Abstract
The adenovirus 12 early region 1B55K (Ad12E1B55K) protein has long been known to cause non-random damage to chromosomes 1 and 17 in human cells. These sites, referred to as Ad12 modification sites, have marked similarities to classic fragile sites. In the present report we have investigated the effects of Ad12E1B55K on the cellular DNA damage response and on DNA replication, considering our increased understanding of the pathways involved. We have compared human skin fibroblasts expressing Ad12E1B55K (55K+HSF), but no other viral proteins, with the parental cells. Appreciable chromosomal damage was observed in 55K+HSFs compared to parental cells. Similarly, an increased number of micronuclei was observed in 55K+HSFs, both in cycling cells and after DNA damage. We compared DNA replication in the two cell populations; 55K+HSFs showed increased fork stalling and a decrease in fork speed. When replication stress was introduced with hydroxyurea the percentage of stalled forks and replication speeds were broadly similar, but efficiency of fork restart was significantly reduced in 55K+HSFs. After DNA damage, appreciably more foci were formed in 55K+HSFs up to 48 h post treatment. In addition, phosphorylation of ATM substrates was greater in Ad12E1B55K-expressing cells following DNA damage. Following DNA damage, 55K+HSFs showed an inability to arrest in cell cycle, probably due to the association of Ad12E1B55K with p53. To confirm that Ad12E1B55K was targeting components of the double-strand break repair pathways, co-immunoprecipitation experiments were performed which showed an association of the viral protein with ATM, MRE11, NBS1, DNA-PK, BLM, TOPBP1 and p53, as well as with components of the replisome, MCM3, MCM7, ORC1, DNA polymerase δ, TICRR and cdc45, which may account for some of the observed effects on DNA replication. We conclude that Ad12E1B55K impacts the cellular DNA damage response pathways and the replisome at multiple points through protein–protein interactions, causing genomic instability.
Highlights
The random damage by Ad5, has been attributed to expression of E1A [21]. In view of these observations, we considered that further analysis of the ability of Ad12E1B55K to cause DNA damage and its effects on cellular DNA damage response (DDR) would be of considerable interest, in view of our greatly enhanced understanding of DNA damage repair pathways since the original investigations were undertaken
We have shown that Ad12E1B55K expression sensitizes cells to different damaging agents, seen as an increased number of DNA damage foci and increased activation of ATM, and, consistent with historical reports, increased genomic instability, seen as an increase in chromosomal damage and micronuclei occurrence
We examined whether inactivation of MRE11 by the the inhibitor mirin would duplicate some of the effects of Ad12E1B55K in the parental inhibitor mirin would duplicate some of the effects of Ad12E1B55K in the parental HSFs
Summary
Human adenoviruses comprise a large family of approximately 90 different types divided into seven species (A to G) They cause relatively mild infections of the respiratory and gastrointestinal tracts and the eye, depending on species. Injection of Ad12E1-transformed rodent cells into the syngeneic host could produce tumors, whereas cells transformed with Ad5 or Ad2 DNA were only tumorigenic in athymic nude mice or immunosuppressed animals [6,7,8,9]. These observations allowed adenoviruses to be categorized as ‘DNA tumor viruses’. Consistent with this, Ad12 has been shown to cause retinoblastoma-like tumors in baboons [10]
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