Enhancement of DNA replication by transcription factors NFI and NFIII/Oct-1 depends critically on the positions of their binding sites in the adenovirus origin of replication

The origin of DNA replication of many human adenoviruses is composed of a highly conserved core origin and an auxiliary region, containing the binding sites for NFI and NFIII/Oct-1. We examined enhancement of DNA replication in vitro by the purified functional DNA-binding domains of NFI (NFI-BD) and NFIII/Oct-1 (the POU domain), using origins in which the positions of the binding sites for these proteins were transposed. Insertion or deletion of two or three base pairs between the core origin and the NFI binding site resulted in a 3-5-fold decrease of stimulation, whereas larger insertions gradually reduced the stimulation further. Mutants in which the NFI binding site was separated approximately one or two helical turns from the core origin by AT-rich sequences could still be stimulated by NFI. In contrast, insertion of two or more base pairs between the NFI and NFIII/Oct-1 binding sites abolished stimulation by NFIII/Oct-1 almost completely. Furthermore, stimulation by this protein was lost when the Ad2 NFIII/Oct-1 binding site was transposed to a position closer to the core origin, destroying the NFI binding site. This shows that the position of the NFIII/Oct-1 binding site is essential for stimulation. Models to explain these position-dependent effects on stimulation are discussed.     

DNA sequences required for the initiation of adenovirus type 4 DNA replication in vitro

In-vivo studies have demonstrated that adenovirus type 2 and adenovirus type 4 have different DNA sequence requirements for the initiation of DNA replication. To investigate the basis of these differences an in-vitro system has been developed which will faithfully initiate adenovirus type 4 DNA replication. A plasmid containing 140 base-pairs of the right terminus of adenovirus type 4 supported initiation of DNA replication in vitro, provided that the plasmid was linearized in such a way as to locate the viral terminal sequences at the molecular ends of the DNA. Initiation by adenovirus type 4-infected cell extracts was also supported by a plasmid containing the complete adenovirus type 2 inverted terminal repeat (ITR). Deletion analysis of both adenovirus types 2 and 4 ITRs revealed that only the terminal 18 base-pairs of the genomes (perfectly conserved between the 2 viruses) were required for initiation in vitro. Thus, initiation was not enhanced by the presence of either the NFI site, the NFIII site or both sites together. Fractionation of a HeLa cell nuclear extract, by ion-exchange chromatography, identified a nuclear factor that stimulated the initiation reaction four- to fivefold. The stimulatory factor did not correspond to either of the cellular proteins NFI or NFIII which stimulate adenovirus type 2 DNA replication in vitro. Initiation in vitro was also supported by single-stranded DNA templates, albeit at a lower efficiency. Studies with synthetic oligonucleotides indicated a surprising specificity for initiation: whereas the strand used as template during initiation in vivo was active as a template for initiation in vitro, the complementary strand was inactive.     

Adenovirus DNA-binding protein forms a multimeric protein complex with double-stranded DNA and enhances binding of nuclear factor I.

The 72-kilodalton adenovirus DNA-binding protein (DBP) binds to single-stranded DNA as well as to RNA and double-stranded DNA and is essential for the replication of viral DNA. We investigated the binding of DBP to double-stranded DNA by gel retardation analysis. By using a 114-base-pair DNA fragment, five or six different complexes were observed by gel retardation. The mobility of these complexes is dependent on the DBP concentration, suggesting that the complexes arise by sequential binding of DBP molecules to the DNA. In contrast to binding to single-stranded DNA, the binding of DBP to double-stranded DNA appears to be noncooperative. DBP binds to linear DNA as well as to circular DNA, while linear DNA containing the adenovirus terminal protein was also recognized. No specificity for adenovirus origin sequences was observed. To study whether the binding of DBP could influence initiation of DNA replication, we analyzed the effect of DBP on the binding of nuclear factor I (NFI) and NFIII, two sequence-specific origin-recognizing proteins that enhance initiation. At subsaturating levels of NFI, DBP increases the rate of binding of NFI considerably, while no effect was seen on NFIII. This stimulation of NFI binding is specific for DBP and was not observed with another protein (NFIV), which forms a similar DNA-multimeric protein complex. In agreement with enhanced NFI binding, DBP stimulates initiation of adenovirus DNA replication in vitro especially strongly at subsaturating NFI concentrations. We explain our results by assuming that DBP forms a complex with origin DNA that promotes formation of an alternative DNA structure, thereby facilitating the binding of NFI as well as the initiation of DNA replication via NFI.     

In vitro replication of DNA containing either the SV40 or the polyoma origin

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro. Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo. The host-cell source of DNA polymerase alpha - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase alpha - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).     

Identification of replication factor C from Saccharomyces cerevisiae: a component of the leading-strand DNA replication complex.

A number of proteins have been isolated from human cells on the basis of their ability to support DNA replication in vitro of the simian virus 40 (SV40) origin of DNA replication. One such protein, replication factor C (RFC), functions with the proliferating cell nuclear antigen (PCNA), replication protein A (RPA), and DNA polymerase delta to synthesize the leading strand at a replication fork. To determine whether these proteins perform similar roles during replication of DNA from origins in cellular chromosomes, we have begun to characterize functionally homologous proteins from the yeast Saccharomyces cerevisiae. RFC from S. cerevisiae was purified by its ability to stimulate yeast DNA polymerase delta on a primed single-stranded DNA template in the presence of yeast PCNA and RPA. Like its human-cell counterpart, RFC from S. cerevisiae (scRFC) has an associated DNA-activated ATPase activity as well as a primer-template, structure-specific DNA binding activity. By analogy with the phage T4 and SV40 DNA replication in vitro systems, the yeast RFC, PCNA, RPA, and DNA polymerase delta activities function together as a leading-strand DNA replication complex. Now that RFC from S. cerevisiae has been purified, all seven cellular factors previously shown to be required for SV40 DNA replication in vitro have been identified in S. cerevisiae.