31 research outputs found
Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.
The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin
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Structure and function of the adenovirus origin of replication
Efficient initiation of adenovirus DNA replication requires the presence of specific terminal nucleotide sequences that collectively constitute the viral origin of replication. Using plasmids with deletions or base substitutions in a cloned segment of DNA derived from the terminus of the adenovirus 2 genome, we have demonstrated that the origin contains two functionally distinct regions. The first 18 bp of the viral genome are sufficient to support a limited degree of initiation. However, the presence of a sequence in the region between nucleotides 19 and 67 greatly enhances the efficiency of the initiation reaction. This region contains a specific binding site for a protein present in uninfected cells (K
D = 2 × 10
−11 M). The bound protein protects the DNA segment between base pairs 19 and 43 from attack by DNAase I. Studies with deletion mutants indicate that binding of the cellular protein is responsible for the enhancement of initiation
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Replication of adenovirus and SV40 chromosomes in vitro
As an approach to studying the mechanisms involved in the replication of eukaryotic chromosomes, we have developed and characterized cell-free replication systems for the animal viruses, adenovirus and SV40. In this report we summarize recent work on the proteins required for the initiation of DNA synthesis in these two systems. The adenovirus origin of DNA replication was shown to consist of three functionally distinct sequence domains. Cellular proteins that specifically recognize each of these domains were purified and characterized. Initiation of adenovirus DNA replication was reconstituted from two virus-encoded and three cell-encoded factors. The SV40 origin of replication consists of a 65 base pair DNA segment that contains a high affinity binding site for the viral initiation protein T antigen. Evidence is presented that the first step in initiation of SV40 DNA replication involves the specific binding of T antigen to the origin, followed by the local unwinding of the two strands of the template. The unwinding reaction is specific for DNA templates containing the SV40 origin and requires ATP hydrolysis. In addition to T antigen, efficient unwinding requires a cellular factor(s) that can be replaced by the single-stranded DNA binding protein of Escherichia coli. These results indicate that the recently discovered helicase activity of T antigen plays a central role in initiation of viral DNA synthesis