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Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primasea.

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Title: Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primasea.
Authors: VaithiyaIingam, Sivaraja1,2, Warren, Eric M.2,3, Eichman, Brandt F.1,2,3 walter.chazin@vanderbitt.edu, Chazin, Walter J.1,2,4 brandteichman@vanderbilt.edu
Source: Proceedings of the National Academy of Sciences of the United States of America. 8/3/2010, Vol. 107 Issue 31, p13684-13689. 6p.
Subjects: DNA replication, Eukaryotic cells, DNA primers, X-ray crystallography, Anisotropy, Nuclear magnetic resonance, Mutagenesis
Abstract: DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-A resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionarily conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysis of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/ dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints frorn NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated 55DNA and regulation of primase activity. [ABSTRACT FROM AUTHOR]
Copyright of Proceedings of the National Academy of Sciences of the United States of America is the property of National Academy of Sciences and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)
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  Data: Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primasea.
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  Data: <searchLink fieldCode="DE" term="%22DNA+replication%22">DNA replication</searchLink><br /><searchLink fieldCode="DE" term="%22Eukaryotic+cells%22">Eukaryotic cells</searchLink><br /><searchLink fieldCode="DE" term="%22DNA+primers%22">DNA primers</searchLink><br /><searchLink fieldCode="DE" term="%22X-ray+crystallography%22">X-ray crystallography</searchLink><br /><searchLink fieldCode="DE" term="%22Anisotropy%22">Anisotropy</searchLink><br /><searchLink fieldCode="DE" term="%22Nuclear+magnetic+resonance%22">Nuclear magnetic resonance</searchLink><br /><searchLink fieldCode="DE" term="%22Mutagenesis%22">Mutagenesis</searchLink>
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  Data: DNA replication requires priming of DNA templates by enzymes known as primases. Although DNA primase structures are available from archaea and bacteria, the mechanism of DNA priming in higher eukaryotes remains poorly understood in large part due to the absence of the structure of the unique, highly conserved C-terminal regulatory domain of the large subunit (p58C). Here, we present the structure of this domain determined to 1.7-A resolution by X-ray crystallography. The p58C structure reveals a novel arrangement of an evolutionarily conserved 4Fe-4S cluster buried deeply within the protein core and is not similar to any known protein structure. Analysis of the binding of DNA to p58C by fluorescence anisotropy measurements revealed a strong preference for ss/ dsDNA junction substrates. This approach was combined with site-directed mutagenesis to confirm that the binding of DNA occurs to a distinctively basic surface on p58C. A specific interaction of p58C with the C-terminal domain of the intermediate subunit of replication protein A (RPA32C) was identified and characterized by isothermal titration calorimetry and NMR. Restraints frorn NMR experiments were used to drive computational docking of the two domains and generate a model of the p58C-RPA32C complex. Together, our results explain functional defects in human DNA primase mutants and provide insights into primosome loading on RPA-coated 55DNA and regulation of primase activity. [ABSTRACT FROM AUTHOR]
– Name: AbstractSuppliedCopyright
  Label:
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  Data: <i>Copyright of Proceedings of the National Academy of Sciences of the United States of America is the property of National Academy of Sciences and its content may not be copied or emailed to multiple sites without the copyright holder's express written permission. Additionally, content may not be used with any artificial intelligence tools or machine learning technologies. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract.</i> (Copyright applies to all Abstracts.)
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        Value: 10.1073/pnas.1002009107
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        Text: English
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      – SubjectFull: DNA replication
        Type: general
      – SubjectFull: Eukaryotic cells
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      – SubjectFull: DNA primers
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      – SubjectFull: X-ray crystallography
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      – SubjectFull: Anisotropy
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      – SubjectFull: Nuclear magnetic resonance
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      – SubjectFull: Mutagenesis
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      – TitleFull: Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primasea.
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              Text: 8/3/2010
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