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An iron-sulfur domain of the eukaryotic primase is essential for RNA primer synthesis

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Abstract

Primases synthesize the RNA primers that are necessary for replication of the parental DNA strands. Here we report that the heterodimeric archaeal/eukaryotic primase is an iron-sulfur (Fe-S) protein. Binding of the Fe-S cluster is mediated by an evolutionarily conserved domain at the C terminus of the large subunit. We further show that the Fe-S domain is essential to the unique ability of the eukaryotic primase to start DNA replication.

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Figure 1: The archaeal/eukaryotic primase is a Fe-S protein.
Figure 2: Functional analysis of the S. cerevisiae primase.

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Acknowledgements

We thank R. Nookala and M. Lyst for early work on the S. solfataricus PriL-CTD, J. Crack (University of East Anglia) for advice on iron-sulfur biochemistry, J. Downs (University of Sussex) for the gift of yeast genomic DNA, I. Gomez-Morillo for help with the microPIXE analysis, S.C. Onuoha for help with the CD analysis, O. Zaid for critical reading of the manuscript and S.D. Bell for comments and advice. S.K. is funded by the Gates Cambridge Trust and the Medical Research Council. This work was supported by a Wellcome Trust Senior Fellowship in Basic Biomedical Sciences to L.P.

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Authors and Affiliations

  1. Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA, UK

    Sebastian Klinge, Joseph D Maman & Luca Pellegrini

  2. Department of Zoology, University of Cambridge, Downing Street, Cambridge, CB2 3EJ, UK

    Sebastian Klinge & Torsten Krude

  3. Medical Research Council Dunn Human Nutrition Unit, Wellcome Trust/Medical Research Council Building, Hills Road, Cambridge, CB2 2XY, UK

    Judy Hirst

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  1. Sebastian Klinge
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  2. Judy Hirst
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  3. Joseph D Maman
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  4. Torsten Krude
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  5. Luca Pellegrini
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Contributions

S.K. and L.P. conceived the project and designed the experiments; S.K. prepared the recombinant proteins and performed the biochemical experiments; J.H. performed the EPR analysis; J.D.M. first suggested that the chromophore in the PriL-CTD might be a Fe-S cluster and performed the CD analysis; T.K. assisted with the primase assays and S.K. and L.P. interpreted the data and wrote the paper.

Corresponding author

Correspondence to Luca Pellegrini.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Tables 1 and 2, Supplementary Methods (PDF 6016 kb)

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Klinge, S., Hirst, J., Maman, J. et al. An iron-sulfur domain of the eukaryotic primase is essential for RNA primer synthesis. Nat Struct Mol Biol 14, 875–877 (2007). https://doi.org/10.1038/nsmb1288

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  • DOI: https://doi.org/10.1038/nsmb1288

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