Adenovirus E1A Oncogene Induces Rereplication of Cellular DNA and Alters DNA Replication Dynamics

Abstract

The oncogenic property of the adenovirus (Ad) transforming E1A protein is linked to its capacity to induce cellular DNA synthesis which occurs as a result of its interaction with several host proteins, including pRb and p300/CBP. While the proteins that contribute to the forced induction of cellular DNA synthesis have been intensively studied, the nature of the cellular DNA replication that is induced by E1A in quiescent cells is not well understood. Here we show that E1A expression in quiescent cells leads to massive cellular DNA rereplication in late S phase. Using a single-molecule DNA fiber assay, we studied the cellular DNA replication dynamics in E1A-expressing cells. Our studies show that the DNA replication pattern is dramatically altered in E1A-expressing cells, with increased replicon length, fork velocity, and interorigin distance. The interorigin distance increased by about 3-fold, suggesting that fewer DNA replication origins are used in E1A-expressing cells. These aberrant replication events led to replication stress, as evidenced by the activation of the DNA damage response. In earlier studies, we showed that E1A induces c-Myc as a result of E1A binding to p300. Using an antisense c-Myc to block c-Myc expression, our results indicate that induction of c-Myc in E1A-expressing cells contributes to the induction of host DNA replication. Together, our results suggest that the E1A oncogene-induced cellular DNA replication stress is due to dramatically altered cellular replication events and that E1A-induced c-Myc may contribute to these events. The adenovirus (Ad) transforming E1A protein [a 243-aminoacid E1A protein, also referred to as small E1A protein [1,2]) has the capacity to induce S phase in quiescent cells, and in the presence of activated ras or virus-encoded E1B19K or 55K proteins, E1A can transform rodent cells in culture (1, 2). The S-phase induction and cell transformation activities of the small E1A protein are genetically linked and are dependent on the N-terminal region of E1A binding to cellular protein complexes, including TRRAP/p400/GCN5, histone acetyltransferase p300/CBP, and the Rb family tumor suppressor proteins (1-4). E1A-Rb interactions result in the release of the progrowth E2F family transcription factors from the Rb-histone deacetylase (HDAC) repressor complexes and the induction of the S phase (1, 5). However, studies have shown that in order for E1A to induce S phase efficiently, it must bind to p300/CBP and Rb family proteins simultaneously, suggesting that E1A must also alter the functions of p300/CBP (3, 6).Although a large number of studies have focused on the cellular proteins that contribute to the forced induction of host DNA synthesis in E1A-expressing cells, the nature of the cellular DNA that replicates in these cells is not well understood. Previous studies have shown that the E1A-expressing cells fail to undergo proper mitosis and that such cells accumulate in the S and G 2 /M phases (7-10). Mammalian cells contain a large number of DNA replication origins, a...

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