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The goal of this study was to investigate the role of host double-strand break repair (DSBR) on the life cycle of Adenovirus (Ad). Ad mutants that lack the entire E4 region activate a cellular DNA damage response accompanied by phosphorylation of several host DSBR and DNA damage response proteins. We find that aspects of the E4 mutant induced DNA damage response occurs at the onset of viral DNA replication and may be activated by physical replication of viral genomes. Genetic analysis of the E4 mutants revealed that the E4-34kDa protein was required to prevent the activation of DNA damage response. Redistribution of MRN complex proteins away from viral replication centers by the E4-11kDa protein was not sufficient to prevent the DNA damage response. Activation of the DNA damage response does not interfere with viral DNA replication in the presence of E4-11kDa protein. E4 mutants are severely defective for late gene expression following concatenation of their genomes by host DSBR proteins. We find that E4 mutant late gene expression improves in MO59J cells that fail to form genome concatemers. DSBR kinase inhibitors interfere with genome concatenation and also stimulate late gene expression. Concatenation of E4 mutant genomes interferes with cytoplasmic accumulation of viral late messages and leads to reduced late protein levels and poor viral yields following high multiplicity infection. However, failure to concatenate viral genomes did not rescue either the DNA replication defect or virus yield following low multiplicity E4 mutant infection. Our results indicate that if the E4 mutant DNA replication defect is overcome by high multiplicity infection, concatenation of the replicated genomes by host DSBR interferes with viral late gene expression. These studies provide insight into the role of host DSBR as an obstacle to a productive Ad infection, and how the virus dismantles this barrier.