Interaction between the octamer-binding protein nuclear factor III and the adenovirus origin of DNA replication.
Nuclear factor III (NFIII) is a HeLa sequence-specific DNA-binding protein that stimulates initiation of adenovirus DNA replication in vitro and may be involved in regulation of transcription of several cellular and viral genes. We have studied the interaction between NFIII and the binding site in the adenovirus type 2 (Ad2) origin in detail by methidiumpropyl-EDTA.iron(II) and hydroxyl radical footprinting and by alkylation interference experiments. Our results indicate that (i) the core of the recognition sequence is 5'-TATGATAAT-3'; (ii) both major and minor groove base contacts are detected, and all base pairs in the core are involved in binding; (iii) many backbone contacts are observed divided into a large domain coinciding with the core and a small domain; (iv) contact points are not confined to one side of the DNA helix in contrast to the nuclear factor I (NFI)-binding site; (v) the binding site overlaps the NFI-binding site for at least one nucleotide. A number of Ad2 mutants as well as related binding sites in the origins of other adenovirus serotypes were systematically compared for binding with NFIII. The results are in good agreement with the contact point studies and show that at least one AT base pair is commonly required by NFI and NFIII for optimal binding. The strongest binding site, which contains the octamer/decanucleotide motif (ATGCAAAT[NA]), was found in the Ad4 origin, which lacks an NFI-binding site. Stimulation of in vitro DNA replication of Ad2, Ad4, and Ad12 by NFIII showed that the maximal level of stimulation is dependent on the affinity of NFIII for the origin.
Pruijn
G J
GJ
Laboratory for Physiological Chemistry, State University of Utrecht, The Netherlands.
van Miltenburg
R T
RT
Claessens
J A
JA
van der Vliet
P C
PC
D000260:Adenoviruses, Human / Q000235:genetics* / Q000502:physiology; D001483:Base Sequence; D003196:Computer Graphics; D004261:DNA Replication; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005816:Genetic Complementation Test; D006367:HeLa Cells; D051863:Host Cell Factor C1; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D050811:Octamer Transcription Factor-1; D014157:Transcription Factors; D014779:Virus Replication
D004279:DNA, Viral; D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D009687:Nuclear Proteins; D050811:Octamer Transcription Factor-1; C494165:POU2F1 protein, human; D014157:Transcription Factors; D004262:DNA Restriction Enzymes