As a method to hide from their host and prevent degradation, bacteriophages (viruses that infect bacteria) will modify functional systems to evade detection. In literature, it has been found that a common modification mechanism employed by bacteriophages is adding a functional group to a DNA base such that restriction systems cannot recognize and cleave viral DNA. One example of these modifications include the addition of a methyl group to generate methylcytosine. Successful modification results in the host restriction enzyme mechanisms failing to recognize the foreign DNA, which allows the phage to thrive. Western Carolina University (WCU) is part of the Howard Hughes Medical Institute’s (HHMI) Science Education Alliance-Phage Hunters Advancing Genomics and Evolutionary Science (SEA-PHAGES) program, and work from WCU undergraduate students suggests that there are novel DNA modification systems present in Actinobacteriophages (viruses that infect Actinobacteria). To help understand what modifications might be present, this thesis focuses on the characterization of a panel of viruses predicted to have modified genomes. Of particular interest is phage TinyTimothy, whose genome is resistant to digestion by a standard panel of enzymes and fails to provide PCR products using Q5® DNA polymerase. Analysis of the nucleotide content of TinyTimothy genomic DNA using liquid chromatography – mass spectrometry (LC-MS) reveals the four canonical nucleotides and an additional unknown peak that elutes at ~18 minutes. Current efforts are focused on identifying the modification that generates this peak, as well as performing LC-MS on other viral genomes believed to be modified. During infection of a bacterial cell, temperate bacteriophages can choose between either the lytic or lysogenic replication cycles. During the lytic cycle, the phage will produce new virions by replicating its DNA using host machinery. In the lysogenic cycle, rather than producing progeny, the phage will incorporate its genome into the host chromosome to remain dormant. Along with nucleotide modifications, viruses have also developed proteins known as immunity repressors that bind the phage genome to prevent transcription of lytic genes and allow the phage to remain dormant and hidden in the host. Our laboratory recently published a manuscript describing a novel repressor protein found in cluster A mycobacteriophages. A unique feature of this repressor is that it uses two domains to bind DNA as a monomer; most repressors described to date bind DNA as higher-ordered oligomers. A detailed bioinformatic analysis of this monomeric repressor revealed novel protein sequences in which the repressor is fused to other protein domains with a variety of predicted functions, thus giving reason that the repressor does more than simply silencing lytic genes. Repressor fusion proteins range in size from 300 to 1,900 amino acids, and the predicted functions of these fusions vary. A few of these repressor fusions are found in pathogenic bacteria, which provides a possible avenue for novel therapeutics. We have expressed and purified the repressor fusion protein from Mycobacterium sp. ENV421, which has a repressor domain fused to a domain of unknown function. Overall, our goal is to better understand the synergy between repressors and the diverse protein domains that make up these novel proteins.