Repair of protein-linked DNA double strand breaks: Using the adenovirus genome as a model substrate in cell-based assays.

The DNA double strand breaks (DSBs) created during meiotic recombination and during some types of chemotherapy contain protein covalently attached to their 5' termini. Removal of the end-blocking protein is a prerequisite to DSB processing by non-homologous end-joining or homologous recombination. One mechanism for removing the protein involves CtIP-stimulated Mre11-catalyzed nicking of the protein-linked strand distal to the DSB terminus, releasing the end-blocking protein while it remains covalently attached to an oligonucleotide. Much of what is known about this repair process has recently been deciphered through in vitro reconstitution studies. We present here a novel model system based on adenovirus (Ad), which contains the Ad terminal protein covalently linked to the 5' terminus of its dsDNA genome, for studying the repair of 5' protein-linked DSBs in vivo. It was previously shown that the genome of Ad mutants that lack early region 4 (E4) can be joined into concatemers in vivo, suggesting that the Ad terminal protein had been removed from the genome termini prior to ligation. Here we show that during infection with the E4-deleted Ad mutant dl1004, the Ad terminal protein is removed in a manner that recapitulates removal of end-blocking proteins from cellular DSBs. In addition to displaying a dependence on CtIP, and Mre11 acting as the endonuclease, the protein-linked oligonucleotides that are released from the viral genome are similar in size to the oligos that remain attached to Spo11 and Top2 after they are removed from the 5' termini of DSBs during meiotic recombination and etoposide chemotherapy, respectively. The single nucleotide resolution that is possible with this assay, combined with the single sequence context in which the lesion is presented, make it a useful tool for further refining our mechanistic understanding of how blocking proteins are removed from the 5' termini of DSBs.

The adenovirus E1b55K/E4orf6 complex induces degradation of the Bloom helicase during infection.

The adenovirus (Ad) E1b55K and E4orf6 gene products assemble an E3 ubiquitin ligase complex that promotes degradation of cellular proteins. Among the known substrates are p53 and the Mre11-Rad50-Nbs1 (MRN) complex. Since members of the RecQ helicase family function together with MRN in genome maintenance, we investigated whether adenovirus affects RecQ proteins. We show that Bloom helicase (BLM) is degraded during adenovirus type 5 (Ad5) infection. BLM degradation is mediated by E1b55K/E4orf6 but is independent of MRN. We detected BLM localized at discrete foci around viral replication centers. These studies identify BLM as a new substrate for degradation by the adenovirus E1b55K/E4orf6 complex.

The MRN complex in double-strand break repair and telomere maintenance.

Genomes are subject to constant threat by damaging agents that generate DNA double-strand breaks (DSBs). The ends of linear chromosomes need to be protected from DNA damage recognition and end-joining, and this is achieved through protein-DNA complexes known as telomeres. The Mre11-Rad50-Nbs1 (MRN) complex plays important roles in detection and signaling of DSBs, as well as the repair pathways of homologous recombination (HR) and non-homologous end-joining (NHEJ). In addition, MRN associates with telomeres and contributes to their maintenance. Here, we provide an overview of MRN functions at DSBs, and examine its roles in telomere maintenance and dysfunction.

Mislocalization of the MRN complex prevents ATR signaling during adenovirus infection.

The protein kinases ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) are activated in response to DNA damage, genotoxic stress and virus infections. Here we show that during infection with wild-type adenovirus, ATR and its cofactors RPA32, ATRIP and TopBP1 accumulate at viral replication centres, but there is minimal ATR activation. We show that the Mre11/Rad50/Nbs1 (MRN) complex is recruited to viral centres only during infection with adenoviruses lacking the early region E4 and ATR signaling is activated. This suggests a novel requirement for the MRN complex in ATR activation during virus infection, which is independent of Mre11 nuclease activity and recruitment of RPA/ATR/ATRIP/TopBP1. Unlike other damage scenarios, we found that ATM and ATR signaling are not dependent on each other during infection. We identify a region of the viral E4orf3 protein responsible for immobilization of the MRN complex and show that this prevents ATR signaling during adenovirus infection. We propose that immobilization of the MRN damage sensor by E4orf3 protein prevents recognition of viral genomes and blocks detrimental aspects of checkpoint signaling during virus infection.

Nuclear export of the human cytomegalovirus tegument protein pp65 requires cyclin-dependent kinase activity and the Crm1 exporter.

We have previously shown that treatment of human cytomegalovirus-infected cells with the cyclin-dependent kinase (cdk) inhibitor roscovitine has significant effects on several stages of the virus life cycle depending on the time of addition (V. Sanchez, A. K. McElroy, J. Yen, S. Tamrakar, C. L. Clark, R. A. Schwartz, and D. H. Spector, J. Virol. 78:11219-11232, 2004; V. Sanchez and D. Spector, J. Virol. 80:5886-5896, 2006). In this report, we add to these findings by demonstrating alterations in the phosphorylation and localization of pp65 (UL83) in cells treated with roscovitine. We observed that inhibition of cdk activity causes the retention of pp65 within the nucleus at late times postinfection. At the same time, we observed a change in the phosphorylation pattern of the protein. Interestingly, mutation of potential cdk phosphorylation sites did not affect the ability of pp65 to localize to the nucleus or to relocalize to the cytoplasm late in infection. However, we found that the cytoplasmic accumulation of pp65 late in infection was sensitive to the Crm1 inhibitor leptomycin B.