Adenovirus-2 early region IA protein synthesized in Escherichia coli extracts indirectly associates with DNA.
The interaction of adenovirus-2 (Ad2) early region IA (EIA) protein (encoded by the 13S mRNA) with DNA was examined using EIA protein synthesized in Escherichia coli extracts directed by a plasmid containing the cloned EIA gene. Without any purification, this protein when chromatographed over calf thymus DNA immobilized on cellulose, showed at least two types of salt-sensitive activities after associating with equal efficiency to both single- and double-stranded DNA; however, a putative C-terminal proteolytic fragment of the EIA protein (identified by immunoprecipitation with anti-serum specific to the EIA carboxy-terminus) showed 10-fold greater affinity to double- versus single-stranded DNA. When examined with Ad2 DNA, the EIA protein had a retention that was at least 2-fold higher compared to calf thymus DNA, suggesting some substrate specificity. It was also found that a 1.0 M salt concentration was required for the elution of the EIA protein from pBR322 DNA containing cloned regulatory sequences of adenovirus early regions II and III. This suggests that the strength of the protein interaction depends on the target DNA sequence. Finally, addition of uninfected HeLa cell extract to bacterial extracts containing EIA-like protein potentiated the association of the protein to double-stranded calf thymus DNA up to 7-fold. These data support the hypothesis that the EIA protein interacts with target DNA, presumably mediated by co-factor(s) in an indirect fashion.
D017456:Adenovirus Early Proteins; D000260:Adenoviruses, Human / Q000235:genetics*; D004269:DNA, Bacterial / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D009856:Oncogene Proteins, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D013462:Sulfur Radioisotopes
D017456:Adenovirus Early Proteins; D004269:DNA, Bacterial; D009856:Oncogene Proteins, Viral; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes