Adenovirus infection induces a cellular DNA damage response that can inhibit viral DNA replication and ligate viral genomes into concatemers. It is not clear if the input virus is sufficient to trigger this response or if viral DNA replication is required. Adenovirus has evolved two mechanisms that target the Mre11-Rad50-Nbs1 (MRN) complex to inhibit the DNA damage response. These include E4-ORF3-dependent relocalization of MRN proteins and E4-ORF6/E1B-55K-dependent degradation of MRN components. The literature suggests that degradation of the MRN complex due to E4-ORF6/E1B-55K does not occur until after viral DNA replication has begun. We show that, by the time viral DNA accumulates, the MRN complex is inactivated by either of the E4-induced mechanisms and that, with E4-ORF6/E1B-55K, this inactivation is due to MRN degradation. Our data are consistent with the conclusion that input viral DNA is sufficient to induce the DNA damage response. Further, we demonstrate that when the DNA damage response is active in E4 mutant virus infections, the covalently attached terminal protein is not cleaved from viral DNAs, and the viral origins of replication are not detectably degraded at a time corresponding to the onset of viral replication. The sequences of concatemeric junctions of viral DNAs were determined, which supports the conclusion that nonhomologous end joining mediates viral DNA ligation. Large deletions were found at these junctions, demonstrating nucleolytic procession of the viral DNA; however, the lack of terminal protein cleavage and terminus degradation at earlier times shows that viral genome deletion and concatenation are late effects.Adenovirus (Ad) has a linear, double-stranded DNA genome with inverted terminal repeats (ITRs) at each extremity that contain the origins of replication. Several viral proteins have been found to be key components in supporting viral DNA replication, and of particular importance here, this includes the E4-ORF3 and E4-ORF6 products. With mutant viruses that lack E4-ORF3 and E4-ORF6, early viral transcription and gene expression are normal; however, there is a significant delay and reduction in viral DNA replication, and virus yield is reduced by ϳ1,000-fold (20, 49). Either the E4-ORF3 or E4-ORF6 protein is individually sufficient to complement the DNA replication defect and virus growth (7, 24); thus, these proteins are considered functionally redundant.An important function of these two Ad E4 proteins is the inhibition of the Mre11-Rad50-Nbs1 (MRN) complex (30, 50). The MRN complex is involved in double-strand break repair (DSBR) and is regarded as the sensor of double-strand breaks (DSBs) (9, 12, 46). When a DSB occurs in the cellular genome due to a multitude of causes, ranging from ionizing radiation (33) to VDJ recombination (10), the MRN complex recognizes the lesion and recruits the protein kinases ataxia-telangiectasia mutated (ATM) and ATM-Rad3 related (ATR) to the site of the break to initiate the process of nonhomologous end joining (NHEJ) (46). These two kinases...