Journal of General Virology 2002-03-01

Analysis of DNA binding by the adenovirus type 5 E1A oncoprotein.

Nikita Avvakumov, Majdina Sahbegovic, Zhiying Zhang, Michael Shuen, Joe S Mymryk

Index: J. Gen. Virol. 83(Pt 3) , 517-24, (2002)

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Abstract

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.