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 α. 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. 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 α. 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. DNA polymerase α-primase (pol-prim) 2The abbreviations used are: pol-prim, DNA polymerase α-primase; p58C, C-terminal domain of p58; HiPIP, high potential iron protein; BME, 2-mer-captoethanol; HSQC, heteronuclear single quantum coherence; MES, 4-morpholineethanesulfonic acid; ssDNA, single-stranded DNA. associates with eukaryotic replication forks in S-phase during the initiation of DNA replication (1Hubscher U. Maga G. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (588) Google Scholar, 2Garg P. Burgers P.M. Crit. Rev. Biochem. Mol. Biol. 2005; 40: 115-128Crossref PubMed Scopus (215) Google Scholar). pol-prim synthesizes a chimeric RNA-DNA primer of ∼30 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 (3Foiani M. Marini F. Gamba D. Lucchini G. Plevani P. Mol. Cell. Biol. 1994; 14: 923-933Crossref PubMed Google Scholar, 4Foiani M. Liberi G. Lucchini G. Plevani P. Mol. Cell. Biol. 1995; 15: 883-891Crossref PubMed Google Scholar) and cell-free SV40 DNA replication (5Ott R.D. Rehfuess C. Podust V.N. Clark J.E. Fanning E. Mol. Cell. Biol. 2002; 22: 5669-5678Crossref PubMed Scopus (22) Google Scholar). In addition, phosphorylation of p68 alters the activity of polprim in SV40 replication (6Schub O. Rohaly G. Smith R.W. Schneider A. Dehde S. Dornreiter I. Nasheuer H.P. J. Biol. Chem. 2001; 276: 38076-38083Abstract Full Text Full Text PDF PubMed Google Scholar, 7Uchiyama M. Wang T.S. Mol. Cell. Biol. 2004; 24: 7419-7434Crossref PubMed Scopus (21) Google Scholar, 8Voitenleitner C. Fanning E. Nasheuer H.P. Oncogene. 1997; 14: 1611-1615Crossref PubMed Scopus (62) Google Scholar, 9Voitenleitner C. Rehfuess C. Hilmes M. O'Rear L. Liao P.C. Gage D.A. Ott R. Nasheuer H.P. Fanning E. Mol. Cell. Biol. 1999; 19: 646-656Crossref PubMed Google Scholar). The two smallest subunits, p48 and p58, together function as the DNA primase by creating an RNA primer of 7–10 nucleotides (10Arezi B. Kuchta R.D. Trends Biochem. Sci. 2000; 25: 572-576Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 11Frick D.N. Richardson C.C. Annu. Rev. Biochem. 2001; 70: 39-80Crossref PubMed Scopus (305) Google Scholar). The p48 subunit contains the catalytic site (12Schneider A. Smith R.W. Kautz A.R. Weisshart K. Grosse F. Nasheuer H.P. J. Biol. Chem. 1998; 273: 21608-21615Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The p58 subunit stabilizes p48 and participates in initiation, elongation, and “counting” the ribonucleotides polymerized (13Zerbe L.K. Kuchta R.D. Biochemistry. 2002; 41: 4891-4900Crossref PubMed Scopus (71) Google Scholar). 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 (1Hubscher U. Maga G. Spadari S. Annu. Rev. Biochem. 2002; 71: 133-163Crossref PubMed Scopus (588) Google Scholar, 14Arezi B. Kirk B.W. Copeland W.C. Kuchta R.D. Biochemistry. 1999; 38: 12899-12907Crossref PubMed Scopus (39) Google Scholar, 15Copeland W.C. Wang T.S. J. Biol. Chem. 1993; 268: 26179-26189Abstract Full Text PDF PubMed Google Scholar). Knowledge of the molecular basis for regulation of the length of RNA portion of the primer and internal transfer to the p180 subunit is very limited. Despite the fundamental importance of primase in DNA replication, the only structural information available for a heterodimeric primase is for an archaeal (Sulfolobus solfataricus) primase that does not form a pol-prim complex (16Lao-Sirieix S.H. Nookala R.K. Roversi P. Bell S.D. Pellegrini L. Nat. Struct. Mol. Biol. 2005; 12: 1137-1144Crossref PubMed Scopus (55) Google Scholar, 17Lao-Sirieix S.H. Pellegrini L. Bell S.D. Trends Genet. 2005; 21: 568-572Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Multiple sequence alignments reveal homology between the p48 subunits and the N-terminal half of the p58 subunit. In the crystal structure of the S. solfataricus primase core, the p48 subunit assembles with the N-terminal half of p58 (16Lao-Sirieix S.H. Nookala R.K. Roversi P. Bell S.D. Pellegrini L. Nat. Struct. Mol. Biol. 2005; 12: 1137-1144Crossref PubMed Scopus (55) Google Scholar). However, in human p58, both the N- and C-terminal regions have contacts with p48 (18Copeland W.C. Protein Expr. Purif. 1997; 9: 1-9Crossref PubMed Scopus (26) Google Scholar). Interestingly, the C-terminal half of p58 also contains a region with homology to a DNA polymerase β domain; this region was determined to be important for primer synthesis (13Zerbe L.K. Kuchta R.D. Biochemistry. 2002; 41: 4891-4900Crossref PubMed Scopus (71) Google Scholar), but how it functions is not known. DNA primase serves as a key target for regulation of DNA replication initiation, telomere maintenance, and response to DNA damage or fork stalling, in part through its physical interactions with other proteins involved in DNA replication and in checkpoint signaling (19Foiani M. Lucchini G. Plevani P. Trends Biochem. Sci. 1997; 22: 424-427Abstract Full Text PDF PubMed Scopus (83) Google Scholar). Primase interacts physically with the viral helicase SV40 large T antigen, eukaryotic replication protein A (20Dornreiter I. Erdile L.F. Gilbert I.U. von Winkler D. Kelly T.J. Fanning E. EMBO J. 1992; 11: 769-776Crossref PubMed Scopus (285) Google Scholar, 21Weisshart K. Forster H. Kremmer E. Schlott B. Grosse F. Nasheuer H.P. J. Biol. Chem. 2000; 275: 17328-17337Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), and GINS, a recently identified component that plays a central role in establishment and progression of eukaryotic and archaeal replication forks (22De Falco M. Ferrari E. De Felice M. Rossi M. Hubscher U. Pisani F.M. EMBO Rep. 2007; 8: 99-103Crossref PubMed Scopus (46) Google Scholar, 23Kamada K. Kubota Y. Arata T. Shindo Y. Hanaoka F. Nat. Struct. Mol. Biol. 2007; 14: 388-396Crossref PubMed Scopus (75) Google Scholar, 24Labib K. Gambus A. Trends Cell Biol. 2007; 17: 271-278Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Primase activity is essential for optimal checkpoint signaling at stalled replication forks (25Michael W.M. Ott R. Fanning E. Newport J. Science. 2000; 289: 2133-2137Crossref PubMed Scopus (160) Google Scholar, 26Byun T.S. Pacek M. Yee M.C. Walter J.C. Cimprich K.A. Genes Dev. 2005; 19: 1040-1052Crossref PubMed Scopus (571) Google Scholar, 27Cortez D. Genes Dev. 2005; 19: 1007-1012Crossref PubMed Scopus (72) Google Scholar, 28Paulsen R.D. Cimprich K.A. DNA Repair (Amst.). 2007; 6: 953-966Crossref PubMed Scopus (207) Google Scholar) and possibly in rescuing stalled replication fork progression (29Lambert S. Froget B. Carr A.M. DNA Repair (Amst.). 2007; 6: 1042-1061Crossref PubMed Scopus (97) Google Scholar), but its interaction partners are not known. To better understand the role of human primase in these pathways, it will be vital to elucidate its structure and interactions with partner proteins. This strategy has been useful in determining the roles of the SV40 large T antigen-replication protein A interaction in the context of SV40 DNA replication (30Arunkumar A.I. Klimovich V. Jiang X. Ott R.D. Mizoue L. Fanning E. Chazin W.J. Nat. Struct. Mol. Biol. 2005; 12: 332-339Crossref PubMed Scopus (68) Google Scholar, 31Jiang X. Klimovich V. Arunkumar A.I. Hysinger E.B. Wang Y. Ott R.D. Guler G.D. Weiner B. Chazin W.J. Fanning E. EMBO J. 2006; 25: 5516-5526Crossref PubMed Scopus (59) Google Scholar). To facilitate similar experiments with human DNA primase, we sought to characterize the domain architecture of DNA primase. The p58 and p48 subunits can be expressed and purified independently of the other two subunits and retain primase activity in vitro at levels similar to those observed for the intact heterotetramer (12Schneider A. Smith R.W. Kautz A.R. Weisshart K. Grosse F. Nasheuer H.P. J. Biol. Chem. 1998; 273: 21608-21615Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 18Copeland W.C. Protein Expr. Purif. 1997; 9: 1-9Crossref PubMed Scopus (26) Google Scholar). Working from bacterially expressed primase protein, a structured domain in the C terminus of the p58 subunit (p58C) was identified. Biophysical analysis of this that the domain is folded and has the characteristics of a 4Fe-4S high potential iron protein The of four a role for the and this was by in vitro experiments that that the 4Fe-4S cluster is required for primase Primase human p48/p58 primase used in this has been (18Copeland W.C. Protein Expr. Purif. 1997; 9: 1-9Crossref PubMed Scopus (26) Google Scholar). p48 was using the dimer as the and it was then into the for which contains an N-terminal A (p58C) was from the dimer into using and This also contains an N-terminal were in the p58C by and by DNA sequencing. fragment of p58C containing the was then used to the corresponding wild-type fragment in the dimer p48/p58 Protein and was expressed in were at in to an of The was then to and the were to for was using were by were in containing of and one were by at was by The primase were purified using The proteins were using a from to containing the primase were and at into containing and The was then purified using a in the and with a to and p48 primase were into containing and using were at using a of the of the an was with and for The were then on the of the were by of were and for and mass spectrometry or were to and for was into and The protein was The CD was at from to using a structure was using the P. F. Protein 1993; 6: PubMed Scopus Google Scholar). p58C, the protein was expressed in with as the The protein was into BME, and heteronuclear single quantum were at on a with a A of was in the for of the in the The data were using F. S. G. J. A. 1995; 6: PubMed Scopus Google Scholar) and and using J. 1994; PubMed Scopus Google Scholar). UV-visible were using a were from to at in containing and were at p58C in and then with were at on a The data were from at using an with The were with a NMR and the was with an Multiple of p58 from and were using T.J. F. F. 1997; 25: PubMed Scopus Google Scholar). Primase activity of wild-type and was on of primase, of in and of were on and at for were with in with and were in at for and by for at using and as The were and by Primase involved in DNA replication are containing domains together Chazin W.J. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In it is to the of these structural domains by the intact protein to very limited then using mass spectrometry and N-terminal to the To DNA primase structured the p48/p58 dimer was subjected to limited with K. were as a function of by using in two stable were produced corresponding to molecular of and were from the and by mass spectrometry, spectrometry, and N-terminal to the of the two The was to be the of a C-terminal of from of p48 produced the fragment which that the fragment is a stable domain of The fragment to the C-terminal half of p58, to of Primase the potential primase domains were into p48 and were into but not protein, the C terminus is an part of the protein. p48 contains a in the of the a stable fragment was not in the experiments for the N-terminal half of p58, sequence analysis it form a structured sequence homology between the N-terminal half of p58 and the region of the large subunit from S. solfataricus that has been (16Lao-Sirieix S.H. Nookala R.K. Roversi P. Bell S.D. Pellegrini L. Nat. Struct. Mol. Biol. 2005; 12: 1137-1144Crossref PubMed Scopus (55) Google Scholar). In an to and characterize a a of experiments was but protein. A similar was for the p58 subunit. In to p58C has homology to proteins structure is known. However, an of structure was for this stable In the p58C domain expressed well and was very to very high A of CD and NMR spectroscopy was used to characterize the structural of observed at and in the CD a of in of the CD using the an of and The NMR of p58C is in The contains and very which are of a well folded structural in the primase dimer or p58C was purified, the protein a The as the protein was very at high A UV-visible of p58C a at similar to from proteins containing iron-sulfur clusters J. V. W.J. Mol. Cell. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). 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DNA not have this and the and to DNA this has a of it we have that the iron-sulfur cluster in DNA primase is required for enzymatic the function of the cluster The and of the in p58C that the cluster does of structural to the protein. However, p58 is structured in the of the iron-sulfur cluster to Interestingly, a yeast that of one of the ligands in p58 a S. A. C. Lucchini G. Plevani P. Sci. U. S. A. PubMed Scopus Google Scholar, L. Plevani P. Lucchini G. 1993; PubMed Google Scholar). This substitution be to in loss of the iron-sulfur cluster from p58. The that the is not in the context of the pol-prim complex in the cluster may a regulatory as to a structural function in p58. The that the DNA primase iron-sulfur cluster active to DNA be The that this for of the of p58, as its to the length of is The as well as both subunits of primase B. Kirk B.W. Copeland W.C. Kuchta R.D. Biochemistry. 1999; 38: 12899-12907Crossref PubMed Scopus (39) Google Scholar) a is to be important for DNA by these proteins. The have domains that the protein to it the of the strand in DNA. It is the iron-sulfur the helicase the its interaction with the is or pol-prim to template into an that to p58 B. Kirk B.W. Copeland W.C. Kuchta R.D. Biochemistry. 1999; 38: 12899-12907Crossref PubMed Scopus (39) Google Scholar). These that the RNA to p58C, on the sequence homology to the domain of DNA that is to regulation of primer length a stable B. Kirk B.W. Copeland W.C. Kuchta R.D. Biochemistry. 1999; 38: 12899-12907Crossref PubMed Scopus (39) Google Scholar, Kuchta R.D. Biochemistry. 1993; PubMed Scopus Google Scholar), that the forms a stable complex with p58C to internal transfer of the primer to the polymerase active site in intact is a in the the transfer the In the of the polymerase subunit, primase activity only the primase from the and to these that p58C interacts with during and primer the iron-sulfur cluster in p58C is important for the primase to to the This also the of the p58 yeast which be to from the DNA more at and in the In of a function for the iron-sulfur cluster in p58C, one that of the may primase to be on the growing the primer is and to p180 for elongation, or primase from the The discovery of an iron-sulfur cluster in DNA primase new horizons in which to investigate the function of 4Fe-4S clusters in DNA replication and machinery. Our in to the structural the cluster in eukaryotic DNA may well function in form of regulatory in the length of the primer is by the of and high structural analysis in the role of the iron-sulfur cluster in DNA primase To this structural and are in in C. A. R. D. H. X. M. R. and S. M. for and also for the and the in