An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human DNA primase.

Weiner BE, Huang H, Dattilo BM, Nilges MJ, Fanning E, Chazin WJ
J Biol Chem. 2007 282 (46): 33444-51

PMID: 17893144 · DOI:10.1074/jbc.M705826200

DNA primase synthesizes short RNA primers that are required to initiate DNA synthesis on the parental template strands during DNA replication. Eukaryotic primase contains two subunits, p48 and p58, and is normally tightly associated with DNA polymerase alpha. Despite the fundamental importance of primase in DNA replication, structural data on eukaryotic DNA primase are lacking. The p48/p58 dimer was subjected to limited proteolysis, which produced two stable structural domains: one containing the bulk of p48 and the other corresponding to the C-terminal fragment of p58. These domains were identified by mass spectrometry and N-terminal sequencing. The C-terminal p58 domain (p58C) was expressed, purified, and characterized. CD and NMR spectroscopy experiments demonstrated that p58C forms a well folded structure. The protein has a distinctive brownish color, and evidence from inductively coupled plasma mass spectrometry, UV-visible spectrophotometry, and EPR spectroscopy revealed characteristics consistent with the presence of a [4Fe-4S] high potential iron protein cluster. Four putative cysteine ligands were identified using a multiple sequence alignment, and substitution of just one was sufficient to cause loss of the iron-sulfur cluster and a reduction in primase enzymatic activity relative to the wild-type protein. The discovery of an iron-sulfur cluster in DNA primase that contributes to enzymatic activity provides the first suggestion that the DNA replication machinery may have redox-sensitive activities. Our results offer new horizons in which to investigate the function of high potential [4Fe-4S] clusters in DNA-processing machinery.

MeSH Terms (15)

Amino Acid Sequence Circular Dichroism Cysteine DNA DNA Primase Electron Spin Resonance Spectroscopy Humans Iron-Sulfur Proteins Magnetic Resonance Spectroscopy Molecular Sequence Data Protein Structure, Tertiary Recombinant Proteins Sequence Homology, Amino Acid Spectrophotometry Spectrophotometry, Ultraviolet

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