Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases

Powers, Kyle T.; Washington, M. Todd

doi:10.1016/bs.mie.2017.04.002

Cited by 12 publications

(15 citation statements)

References 56 publications

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“…The most straightforward model of non-classical polymerase selection is the kinetic sampling model ( 11 ). According to this model, multiple non-classical polymerases directly compete with one another for binding the DNA substrate.…”

Section: Discussionmentioning

confidence: 99%

The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility

Powers

Elcock

Washington

2018

Nucleic Acids Research

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Eukaryotic DNA polymerase η catalyzes translesion synthesis of thymine dimers and 8-oxoguanines. It is comprised of a polymerase domain and a C-terminal region, both of which are required for its biological function. The C-terminal region mediates interactions with proliferating cell nuclear antigen (PCNA) and other translesion synthesis proteins such as Rev1. This region contains a ubiquitin-binding/zinc-binding (UBZ) motif and a PCNA-interacting protein (PIP) motif. Currently little structural information is available for this region of polymerase η. Using a combination of approaches—including genetic complementation assays, X-ray crystallography, Langevin dynamics simulations, and small-angle X-ray scattering—we show that the C-terminal region is partially unstructured and has high conformational flexibility. This implies that the C-terminal region acts as a flexible tether linking the polymerase domain to PCNA thereby increasing its local concentration. Such tethering would facilitate the sampling of translesion synthesis polymerases to ensure that the most appropriate one is selected to bypass the lesion.

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Section: Discussionmentioning

confidence: 99%

The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility

Powers

Elcock

Washington

2018

Nucleic Acids Research

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“…Pre-steady-state kinetic parameters of tc TERT were obtained using established pre-steady-state kinetics protocols for DNA polymerases, also known as single turnover kinetics 70 , 71 . Briefly, we preincubated 2 μM tc TERT with 200 nM annealed DNA:RNA hybrid substrate, with a 6-FAM label on the 5′ end of the DNA component.…”

Section: Methodsmentioning

confidence: 99%

Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs

et al. 2020

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Telomerase is a specialized reverse transcriptase that adds GGTTAG repeats to chromosome ends and is upregulated in most human cancers to enable limitless proliferation. Here, we uncover two distinct mechanisms by which naturally occurring oxidized dNTPs and therapeutic dNTPs inhibit telomerase-mediated telomere elongation. We conduct a series of direct telomerase extension assays in the presence of modified dNTPs on various telomeric substrates. We provide direct evidence that telomerase can add the nucleotide reverse transcriptase inhibitors ddITP and AZT-TP to the telomeric end, causing chain termination. In contrast, telomerase continues elongation after inserting oxidized 2-OH-dATP or therapeutic 6-thio-dGTP, but insertion disrupts translocation and inhibits further repeat addition. Kinetics reveal that telomerase poorly selects against 6-thio-dGTP, inserting with similar catalytic efficiency as dGTP. Furthermore, telomerase processivity factor POT1-TPP1 fails to restore processive elongation in the presence of inhibitory dNTPs. These findings reveal mechanisms for targeting telomerase with modified dNTPs in cancer therapy.

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“…Simultaneous rather than sequential or competitive binding of TLS polymerases to PCNA would allow them to act together to bypass DNA lesions and would facilitate the selection of the appropriate polymerase and polymerase switching events [ 72 , 73 ]. The selection of the appropriate polymerase is most likely determined by the kinetics of nucleotide incorporation, as described by the ‘kinetic selection’ model [ 74 ]. While PCNA monoubiquitination does not interfere with the binding of Pol δ or RFC (replication factor C) and thus allows for the co-existence of TLS and replicative polymerases on the PCNA clamp, it hinders the binding of FEN1 [ 46 ], consistent with the existence of additional FEN1 contacts with PCNA that extend outside of its PIP-box [ 38 , 43 ].…”

Section: Protein Interaction Interfaces On Proliferating Cell Nuclmentioning

confidence: 99%

Maneuvers on PCNA Rings during DNA Replication and Repair

Slade

2018

Genes

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DNA replication and repair are essential cellular processes that ensure genome duplication and safeguard the genome from deleterious mutations. Both processes utilize an abundance of enzymatic functions that need to be tightly regulated to ensure dynamic exchange of DNA replication and repair factors. Proliferating cell nuclear antigen (PCNA) is the major coordinator of faithful and processive replication and DNA repair at replication forks. Post-translational modifications of PCNA, ubiquitination and acetylation in particular, regulate the dynamics of PCNA-protein interactions. Proliferating cell nuclear antigen (PCNA) monoubiquitination elicits ‘polymerase switching’, whereby stalled replicative polymerase is replaced with a specialized polymerase, while PCNA acetylation may reduce the processivity of replicative polymerases to promote homologous recombination-dependent repair. While regulatory functions of PCNA ubiquitination and acetylation have been well established, the regulation of PCNA-binding proteins remains underexplored. Considering the vast number of PCNA-binding proteins, many of which have similar PCNA binding affinities, the question arises as to the regulation of the strength and sequence of their binding to PCNA. Here I provide an overview of post-translational modifications on both PCNA and PCNA-interacting proteins and discuss their relevance for the regulation of the dynamic processes of DNA replication and repair.

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Analyzing the Catalytic Activities and Interactions of Eukaryotic Translesion Synthesis Polymerases

Cited by 12 publications

References 56 publications

The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility

The C-terminal region of translesion synthesis DNA polymerase η is partially unstructured and has high conformational flexibility

Mechanisms of telomerase inhibition by oxidized and therapeutic dNTPs

Maneuvers on PCNA Rings during DNA Replication and Repair

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