From single amino acid to environmental ecology and back: the saga of DNA polymerase I
Date
2024
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Publisher
University of Delaware
Abstract
Viruses, comprising a vast array of genetic diversity, are pivotal components of Earth’s ecosystems. Bacteriophages (phages), or viruses infecting bacteria, outnumber host populations by a factor of ten in marine environments and therefore have outsized impacts on microbially-mediated ecosystem dynamics. Metagenomic sequencing of viral communities (viromes) offers insights into this diversity and unveils the biochemical repertoire of novel viral enzymes. This dissertation investigates the biochemical characteristics of DNA polymerase I (PolA) enzymes encoded by environmental phages. PolA is encoded by an estimated 25% of dsDNA phages and is solely responsible for the faithful replication of the phage genome. Historical biochemical analyses and metagenome mining have shown that three variants of amino acid residues at a critical position responsible for nucleotide incorporation (762 in E. coli and 526 in Coliphage T7), result in distinct enzyme characteristics. Tyrosine at this position leads to very fast replication with a moderate mutation rate and is believed to be a marker of lytic phages. Phenylalanine slows DNA replication by 10-fold compared to tyrosine and is found in many bacteria and a wide range of viruses. Leucine, prevalent in many lysogenic phages and abundantly found in metagenome sequences, has been shown to slow replication by 1000-fold and decrease mutation rates. This study employs a multidisciplinary approach to explore these hypotheses. Biochemical assays were conducted on single-point mutant proteins and environmental proteins to characterize their activity, temperature stability, and fidelity. Genetic engineering techniques were utilized to create mutant strains of bacteriophage T7 with altered PolA residues, which were then tested for their effects on phage replication dynamics. These experiments confirmed that tyrosine results in faster replication, but with a much lower mutation rate than expected, while phenylalanine and leucine significantly slow replication, with leucine exhibiting the most dramatic decrease (97%). Additionally, computational analysis of virome data was used to classify viral populations across a depth gradient in the pelagic ocean, revealing novel 762 position identities such as histidine and glutamine, which are currently of unknown biochemical function but suggest diverse ecological adaptations. These findings not only advance our understanding of viral enzyme biochemistry and phage ecology but also have significant implications for both fundamental virology research and practical applications in biotechnology and environmental science.
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Keywords
Viruses, Ecosystems, Bacteriophages, Amino acid