts with the four direct repeats in oriX to generate a specific nucleoprotein structure. If P and DnaB proteins are added in addition to 0 protein, we observe a larger and more asymmetric nucleoprotein structure, which we presume to be the second stage in the pathway to assembly of an initiating complex. We consider possible general functions for specialized nucleoprotein structures in DNA transactions requiring exceptional precision. MATERIALS AND METHODSProteins. Phage X 0 protein was purified by a modification of the procedure of Roberts and McMacken (13); the specific activity is 1 x 105 units/mg. Phage X P protein was purified as described by McMacken et al. (21); the specific activi...

T-antigen-DNA polymerase alpha complex implicated in simian virus 40 DNA replication.

“…This protein is also thought to interact with the host dnaB gene product and the phage P protein before replication initiation (15). E. coli oriC replication initiates through the binding of dnaA protein to the origin (19), and genetic evidence suggests that RNA polymerase may interact directly with dnaA (3,4,45).…”

The herpes simplex virus type 1 origin-binding protein interacts specifically with the viral UL8 protein

“…The possibility that separate domains of the UL9 protein are involved in DNA binding and the recruitment of proteins to the origin has a close parallel in bacteriophage 2 DNA replication. Here the N terminus of the gpO protein binds to the origin whilst the C-terminus interacts with the gpP/dnaB complex directing the host replication machinery to the origin (Dodson et al, 1985(Dodson et al, , 1989Wickner & Zahn, 1986).…”

Nucleoprotein complex formed between herpes simplex virus UL9 protein and the origin of DNA replication: inter- and intramolecular interactions.

“…DNA-bound proteins may interact to form specialized nucleoprotein structures that are visible in the electron microscope (21). Phage A 0 protein binds as an octamer, condensing the DNA by forming a DNA-wound nucleoprotein complex ("O-some") visible by electron microscopy (22). Simian virus 40 large tumor (T) antigen, in the presence of ATP, binds as a double-hexamer structure, covering about 90 bp of DNA at the core origin (23,24), but does not condense or wrap the DNA upon binding.…”