Keown Rachel A, Dums Jacob T, Brumm Phillip J, MacDonald Joyanne, Mead David A, Ferrell Barbra D, Moore Ryan M, Harrison Amelia O, Polson Shawn W, Wommack K Eric
Department of Biological Sciences, College of Arts and Sciences, University of Delaware, Newark, DE, United States.
Biotechnology Program, North Carolina State University, Raleigh, NC, United States.
Front Microbiol. 2022 Apr 21;13:858366. doi: 10.3389/fmicb.2022.858366. eCollection 2022.
Viruses are the most abundant and diverse biological entities on the planet and constitute a significant proportion of Earth's genetic diversity. Most of this diversity is not represented by isolated viral-host systems and has only been observed through sequencing of viral metagenomes (viromes) from environmental samples. Viromes provide snapshots of viral genetic potential, and a wealth of information on viral community ecology. These data also provide opportunities for exploring the biochemistry of novel viral enzymes. The biochemical characteristics of novel viral DNA polymerases were explored, testing hypothesized differences in polymerase biochemistry according to protein sequence phylogeny. Forty-eight viral DNA Polymerase I (PolA) proteins from estuarine viromes, hot spring metagenomes, and reference viruses, encompassing a broad representation of currently known diversity, were synthesized, expressed, and purified. Novel functionality was shown in multiple PolAs. Intriguingly, some of the estuarine viral polymerases demonstrated moderate to strong innate DNA strand displacement activity at high enzyme concentration. Strand-displacing polymerases have important technological applications where isothermal reactions are desirable. Bioinformatic investigation of genes neighboring these strand displacing polymerases found associations with SNF2 helicase-associated proteins. The specific function of SNF2 family enzymes is unknown for prokaryotes and viruses. In eukaryotes, SNF2 enzymes have chromatin remodeling functions but do not separate nucleic acid strands. This suggests the strand separation function may be fulfilled by the DNA polymerase for viruses carrying SNF2 helicase-associated proteins. Biochemical data elucidated from this study expands understanding of the biology and ecological behavior of unknown viruses. Moreover, given the numerous biotechnological applications of viral DNA polymerases, novel viral polymerases discovered within viromes may be a rich source of biological material for further DNA amplification advancements.
病毒是地球上数量最多、种类最丰富的生物实体,占地球遗传多样性的很大比例。这种多样性的大部分并非由孤立的病毒-宿主系统所代表,而仅通过对环境样本中的病毒宏基因组(病毒组)进行测序才得以观察到。病毒组提供了病毒遗传潜力的快照,以及关于病毒群落生态学的丰富信息。这些数据还为探索新型病毒酶的生物化学提供了机会。我们探索了新型病毒DNA聚合酶的生化特性,根据蛋白质序列系统发育来测试聚合酶生化方面的假设差异。我们合成、表达并纯化了来自河口病毒组、温泉宏基因组和参考病毒的48种病毒DNA聚合酶I(PolA)蛋白,这些蛋白广泛代表了目前已知的多样性。多种PolA显示出了新的功能。有趣的是,一些河口病毒聚合酶在高酶浓度下表现出中度至强的固有DNA链置换活性。链置换聚合酶在需要等温反应的技术应用中具有重要作用。对这些链置换聚合酶附近基因的生物信息学研究发现它们与SNF2解旋酶相关蛋白有关联。对于原核生物和病毒而言,SNF2家族酶的具体功能尚不清楚。在真核生物中,SNF2酶具有染色质重塑功能,但不分离核酸链。这表明对于携带SNF2解旋酶相关蛋白的病毒,链分离功能可能由DNA聚合酶来完成。这项研究阐明的生化数据扩展了我们对未知病毒生物学和生态行为的理解。此外,鉴于病毒DNA聚合酶有众多生物技术应用,在病毒组中发现的新型病毒聚合酶可能是进一步推动DNA扩增进展的丰富生物材料来源。