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PMID: 3821731 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

Molecular and cellular biology ·Vol. 7 ·No. 2 ·1987-02-00 ·Pages 875-86

Rosenfeld PJ, O'Neill EA, Wides RJ, Kelly TJ

Abstract

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.

MeSH Terms
Adenoviruses, Human/genetics Base Sequence Binding Sites DNA Replication DNA-Binding Proteins/physiology Genetic Complementation Test HeLa Cells Structure-Activity Relationship Viral Proteins/physiology Virus Replication
Chemicals
DNA-Binding Proteins Viral Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Rosenfeld P J
O'Neill E A
Wides R J
Kelly T J
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44 references, click to expand
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.7.2.875-886.1987
Published
1987-02-00
Pages
875-86
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC365146
Subset
IM
Grants
NCI NIH HHS · CA16519 · United States
NIGMS NIH HHS · GM07309 · United States
External Links
PubMed source DOI: 10.1128/mcb.7.2.875-886.1987 PMC Full Text View journal details
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