Analysis of DNA binding by the adenovirus type 5 E1A oncoprotein.
Adenovirus type 5 E1A proteins interact with cellular regulators of transcription to reprogram gene expression in the infected or transformed cell. Although E1A also interacts with DNA directly in vitro, it is not clear how this relates to its function in vivo. The N-terminal conserved regions 1, 2 and 3 and the C-terminal portions of E1A were prepared as purified recombinant proteins and analyses showed that only the C-terminal region bound DNA in vitro. Deletion of E1A amino acids 201-220 inhibited binding and a minimal fragment encompassing amino acids 201-218 of E1A was sufficient for binding single- and double-stranded DNA. This portion of E1A also bound the cation-exchange resins cellulose phosphate and carboxymethyl Sepharose. As this region contains six basic amino acids, in vitro binding of E1A to DNA probably results from an ionic interaction with the phosphodiester backbone of DNA. Studies in Saccharomyces cerevisiae have shown that expression of a strong transcriptional activation domain fused to a DNA-binding domain can inhibit growth. Although fusion of the C-terminal region of E1A to a strong transcriptional activation domain inhibited growth when expressed in yeast, this was not mediated by the DNA-binding domain identified in vitro. These data suggest that E1A does not bind DNA in vivo.
Avvakumov
Nikita
N
Departments of Microbiology and Immunology1, Biochemistry2, Pharmacology and Toxicology3 and Oncology4, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, CanadaN6A 4L6.
Sahbegovic
Majdina
M
Departments of Microbiology and Immunology1, Biochemistry2, Pharmacology and Toxicology3 and Oncology4, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, CanadaN6A 4L6.
Zhang
Zhiying
Z
Departments of Microbiology and Immunology1, Biochemistry2, Pharmacology and Toxicology3 and Oncology4, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, CanadaN6A 4L6.
Shuen
Michael
M
Departments of Microbiology and Immunology1, Biochemistry2, Pharmacology and Toxicology3 and Oncology4, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, CanadaN6A 4L6.
Mymryk
Joe S
JS
Departments of Microbiology and Immunology1, Biochemistry2, Pharmacology and Toxicology3 and Oncology4, The University of Western Ontario, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, CanadaN6A 4L6.
D017500:Adenovirus E1A Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000260:Adenoviruses, Human; D001665:Binding Sites; D002455:Cell Division; D002482:Cellulose / Q000031:analogs & derivatives* / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D012685:Sepharose / Q000378:metabolism; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D017500:Adenovirus E1A Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D002482:Cellulose; D004247:DNA; D012685:Sepharose; C008881:phosphocellulose