Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner.

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Two novel sequence-specific endonucleases have been isolated from Proteus vulgaris, ATCC 13315. PvuI recognizes the sequence: 5' C G A T decrease C G 3' 3' G C increase T A G C 5' and PvuII recognizes the sequence: 5' C A G decrease C T G 3' 3' G T C increase G A C 5' and cleave as indicated by the arrow (decrease). PvuI is an isoschizomer of XorII, RshI, and XniI. No enzyme with the specificity of PvuII has been described previously.

Background: During replication, the lagging strand is synthesized discontinuously from a series of Okazaki fragments.Results: Okazaki fragment maturation was reconstituted using purified proteins from Thermococcus.Conclusion: In Thermococcus, efficient Okazaki fragment processing requires DNA polymerase B, flap endonuclease, and DNA ligase.Significance: Okazaki fragment maturation in Thermococcus shares similarities to both bacterial and eukaryotic systems.

Accurate DNA replication is essential for maintenance of every genome. All archaeal genomes except Crenarchaea, encode for a member of Family B (polB) and Family D (polD) DNA polymerases. Gene deletion studies in Thermococcus kodakaraensis and Methanococcus maripaludis show that polD is the only essential DNA polymerase in these organisms. Thus, polD may be the primary replicative DNA polymerase for both leading and lagging strand synthesis. To understand this unique archaeal enzyme, we report the biochemical characterization of a heterodimeric polD from Thermococcus. PolD contains both DNA polymerase and proofreading 3′–5′ exonuclease activities to ensure efficient and accurate genome duplication. The polD incorporation fidelity was determined for the first time. Despite containing 3′–5′ exonuclease proofreading activity, polD has a relatively high error rate (95 × 10−5) compared to polB (19 × 10−5) and at least 10-fold higher than the polB DNA polymerases from yeast (polε and polδ) or Escherichia coli DNA polIII holoenzyme. The implications of polD fidelity and biochemical properties in leading and lagging strand synthesis are discussed.Electronic supplementary materialThe online version of this article (doi:10.1007/s00792-014-0646-9) contains supplementary material, which is available to authorized users.