An iron-sulfur cluster in the C-terminal domain of the p58 subunit of human DNA primase.
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.
Weiner
Brian E
BE
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, 37232; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232.
Huang
Hao
H
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, 37232.
Dattilo
Brian M
BM
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, 37232; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232.
Nilges
Mark J
MJ
Illinois EPR Research Center, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801.
Fanning
Ellen
E
Department of Biological Sciences, Vanderbilt University, Nashville, Tennessee, 37232.
Chazin
Walter J
WJ
Department of Biochemistry, Vanderbilt University, Nashville, Tennessee, 37232; Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37232; Department of Chemistry, Vanderbilt University, Nashville, Tennessee, 37232. Electronic address: walter.chazin@vanderbilt.edu.
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry; D004247:DNA / Q000737:chemistry; D019915:DNA Primase / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry / Q000379:methods; D013056:Spectrophotometry, Ultraviolet
D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D004247:DNA; D019915:DNA Primase; C525356:PRIM2 protein, human; D003545:Cysteine