RESEARCH ARTICLE

Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation

  • Lili Liu 1,2 ,
  • Mingxia Huang , 2
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  • 1. Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA
  • 2. Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, CO 80045, USA

Received date: 09 Dec 2014

Accepted date: 04 Jan 2015

Published date: 01 Apr 2015

Copyright

2014 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

DNA primase catalyzes de novo synthesis of a short RNA primer that is further extended by replicative DNA polymerases during initiation of DNA replication. The eukaryotic primase is a heterodimeric enzyme comprising a catalytic subunit Pri1 and a regulatory subunit Pri2. Pri2 is responsible for facilitating optimal RNA primer synthesis by Pri1 and mediating interaction between Pri1 and DNA polymerase α for transition from RNA synthesis to DNA elongation. All eukaryotic Pri2 proteins contain a conserved C-terminal iron-sulfur (Fe-S) cluster-binding domain that is critical for primase catalytic activity in vitro. Here we show that mutations at conserved cysteine ligands for the Pri2 Fe-S cluster markedly decrease the protein stability, thereby causing S phase arrest at the restrictive temperature. Furthermore, Pri2 cysteine mutants are defective in loading of the entire DNA pol α-primase complex onto early replication origins resulting in defective initiation. Importantly, assembly of the Fe-S cluster in Pri2 is impaired not only by mutations at the conserved cysteine ligands but also by increased oxidative stress in the sod1Δ mutant lacking the Cu/Zn superoxide dismutase. Together these findings highlight the critical role of Pri2’s Fe-S cluster domain in replication initiation in vivo and suggest a molecular basis for how DNA replication can be influenced by changes in cellular redox state.

Cite this article

Lili Liu , Mingxia Huang . Essential role of the iron-sulfur cluster binding domain of the primase regulatory subunit Pri2 in DNA replication initiation[J]. Protein & Cell, 2015 , 6(3) : 194 -210 . DOI: 10.1007/s13238-015-0134-8

1
Aparicio OM, Stout AM, Bell SP (1999) Differential assembly of Cdc45p and DNA polymerases at early and late origins of DNA replication. Proc Natl Acad Sci USA96: 9130-9135

DOI

2
Aparicio O, Geisberg JV, Struhl K (2004) Chromatin immunoprecipitation for determining the association of proteins with speciflc genomic sequences in vivo. Current protocols in cell biology, Chapter17: Unit 17 7

3
Arezi B, Kuchta RD (2000) Eukaryotic DNA primase. Trends Biochem Sci25: 572-576

DOI

4
Arezi B, Kirk BW, Copeland WC, Kuchta RD (1999) Interactions of DNA with human DNA primase monitored with photoactivatable cross-linking agents: implications for the role of the p58 subunit. Biochemistry38: 12899-12907

DOI

5
Baranovskiy AG, Lada AG, Siebler H, Zhang Y, Pavlov YI (2012) DNA polymerases delta and zeta switching by sharing the accessory subunits of DNA polymerase delta. J Biol Chem287 (21): 17281-17287

DOI

6
Brazzolotto X, Gaillard J, Pantopoulos K, Hentze MW, Moulis JM (1999) Human cytoplasmic aconitase (Iron regulatory protein 1) is converted into its [3Fe-4S] form by hydrogen peroxide in vitro but is not activated for iron-responsive element binding. J Biol Chem274: 21625-21630

DOI

7
Budd M, Campbell JL (1987) Temperature-sensitive mutations in the yeast DNA polymerase I gene. Proc Natl Acad Sci USA 84: 2838-2842

DOI

8
Burgers PM (1998) Eukaryotic DNA polymerases in DNA replication and DNA repair. Chromosoma107: 218-227

DOI

9
Burke D, Dawson D, Stearns T (2000) Methods in yeast genetics: a cold spring harbor laboratory course manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

10
Carter CD, Kitchen LE, Au WC, Babic CM, Basrai MA (2005) Loss of SOD1 and LYS7 sensitizes Saccharomyces cerevisiae to hydroxyurea and DNA damage agents and downregulates MEC1 pathway effectors. Mol Cell Biol25: 10273-10285

DOI

11
Casper AM, Mieczkowski PA, Gawel M, Petes TD (2008) Low levels of DNA polymerase alpha induce mitotic and meiotic instability in the ribosomal DNA gene cluster of Saccharomyces cerevisiae. PLoS Genet4: e1000105

DOI

12
Copeland WC (1997) Expression, puriflcation, and characterization of the two human primase subunits and truncated complexes from Escherichia coli. Protein Expr Purif9: 1-9

DOI

13
Copeland WC, Wang TS (1993) Enzymatic characterization of the individual mammalian primase subunits reveals a biphasic mechanism for initiation of DNA replication. J Biol Chem268: 26179-26189

14
De Freitas JM, Liba A, Meneghini R, Valentine JS, Gralla EB (2000) Yeast lacking Cu-Zn superoxide dismutase show altered iron homeostasis. Role of oxidative stress in iron metabolism. J Biol Chem275: 11645-11649

DOI

15
Djaman O, Outten FW, Imlay JA (2004) Repair of oxidized iron-sulfur clusters in Escherichia coli. J Biol Chem279: 44590-44599

DOI

16
Ferrari M, Lucchini G, Plevani P, Foiani M(1996) Phosphorylation of the DNA polymerase alpha-primase B subunit is dependent on its association with the p180 polypeptide. J Biol Chem271: 8661-8666

DOI

17
Foiani M, Santocanale C, Plevani P, Lucchini G (1989) A single essential gene, PRI2, encodes the large subunit of DNA primase in Saccharomyces cerevisiae. Mol Cell Biol9: 3081-3087

18
Foiani M, Marini F, Gamba D, Lucchini G, Plevani P (1994) The B subunit of the DNA polymerase alpha-primase complex in Saccharomyces cerevisiae executes an essential function at the initial stage of DNA replication. Mol Cell Biol14: 923-933

19
Foiani M, Lucchini G, Plevani P (1997) The DNA polymerase alphaprimase complex couples DNA replication, cell-cycle progression and DNA-damage response. Trends Biochem Sci22: 424-427

DOI

20
Francesconi S, Longhese MP, Piseri A, Santocanale C, Lucchini G (1991) Mutations in conserved yeast DNA primase domains impair DNA replication in vivo. Proc Natl Acad Sci USA88: 3877-3881

DOI

21
Frick DN, Richardson CC (2001) DNA primases. Annu Rev Biochem70: 39-80

DOI

22
Fridovich I (1995) Superoxide radical and superoxide dismutases. Annu Rev Biochem64: 97-112

DOI

23
Gardner PR, Fridovich I (1991) Superoxide sensitivity of the Escherichia coli aconitase. J Biol Chem266: 19328-19333

24
Hubscher U, Maga G, Spadari S (2002) Eukaryotic DNA polymerases. Annu Rev Biochem71: 133-163

DOI

25
Kaplan J, McVey Ward D, Crisp RJ, Philpott CC (2006) Irondependent metabolic remodeling in S. cerevisiae. Biochim Biophys Acta1763: 646-651

DOI

26
Kawasaki Y, Sugino A (2001) Yeast replicative DNA polymerases and their role at the replication fork. Mol Cells12: 277-285

27
Kispal G, Csere P, Prohl C, Lill R (1999) The mitochondrial proteins Atm1p and Nfs1p are essential for biogenesis of cytosolic Fe-S proteins. EMBO J18: 3981-3989

DOI

28
Klinge S, Hirst J, Maman JD, Krude T, Pellegrini L (2007) An ironsulfur domain of the eukaryotic primase is essential for RNA primer synthesis. Nat Struct Mol Biol14: 875-877

DOI

29
Lao-Sirieix SH, Nookala RK, Roversi P, Bell SD, Pellegrini L (2005) Structure of the heterodimeric core primase. Nat Struct Mol Biol12: 1137-1144

DOI

30
Lemoine FJ, Degtyareva NP, Kokoska RJ, Petes TD (2008) Reduced levels of DNA polymerase delta induce chromosome fragile site instability in yeast. Mol Cell Biol28: 5359-5368

DOI

31
Liu H, Rudolf J, Johnson KA, McMahon SA, Oke M (2008) Structure of the DNA repair helicase XPD. Cell133: 801-812

DOI

32
Longhese MP, Jovine L, Plevani P, Lucchini G (1993) Conditional mutations in the yeast DNA primase genes affect different aspects of DNA metabolism and interactions in the DNA polymerase alpha-primase complex. Genetics133: 183-191

33
Longo VD, Liou LL, Valentine JS, Gralla EB (1999) Mitochondrial superoxide decreases yeast survival in stationary phase. Arch Biochem Biophys365: 131-142

DOI

34
Longtine MS, McKenzie A 3rd, Demarini DJ, Shah NG, Wach A (1998) Additional modules for versatile and economical PCRbased gene deletion and modiflcation in Saccharomyces cerevisiae. Yeast14: 953-961

DOI

35
Lu C, Cortopassi G (2007) Frataxin knockdown causes loss of cytoplasmic iron-sulfur cluster functions, redox alterations and induction of heme transcripts. Arch Biochem Biophys457: 111-122

DOI

36
Lucchini G, Francesconi S, Foiani M, Badaracco G, Plevani P (1987) Yeast DNA polymerase-DNA primase complex; cloning of PRI 1, a single essential gene related to DNA primase activity. EMBO J6: 737-742

37
Mantiero D, Mackenzie A, Donaldson A, Zegerman P (2011) Limiting replication initiation factors execute the temporal programme of origin flring in budding yeast. EMBO J30: 4805-4814

DOI

38
Messick TE, Chmiel NH, Golinelli MP, Langer MR, Joshua-Tor L (2002) Noncysteinyl coordination to the [4Fe-4S]2+ cluster of the DNA repair adenine glycosylase MutY introduced via sitedirected mutagenesis. Structural characterization of an unusual histidinyl-coordinated cluster. Biochemistry41: 3931-3942

DOI

39
Missirlis F, Hu J, Kirby K, Hilliker AJ, Rouault TA (2003) Compartment-speciflc protection of iron-sulfur proteins by superoxide dismutase. J Biol Chem278: 47365-47369

DOI

40
Mizuno T, Yamagishi K, Miyazawa H, Hanaoka F (1999) Molecular architecture of the mouse DNA polymerase alpha-primase complex. Mol Cell Biol19: 7886-7896

41
Muzi Falconi M, Piseri A, Ferrari M, Lucchini G, Plevani P (1993) De novo synthesis of budding yeast DNA polymerase alpha and POL1 transcription at the G1/S boundary are not required for entrance into S phase. Proc Natl Acad Sci USA90: 10519-10523

DOI

42
Muzi-Falconi M, Giannattasio M, Foiani M, Plevani P (2003) The DNA polymerase alpha-primase complex: multiple functions and interactions. Sci World J3: 21-33

DOI

43
Netz DJ, Stumpflg M, Dore C, Muhlenhoff U, Pierik AJ (2010) Tah18 transfers electrons to Dre2 in cytosolic iron-sulfur protein biogenesis. Nat Chem Biol6: 758-765

DOI

44
Netz DJ, Stith CM, Stumpflg M, Kopf G, Vogel D (2012) Eukaryotic DNA polymerases require an iron-sulfur cluster for the formation of active complexes. Nat Chem Biol8: 125-132

DOI

45
Nunez-Ramirez R, Klinge S, Sauguet L, Melero R, Recuero-Checa MA (2011) Flexible tethering of primase and DNA Pol alpha in the eukaryotic primosome. Nucleic Acids Res39: 8187-8199

DOI

46
Ricke RM, Bielinsky AK (2004) Mcm10 regulates the stability and chromatin association of DNA polymerase-alpha. Mol Cell 16: 173-185

DOI

47
Ricke RM, Bielinsky AK (2006) A conserved Hsp10-like domain in Mcm10 is required to stabilize the catalytic subunit of DNA polymerase-alpha in budding yeast. J Biol Chem281: 18414-18425

DOI

48
Rudolf J, Makrantoni V, Ingledew WJ, Stark MJ, White MF (2006) The DNA repair helicases XPD and FancJ have essential ironsulfur domains. Mol Cell23: 801-808

DOI

49
Sauguet L, Klinge S, Perera RL, Maman JD, Pellegrini L (2010) Shared active site architecture between the large subunit of eukaryotic primase and DNA photolyase. PLoS One5: e10083

DOI

50
Srinivasan C, Liba A, Imlay JA, Valentine JS, Gralla EB (2000) Yeast lacking superoxide dismutase(s) show elevated levels of “free iron” as measured by whole cell electron paramagnetic resonance. J Biol Chem275: 29187-29192

DOI

51
Stearman R, Yuan DS, Yamaguchi-Iwai Y, Klausner RD, Dancis A (1996) A permease-oxidase complex involved in high-afflnity iron uptake in yeast. Science271: 1552-1557

DOI

52
Tan S, Wang TS (2000) Analysis of flssion yeast primase deflnes the checkpoint responses to aberrant S phase initiation. Mol Cell Biol20: 7853-7866

DOI

53
Tanaka T, Nasmyth K (1998) Association of RPA with chromosomal replication origins requires an Mcm protein, and is regulated by Rad53, and cyclin- and Dbf4-dependent kinases. EMBO J17: 5182-5191

DOI

54
Tanaka S, Nakato R, Katou Y, Shirahige K, Araki H (2011) Origin association of Sld3, Sld7, and Cdc45 proteins is a key step for determination of origin-flring timing. Curr Biol21: 2055-2063

DOI

55
Urzica E, Pierik AJ, Muhlenhoff U, Lill R (2009) Crucial role of conserved cysteine residues in the assembly of two iron-sulfur clusters on the CIA protein Nar1. Biochemistry48: 4946-4958

DOI

56
Vaithiyalingam S, Warren EM, Eichman BF, Chazin WJ (2010) Insights into eukaryotic DNA priming from the structure and functional interactions of the 4Fe-4S cluster domain of human DNA primase. Proc Natl Acad Sci USA107: 13684-13689

DOI

57
Vernis L, Facca C, Delagoutte E, Soler N, Chanet R (2009) A newly identifled essential complex, Dre2-Tah18, controls mitochondria integrity and cell death after oxidative stress in yeast. PLoS One4: e4376

DOI

58
Wallace MA, Liou LL, Martins J, Clement MH, Bailey S (2004) Superoxide inhibits 4Fe-4S cluster enzymes involved in amino acid biosynthesis. Cross-compartment protection by CuZn-superoxide dismutase. J Biol Chem279: 32055-32062

DOI

59
Wang TSF (1996) Cellular DNA polymerases. In: DePamphilis ML (ed) DNA replication in eukaryotic cells. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 461-493

60
Wu X, Liu L, Huang M (2011) Analysis of changes in protein level and subcellular localization during cell cycle progression using the budding yeast Saccharomyces cerevisiae. Methods Mol Biol782: 47-57

DOI

61
Yeeles JT, Cammack R, Dillingham MS (2009) An iron-sulfur cluster is essential for the binding of broken DNA by AddAB-type helicase-nucleases. J Biol Chem284: 7746-7755

DOI

62
Zerbe LK, Kuchta RD (2002) The p58 subunit of human DNA primase is important for primer initiation, elongation, and counting. Biochemistry41: 4891-4900

DOI

63
Zhang Y, Liu L, Wu X, An X, Stubbe J (2011) Investigation of in vivo diferric tyrosyl radical formation in Saccharomyces cerevisiae Rnr2 protein: requirement of Rnr4 and contribution of Grx3/4 AND Dre2 proteins. J Biol Chem286: 41499-41509

DOI

64
Zhu W, Ukomadu C, Jha S, Senga T, Dhar SK (2007) Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. Genes Dev21: 2288-2299

DOI

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