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Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ?.

ABSTRACT: Numerous genetic studies have provided compelling evidence to establish DNA polymerase ? (Pol?) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Pol? is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'?5' exonuclease domain common to many replicative polymerases. In addition, Pol? possesses three small subunits that lack a known catalytic activity but associate with components involved in a variety of DNA replication and maintenance processes. Previous enzymatic characterization of the Pol? heterotetramer from budding yeast suggested that the small subunits slightly enhance DNA synthesis by Pol? in vitro. However, similar studies of the human Pol? heterotetramer (hPol?) have been limited by the difficulty of obtaining hPol? in quantities suitable for thorough investigation of its catalytic activity. Utilization of a baculovirus expression system for overexpression and purification of hPol? from insect host cells has allowed for isolation of greater amounts of active hPol?, thus enabling a more detailed kinetic comparison between hPol? and an active N-terminal fragment of the hPol? catalytic subunit (p261N), which is readily overexpressed in Escherichia coli. Here, we report the first pre-steady-state studies of fully-assembled hPol?. We observe that the small subunits increase DNA binding by hPol? relative to p261N, but do not increase processivity during DNA synthesis on a single-stranded M13 template. Interestingly, the 3'?5' exonuclease activity of hPol? is reduced relative to p261N on matched and mismatched DNA substrates, indicating that the presence of the small subunits may regulate the proofreading activity of hPol? and sway hPol? toward DNA synthesis rather than proofreading.

SUBMITTER: Zahurancik WJ

PROVIDER: S-EPMC4426069 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ.

Zahurancik Walter J WJ Baranovskiy Andrey G AG Tahirov Tahir H TH Suo Zucai Z

DNA repair 20150131

Numerous genetic studies have provided compelling evidence to establish DNA polymerase ɛ (Polɛ) as the primary DNA polymerase responsible for leading strand synthesis during eukaryotic nuclear genome replication. Polɛ is a heterotetramer consisting of a large catalytic subunit that contains the conserved polymerase core domain as well as a 3'→5' exonuclease domain common to many replicative polymerases. In addition, Polɛ possesses three small subunits that lack a known catalytic activity but ass ...[more]

PMID: 25684708

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Project description:Pseudorabies virus (PRV) DNA replication occurs in the nuclei of infected cells and requires the viral DNA polymerase. The PRV DNA polymerase comprises a catalytic subunit, UL30, and an accessory subunit, UL42, that confers processivity to the enzyme. Its nuclear localization is a prerequisite for its enzymatic function in the initiation of viral DNA replication. However, the mechanisms by which the PRV DNA polymerase holoenzyme enters the nucleus have not been determined. In this study, we characterized the nuclear import pathways of the PRV DNA polymerase catalytic and accessory subunits. Immunofluorescence analysis showed that UL42 localizes independently in the nucleus, whereas UL30 alone predominantly localizes in the cytoplasm. Intriguingly, the localization of UL30 was completely shifted to the nucleus when it was coexpressed with UL42, demonstrating that nuclear transport of UL30 occurs in an UL42-dependent manner. Deletion analysis and site-directed mutagenesis of the two proteins showed that UL42 contains a functional and transferable bipartite nuclear localization signal (NLS) at amino acids 354-370 and that K(354), R(355), and K(367) are important for the NLS function, whereas UL30 has no NLS. Coimmunoprecipitation assays verified that UL42 interacts with importins α3 and α4 through its NLS. In vitro nuclear import assays demonstrated that nuclear accumulation of UL42 is a temperature- and energy-dependent process and requires both importins α and β, confirming that UL42 utilizes the importin α/β-mediated pathway for nuclear entry. In an UL42 NLS-null mutant, the UL42/UL30 heterodimer was completely confined to the cytoplasm when UL42 was coexpressed with UL30, indicating that UL30 utilizes the NLS function of UL42 for its translocation into the nucleus. Collectively, these findings suggest that UL42 contains an importin α/β-mediated bipartite NLS that transports the viral DNA polymerase holoenzyme into the nucleus in an in vitro expression system.

Dataset Information

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ?.

Publications

Comparison of the kinetic parameters of the truncated catalytic subunit and holoenzyme of human DNA polymerase ɛ.

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