Bioinformatic and in vitro characterization of primase-polymerase enzymes from viruses that infect actinobacterial hosts

WCU Author/Contributor (non-WCU co-authors, if there are any, appear on document)
Nathan Benjamin Folse (Creator)
Institution
Western Carolina University (WCU )
Web Site: http://library.wcu.edu/
Advisor
Jaime R. Wallen

Abstract: Primase-polymerases (prim-pols) are enzymes that exhibit primase, polymerase, and potential helicase-like activities by way of a bifunctional N-terminal prim-pol domain and a C-terminal ATPase domain. Presented is a multifaceted analysis of prim-pols encoded by actinophages, or viruses that infect Actinobacterial hosts. The aims of this study are to bioinformatically characterize all identifiable actinophage-encoded prim-pols and to biochemically characterize the prim-pol encoded by mycobacteriophage Larva, including determining protein-protein interactions between Larva’s prim-pol and host Mycobacterium smegmatis proteins using a bacterial two-hybrid system. Bioinformatic analyses reveal nearly 600 actinophages encoding prim-pols that span a variety of host types, genome sizes, conserved domains, and encoded genetic metabolism proteins. A novel class of truncated prim-pols that contain an intact prim-pol domain but lack any additional C-terminal domain has been identified. Most phages encoding truncated prim-pols also encode a separate protein resembling the ATPase domain of full-length prim-pols. Interestingly, the C-terminal functional domains of full-length prim-pols vary from phage to phage aside from a conserved nucleotide binding motif. The C-terminal domains of prim-pols allow them to be grouped more narrowly than by their N-terminal prim-pol domain alone. To begin to understand the importance of prim-pols in actinophage DNA replication, a detailed characterization has been performed of a prim-pol from mycobacteriophage Larva, which is the virus’ only encoded DNA polymerase. Larva’s prim-pol exhibits Mg2+-dependent primase/polymerase activity on an unprimed ssDNA substrate in the presence of dNTPs. It also binds multiple DNA substrates and translocates on ssDNA in the presence of ATP. CRISPR interference silencing of prim-pol is lethal to Larva, indicating it is essential for viral survival. Bacterial two-hybrid analysis reveals interactions between Larva’s prim-pol and at least five M. smegmatis proteins, including transcription and nucleic acid synthesis proteins. Based on these results, it is hypothesized that despite sharing a conserved prim-pol domain, actinophage-encoded prim-pols fulfill a variety of functions in the replication of phage DNA, some critical to viral survival, depending on the domain organization of the proteins’ C-termini and on the phages’ individual genomic architectures.

Additional Information

Publication
Thesis
Language: English
Date: 2020
Keywords
ATPase, bioinformatics, DNA replication, phage, prim-pol, primase-polymerase

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