Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

Agudo, Rubén; Calvo, Patricia A; Martínez‐Jiménez, María I.; Blanco, Luis

doi:10.1093/nar/gkx633

Cited by 10 publications

(7 citation statements)

References 58 publications

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“…For this aim, we attemped to establish an assay to quantify RNA synthesis activity as the relative increase in fluorescence emitted by SYBR Green II dye after binding to dsRNA. This procedure was adapted from methods previously described to detect dsDNA synthesis by the human primase-polymerase PrimPol 38 . We anticipated that binding of this intercalating agent to dsRNA generated by ZIKV RdRp polymerization activity would lead to an increase in the emitted fluorescence.…”

Section: Resultsmentioning

confidence: 99%

“…S1). Previous studies have documented that an excess of SYBR Green I, chemically related to SYBR Green II, can inhibit other polymerase activities, such as those of Taq polymerase 43 or human PrimPol 38 . Our resulted suggested that SYBR Green II acts as an inhibitor of ZIKV RdRp activity.…”

Section: Resultsmentioning

confidence: 99%

“…The assay records the synthesis of dsRNA in a reaction using a poly-U molecule as a template and ATP as the nucleotide substrate. This technique has been adapted from methods previously documented for the detection of DNA synthesis 38 .…”

Section: Methodsmentioning

confidence: 99%

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Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Sáez-Álvarez

Arias

Águila

et al. 2019

Sci Rep

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Zika virus (ZIKV) is an emerging pathogen that has been associated with large numbers of cases of severe neurologic disease, including Guillain-Barré syndrome and microcephaly. Despite its recent establishment as a serious global public health concern there are no licensed therapeutics to control this virus. Accordingly, there is an urgent need to develop methods for the high-throughput screening of antiviral agents. We describe here a fluorescence-based method to monitor the real-time polymerization activity of Zika virus RNA-dependent RNA polymerase (RdRp). By using homopolymeric RNA template molecules, de novo RNA synthesis can be detected with a fluorescent dye, which permits the specific quantification and kinetics of double-strand RNA formation. ZIKV RdRp activity detected using this fluorescence-based assay positively correlated with traditional assays measuring the incorporation of radiolabeled nucleotides. We also validated this method as a suitable assay for the identification of ZIKV inhibitors targeting the viral polymerase using known broad-spectrum inhibitors. The assay was also successfully adapted to detect RNA polymerization activity by different RdRps, illustrated here using purified RdRps from hepatitis C virus and foot-and-mouth disease virus. The potential of fluorescence-based approaches for the enzymatic characterization of viral polymerases, as well as for high-throughput screening of antiviral drugs, are discussed.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Sáez-Álvarez

Arias

Águila

et al. 2019

Sci Rep

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“…Therefore, the Y89D mutation appears to be structurally conservative, explaining the moderate effect of this alteration on PrimPol in vitro activities and the absence of an effect on the tested PrimPol in vivo activities. Interestingly, a single mutation of Tyr 89 to Arg (Y89R), a change that is invariantly present in the WxRY motif of plant PrimPols (see Supplementary Figure S1 ), enhanced the capacity of human PrimPol as an efficient RNA-dependent-DNA primase/polymerase ( 34 ). Strikingly, this randomly selected mutation (Y89R) displayed an increased stabilization of the preternary complex (protein:template DNA:incoming nucleotide), the specific step preceding dimer formation.…”

Section: Resultsmentioning

confidence: 99%

Motif WFYY of human PrimPol is crucial to stabilize the incoming 3′-nucleotide during replication fork restart

Calvo

Martínez‐Jiménez

Díaz

et al. 2021

Nucleic Acids Research

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PrimPol is the second primase in human cells, the first with the ability to start DNA chains with dNTPs. PrimPol contributes to DNA damage tolerance by restarting DNA synthesis beyond stalling lesions, acting as a TLS primase. Multiple alignment of eukaryotic PrimPols allowed us to identify a highly conserved motif, WxxY near the invariant motif A, which contains two active site metal ligands in all members of the archeo-eukaryotic primase (AEP) superfamily. In vivo and in vitro analysis of single variants of the WFYY motif of human PrimPol demonstrated that the invariant Trp87 and Tyr90 residues are essential for both primase and polymerase activities, mainly due to their crucial role in binding incoming nucleotides. Accordingly, the human variant F88L, altering the WFYY motif, displayed reduced binding of incoming nucleotides, affecting its primase/polymerase activities especially during TLS reactions on UV-damaged DNA. Conversely, the Y89D mutation initially associated with High Myopia did not affect the ability to rescue stalled replication forks in human cells. Collectively, our data suggest that the WFYY motif has a fundamental role in stabilizing the incoming 3′-nucleotide, an essential requisite for both its primase and TLS abilities during replication fork restart.

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“…Although partial kinetic characterizations have been reported [23,27,28], a complete kinetic pathway of PrimPol-mediated DNA elongation and the rate-limiting step(s) of the reaction are unknown. Such information is not only fundamental for further understanding the enzymatic activities of PrimPol and other primase-polymerases, but also useful for exploring novel approaches to modulate PrimPol's activities for biotechnological or therapeutic applications [25,29,30]. In this study, we conducted in-depth kinetic analyses and computer simulations to dissect the elemental steps of PrimPol's DNA polymerase activity.…”

Section: Introductionmentioning

confidence: 99%

Divalent Cations Alter the Rate-Limiting Step of PrimPol-Catalyzed DNA Elongation

Zhao

Morehouse

et al. 2019

Journal of Molecular Biology

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PrimPol is the most recently discovered human DNA polymerase/primase and plays an emerging role in nuclear and mitochondrial genomic maintenance. As a member of archaeo-eukaryotic primase (AEP) superfamily enzymes, PrimPol possesses DNA polymerase and primase activities that are important for replication fork progression in vitro and in cellulo. The enzymatic activities of PrimPol are critically dependent on the nucleotidyl-transfer reaction to incorporate deoxyribonucleotides successively; however, our knowledge concerning the kinetic mechanism of the reaction remains incomplete. Using enzyme kinetic analyses and computer simulations, we dissected the mechanism by which PrimPol transfers a nucleotide to a primer-template DNA, which comprises DNA binding, conformational transition, nucleotide binding, phosphoester bond formation, and dissociation steps. We obtained the rate constants of the steps by steady-state and pre-steady-state kinetic analyses and simulations. Our data demonstrate that the rate-limiting step of PrimPol-catalyzed DNA elongation depends on the metal cofactor involved. In the presence of Mn 2+ , a conformational transition step from non-productive to productive PrimPol:DNA complexes limits the enzymatic turnover, whereas, in the presence of Mg 2+ , the chemical step becomes rate-limiting. As evidenced from our kinetic and simulation data, PrimPol maintains the same kinetic mechanism under either millimolar or physiological micromolar Mn 2+ concentration. Our study revealed the underlying mechanism by which PrimPol catalyzes nucleotide incorporation with two common metal cofactors, and provides a kinetic basis for further understanding the regulatory mechanism of this functionally diverse primase-polymerase.

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Engineering human PrimPol into an efficient RNA-dependent-DNA primase/polymerase

Cited by 10 publications

References 58 publications

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Development of a fluorescence-based method for the rapid determination of Zika virus polymerase activity and the screening of antiviral drugs

Motif WFYY of human PrimPol is crucial to stabilize the incoming 3′-nucleotide during replication fork restart

Divalent Cations Alter the Rate-Limiting Step of PrimPol-Catalyzed DNA Elongation

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