An Iron-Sulfur Cluster in the C-terminal Domain of the p58 Subunit of Human DNA Primase

Abstract

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 ␣. 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 wildtype 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.DNA polymerase ␣-primase (pol-prim) 2 associates with eukaryotic replication forks in S-phase during the initiation of DNA replication (1, 2). pol-prim synthesizes a chimeric RNA-DNA primer of ϳ30 nucleotides that is then extended by more processive DNA polymerases that synthesize the leading and lagging strands. pol-prim is composed of four subunits (p180, p68, p58, and p48). The p180 subunit has the DNA polymerase catalytic activity and binds to both the p68 and p58 subunits. The p68 subunit has a regulatory function that is not completely understood. It is required for initiation of yeast chromosomal replication (3, 4) and cell-free SV40 DNA replication (5). In addition, phosphorylation of p68 alters the activity of polprim in SV40 replication (6 -9).The two smallest subunits, p48 and p58, together function as the DNA primase by creating an RNA primer of 7-10 nucleotides (10, 11). The p48 subunit contains the catalytic site (12). The p58 subunit stabilizes p48 and participates in initiation, elongation, and "counting" the ribonucleotides polymerized (13). Interestingly, p58 is also involved in transferring the RNA strand directly into the active site of the associated p180 subunit, which extends the growing nucleotide with dNTPs to complete the formation of the RNA-DNA primer (1, 14, 15). Knowledge of the molecular basis for regulation of the length of RNA portion of the primer an...

DNA Polymerase δ and ζ Switch by Sharing Accessory Subunits of DNA Polymerase δ

“…S1) (26,27). Such stoichiometry is close to what is typically observed for proteins containing [4Fe-4S] clusters, taking into account that one or two iron atoms per molecule were lost during purification (28,29). Our data are consistent with the recent finding of [4Fe-4S] clusters in yeast Pol ␦ and Pol (30), in human and yeast primases (29,31), and in several proteins involved in DNA repair (26 -28, 32).…”

Structures of human primase reveal design of nucleotide elongation site and mode of Pol α tethering

“…5B). In the Pol α/primase complex, residues 1,447-SEVN-1,450 of Pol α form an antiparallel β-strand that pairs with residues 35-ENIS-38 of PriL, whereas residues 1,451-LSKLF-1,455 fold in one turn of 3 10 helix that packs against the last two turns of PriL helix α3. Hot-spot residues that were shown to be important for primase binding, such as F1455 and L1451, become buried at the hydrophobic proteinpeptide interface.…”

“…Although the primary active site for RNA primer synthesis resides in the PriS subunit, PriL is important for regulation of primase activity and primer transfer to Pol α (5-8). Further insight into PriL function was provided by the observation that a conserved C-terminal Fe-S domain of PriL, PriL-CTD, is essential for initiation of primer synthesis, but dispensable for its subsequent elongation (9)(10)(11)(12)(13).…”

Formation of dAMP-glycerol and dAMP-Tris Derivatives by Thermococcus kodakaraensis DNA Primase

“…Similar to eukaryotic DNA primases, the T. kodakaraensis primase is a two-subunit complex (p41-p46) in which the catalytic activity resides within the small subunit. The large subunit of both primases (T. kodakaraensis p46 and eukaryotic p58) contains a Fe-S cluster (2,3), and sequence alignments between the two enzymes reveal homologies between both small and large subunits (4). In the small subunit, the region around the catalytic aspartate residues of the eukaryotic primase closely resemble the archaeal primase, suggesting that it is biologically important.…”