Simian virus 40 (SV40) replicates in nuclei of human and monkey cells. One viral protein, large tumour (T) antigen, is required for the initiation of DNA replication. The development of in vitro replication systems which retain this property has facilitated the identification of the cellular components required for replication. T antigen recognizes the pentanucleotide 5'-GAGGC-3' which is present in four copies within the 64 base-pairs (bp) of the core origin. In the presence of ATP it binds with increased affinity forming a distinctive, bilobed structure visible in electron micrographs. As a helicase, it unwinds SV40 DNA bidirectionally from the origin. We report here that in vitro and in the presence of ATP, T antigen assembles a double hexamer, centred on the core origin and extending beyond it by 12 bp in each direction. The assembly of this dodecamer initiates an untwisting of the duplex by 2-3 turns. In the absence of ATP, a tetrameric structure is the largest found at the core origin. In the absence of DNA, but in the presence of ATP or its non-hydrolysable analogues, T antigen assembles into hexamers. This suggests that ATP effects an allosteric change in the monomer. The change alters protein-protein interactions and allows the assembly of a double hexamer, which initiates replication at the core origin.

The T antigen specified by SV40 virus is the only viral-encoded protein required for replication of SV40 DNA. T antigen has two activities that appear to be essential for viral DNA replication: specific binding to duplex DNA at the origin of replication and helicase activity that unwinds the two DNA strands. As judged by electron microscopy, DNA unwinding is initiated at the origin of replication and proceeds bidirectionally. Either linear or circular DNA molecules containing the origin of replication are effective substrates; with closed circular DNA, a topoisomerase capable of removing positive superhelical turns is required for an efficient reaction. Presence of an origin sequence on duplex DNA and a single-strand DNA-binding protein appear to be the only requirements for T antigen to catalyze unwinding. This reaction mediated by T antigen defines a likely pathway to precise initiation of DNA replication: (i) the sequence-specific binding activity locates the origin sequence, (ii) the duplex DNA is unwound at this site, and (iii) the DNA polymerase and primase begin DNA replication. A similar pathway has been inferred for the localized initiation of DNA replication by bacteriophage lambda and by Escherichia coli in which a sequence-specific binding protein locates the origin and directs the DnaB helicase to this site. Observations with the SV40 system indicate that localized initiation of duplex DNA replication may be similar for prokaryotes and eukaryotes.

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 ...

The large tumor antigen (T antigen) specified by simian virus 40 (SV40) is required for viral DNA replication. To carry out its function, T antigen binds to duplex DNA at the origin of replication (oriSV40) and exerts a helicase activity that unwinds the two DNA strands. Previous work has defined two binding sites for T antigen near onSV40, designated sites I and H; site II is within the 64-base-pair core sequence absolutely required for viral DNA replication. We have used electron microscopy and gel electrophoresis to characterize the interaction of T antigen with the origin region. We have found that effective binding to site II under conditions that support DNA replication requires ATP or a nonhydrolyzable analog. In the absence ofATP, T antigen binds mainly to site I; in the presence of ATP, both sites I and II are occupied, and binding is markedly increased. The ATP-dependent reaction generates a complex multimeric structure for T antigen. We conclude that T antigen forms an ATP-dependent nucleoprotein structure at oriSV40. We suggest that this nucleoprotein complex provides for the precise initiation of SV40 DNA replication.The only protein encoded by simian virus 40 (SV40) that is required for viral DNA replication is the large tumor antigen (T antigen). The initiation of replication depends on the binding of T antigen to the SV40 replication origin (oriSV40) (1)(2)(3). DNA replication dependent on oriSV40 and on T antigen can be carried out in vitro using HeLa cell extracts (4-6) or with purified enzyme fractions derived from HeLa cells (7). SV40 T antigen carries out an origin-dependent duplex DNA unwinding reaction in vitro in the presence of ATP, Mg2+, and single-stranded-DNA-binding protein (SSB) (8)(9)(10)(11). Covalently closed circular DNA molecules are unwound in the presence of any topoisomerase capable of relaxing positive supercoils (8); linear substrates are efficiently unwound in the absence of a topoisomerase (9). The ability of T antigen to unwind DNA is due to its intrinsic helicase activity (8,10,12,13). Oligonucleotides hybridized to long single-stranded DNA molecules are displaced by T antigen in a non-sequence-specific manner. In contrast, the unwinding of duplex DNA lacking single-stranded regions can be shown to require a functional origin of replication (8-11). A likely pathway by which T antigen mediates the initiation of DNA replication is (i) the sequence-specific binding activity locates oriSV40 in duplex DNA, (ii) the helicase activity initiates DNA unwinding in the presence of SSB, and (iii) the DNA primase and polymerase begin DNA replication.The DNA region essential in initiating SV40 DNA replication has been defined (14-18); it consists of a 64-base-pair (bp) segment, termed the core origin (16). There are two T-antigen binding sites, designated I and II, located near the SV40 replication origin (19-21). Site II corresponds to the core origin (15), whereas site I lies outside the core origin and is not required for DNA replication in vivo or in vitro (17, 18). DNas...

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...