The Development and Testing of a System for Monitoring Site-Specific Lesions In Vivo

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2013

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Kreuzer, Kenneth N

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Abstract

Every day, cells face agents that generate lesions in genomic DNA, which can interfere with the processes of DNA replication and gene expression. These lesions can range from small abasic sites to alkylated bases to large proteins frozen on the DNA and can be caused by both endogenous and exogenous agents. These lesions must be repaired to maintain genomic stability, and multiple pathways exist to perform the necessary repairs or to bypass the damage. These pathways have been discovered and studied using a variety of experimental techniques, both in vitro and in vivo. While these studies have contributed valuable information about many of cellular processes, there are still gaps in the DNA repair field.

The goal of this study is to bridge some of those gaps by constructing a system to introduce DNA containing a site-specific lesion into Escherichia coli cells at high enough levels to monitor the lesion's fate in vivo and in real time. This system combines two separate DNA molecules to simplify the introduction of a site-specific lesion. The first molecule is the DNA from bacteriophage λ, a virus that is able to infect E. coli cells at a high level of efficiency. A typical commercial packaging reaction can yield titers of approximately 1.0 x 109 plaque forming units (PFU)/mL. However, bacteriophage λ has a large genome of approximately 48.5 kb, which makes it a difficult substrate for extensive cloning and manipulation. In contrast, cloning and manipulation of a small plasmid (~4 kb) is a much simpler endeavor, and small plasmids have been used previously to produce DNA containing a site-specific lesion. The problem with using a plasmid occurs when attempting to introduce it into cells, as the process of transformation is not very efficient and can cause unintended consequences in the cells. This new system allows for the incorporation of the lesion into the plasmid, which is then integrated into a bacteriophage λ vector, λ Kytos. The combination of these two molecules produces bacteriophage λ DNA containing a site-specific lesion, which can infect the cells at high efficiency, allowing the fate of the DNA to be monitored in real-time. Interestingly, repair of a single EthenoA lesion after infection appears to be a very inefficient process. Even if the repair system is activated by induction with methyl methanesulfonate (MMS) or if individual repair proteins are overexpressed, little to no repair occurs. As methylation occurs upon injection, it does appear that the DNA is exposed to proteins in the cell, including any repair proteins present. These results indicate that other processes, perhaps replication or transcription, are required to repair a single EthenoA lesion in vivo.

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Asllani, Melissa (2013). The Development and Testing of a System for Monitoring Site-Specific Lesions In Vivo. Dissertation, Duke University. Retrieved from https://hdl.handle.net/10161/7107.

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