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J. Biol. Chem., Vol. 281, Issue 30, 21422-21432, July 28, 2006

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GINS Is a DNA Polymerase Accessory Factor during Chromosomal DNA Replication in Budding Yeast^*

Takashi Seki, Masaki Akita, Yoichiro Kamimura^¶, Sachiko Muramatsu^¶, Hiroyuki Araki^¶, and Akio Sugino¹

From the Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, the Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Aichi 464-8602, and the ^¶Division of Microbial Genetics, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan

Received for publication, April 11, 2006 , and in revised form, May 17, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
GINS is a protein complex found in eukaryotic cells that is composed of Sld5p, Psf1p, Psf2p, and Psf3p. GINS polypeptides are highly conserved in eukaryotes, and the GINS complex is required for chromosomal DNA replication in yeasts and Xenopus egg. This study reports purification and biochemical characterization of GINS from Saccharomyces cerevisiae. The results presented here demonstrate that GINS forms a 1:1 complex with DNA polymerase (Pol ) holoenzyme and greatly stimulates its catalytic activity in vitro. In the presence of GINS, Pol is more processive and dissociates more readily from replicated DNA, while under identical conditions, proliferating cell nuclear antigen slightly stimulates Pol in vitro. These results strongly suggest that GINS is a Pol accessory protein during chromosomal DNA replication in budding yeast. Based on these results, we propose a model for molecular dynamics at eukaryotic chromosomal replication fork.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Three structurally and functionally distinct DNA polymerases, known as DNA polymerases , , and , are required for chromosomal DNA replication in budding yeast (1, 2). DNA polymerase (Pol ),² is composed of the 180-kDa catalytic subunit and an 86-kDa subunit of unknown function. Pol forms a complex with DNA primase and the Pol -primase complex is essential for chromosomal replication in vivo. DNA polymerase (Pol ) from budding yeast is composed of a 125-kDa catalytic subunit as well as 58- and 55-kDa subunits, encoded by POL3 (CDC2), HYS2 (POL31), and POL32, respectively (3, 4). Pol from other eukaryotic systems including fission yeast has a different subunit structure (5-7) from budding yeast Pol . DNA polymerase (Pol ) is composed of four subunits (265-, 80-, 34-, and 31-kDa) encoded by POL2, DPB2, DPB3, and DPB4, respectively (1, 8, 9).

DNA primase initiates DNA replication by synthesizing a short oligoribonucleotides primer, which is immediately elongated by Pol to form short RNA-DNA fragments on both leading and lagging strand of DNA. Pol elongates the short RNA-DNA fragment initiated by Pol -primase to make a mature Okazaki fragment (2, 10). To carry out processive DNA synthesis in vitro, Pol requires PCNA and its loader, replication factor C (RF-C) (10). In cooperation with Fen1p (Rad27p), Dna2p and RPA, Pol also plays a crucial role in processing RNA-linked Okazaki fragments (10).

Although the precise role of Pol in vivo is still unclear, it has been implicated in DNA replication, repair, recombination, and mitosis (1, 2). Pol has at least one essential function in both budding and fission yeasts (11, 12), and several lines of evidence suggest that Pol plays an essential catalytic role during chromosomal DNA replication. Yeast cells harboring a temperature-sensitive pol2 allele are temperature sensitive for growth and express a thermolabile Pol DNA polymerase activity (13). Furthermore, Pol2p is associated with replication forks during S phase (14) and pol2 mutants fail to complete chromosomal DNA replication (13, 15). Furthermore, 3'-5' exonuclease-deficient mutants of POL2 and POL3 (Pol ) accumulate strand-specific lesions in chromosomal DNA (16, 17, 18, 19). These observations support models for chromosomal DNA replication in which Pol and Pol play leading strand- and lagging strand-specific roles during chromosomal DNA replication, respectively. Pol has been proposed as the leading strand DNA polymerase because Pol is a highly processive polymerase without PCNA (1, 8) and pol3 mutants have defects in maturation of Okazaki fragments (10). Nevertheless, it has been reported that the N-terminal portion of budding yeast Pol (Pol2p), which includes motifs required for DNA polymerase and exonuclease activities, is dispensable for DNA replication, DNA repair, and viability (20, 21). However, this conclusion is controversial, because other studies suggest that deletion of the N-terminal region of Pol confers temperature sensitivity for growth, a defect in DNA elongation, premature senescence, and short telomeres. Furthermore, this pol2p deletion is lethal in combination with temperature-sensitive cdc2 and with exonucleasedeficient Pol (pol3-01). These results suggest that Pol plays a crucial role in maintaining genomic integrity (22, 23).

In a previous study, various sld (synthetic lethality with dpb11-1) mutants (24) were identified as yeast mutants that are lethal in combination with temperature-sensitive dpb11-1 (25). DPB11 encodes Dpb11p, a yeast protein that interacts with Pol that is required for initiation of chromosomal DNA replication. Sld1p is identical to Dpb3p, the third subunit of Pol . Sld2p binds to Dpb11p, and the Sld2p Dpb11p heterodimer facilitates loading of Pol -primase and Pol onto replication origins during S phase (26). Sld2p is phosphorylated by S-phase cyclin-dependent protein kinase at the beginning of S phase, and is required for initiation of chromosomal DNA replication (27). Sld3p interacts with Sld4p, which is identical to Cdc45p. Previous studies show that Cdc45p is required for initiation and elongation of chromosomal DNA replication (reviewed in Ref. 2). It has been reported that Sld3 is also important for the progression of DNA replication forks after the initiation step (28), as are Cdc45. In contrast, it does not move with DNA replication forks and only associates with MCM in an unstable manner before initiation. After the establishment of DNA replication forks from early origins, it is no longer essential for the completion of chromosome replication (29). Slp5p is a component of GINS, which also includes Psf1p, Psf2p and Psf3p. GINS is essential for chromosomal DNA replication in yeast (30, 31). Finally, Sld6p is identical to Rad53p, which is required for cell cycle checkpoints (32). GINS associates with replication origins during S phase (30, 31). It has been suggested that GINS promotes an interaction between the Sld3p·Cdc45p complex and the Sld2·Dpb11·Pol , and that this could facilitate initiation of DNA replication (27). GINS polypeptides are conserved in eukaryotic cells. Xenopus egg extracts have a complex that resembles GINS, which is also required for the initiation of chromosomal DNA replication (33). Electron microscopy of the Xenopus GINS complex suggests that GINS forms a ring structure that resembles a trimeric PCNA, which is a Pol clamp. These data are consistent with the idea that GINS is a clamp for Pol (33).

This study reports purification and characterization of GINS from yeast cell extracts. GINS does not bind DNA directly, but it forms a 1:1 complex with Pol and greatly stimulates its catalytic activity in vitro. In the presence of GINS, Pol is more processive and dissociates more readily from replicated DNA. These results suggest that GINS is an accessory protein for Pol during chromosomal DNA replication in yeast.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Yeast Strains—The yeast strains used in this study are YTS12 (MATa ura3-52 trp1 prb1-1122 prc1-407 pep4-3 leu2-3, 112 nuc1::LEU2 bar1::hisG), YTS28 (MATa ura3-52 trp1 prb1-1122 prc1-407 pep4-3 leu2-3,-112 nuc1::LEU2 bar1::hisG 6FLAG-PSF1::URA3), YYK61 (MATa ade2-1 bar1::hisG can1-100 his3-11,15 leu2-3,112 6FLAG-PSF1 SLD3-9myc::URA3 trp1-1 ura3-1) and YYK62 (MATa ade2-1 bar1::hisG can1-100 his3-11,15 leu2-3,112 6FLAG-PSF1 SLD3-9myc::URA3 GALp-CDC6(TRP1) trp1-1 ura3-1). Other yeast strains are previously described (8, 22, 23, 24, 30).

Template Primer DNA—Oligonucleotides used in this study (Gene Design, Inc.) were purified by polyacrylamide gel electrophoresis. The 34-nucleotide primer (5'-CTAGTTACA GAGTTATGGTGACGATACAAACTAT-3') was ³²P-labeled at the 5'-end using [-³²P]ATP and T4 polynucleotide kinase and annealed to the 65-mer (3'-GATCAATGTCTCAATACCACTGCTATGTTTGATATCTCGCCTAATGATATGATGTAATCTTAAGT-5') oligonucleotide template to make a replication substrate for DNA polymerase assays.

Construction of Singly Primed Linear X174 Single-stranded DNA—X174 viral sscDNA (New England Biolabs) was annealed with a 30-mer oligonucleotide (5'-AGCGATAAAACTCTGCAGGTTGGATACGCC-3'; a PstI site is underlined), and digested with the restriction endonuclease PstI to make a sslDNA. Then, a 30-mer primer (5'-TGCAGGTTGGATACGCCAATCATTTTTATC-3') was annealed to X174 sslDNA as published (34).

Purification of GINS—Yeast strain YTS28 was grown in 60 liters of YPD (1% yeast extracts, 2% polypeptone, 2% glucose) medium at 30 °C to about 2 x 10⁸ cells/ml, harvested and stored at -80 °C until used. Cells (450 g) were thawed in 2 volume of lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 10% (v/v) glycerol, 10 mM 2-mercaptoethanol) containing 1% (v/v) protease inhibitor mixture (Sigma) and disrupted as published (8). To cell lysate, 0.3 M KCl (final concentration) was added, and incubation was continued at 4 °C for 30 min with stirring, followed by centrifugation at 10,000 x g for 30 min at 4 °C. The resultant lysate was further supplemented with Triton X-100 and 10% polyethyleneimine solution to give a final concentration of 0.01% (v/v) and 0.4% (v/v), respectively, incubated at 4 °C for 30 min, and centrifuged at 10,000 x g for 30 min at 4 °C. The resultant pellets were dissolved in buffer A (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.01% Triton X-100, and 10 mM 2-mercaptoethanol) containing 1% protease inhibitor mixture and 0.5 M ammonium sulfate, incubated at 4 °C for 30 min, and centrifuged at 10,000 x g for 30 min at 4 °C. The supernatant, which contained more than 90% of GINS complex, was recovered, and protein was precipitated by addition of ammonium sulfate to a final concentration of 50% saturation. After incubating at 4 °C for 30 min, the precipitated protein was collected by centrifugation at 10,000 x g for 30 min at 4 °C. The pellet was dissolved in buffer A containing 1% protease inhibitor mixture and 0.1 M NaCl, dialyzed against the same buffer and centrifuged at 100,000 x g for 30 min at 4 °C. The supernatant was applied to a Mono Q column (HR16/10, GE). After washing the column with buffer A containing 150 mM NaCl, proteins were eluted with a linear gradient from 150 to 600 mM NaCl in buffer A without 2-mercaptoethanol. 6x FLAG-tagged Psf1p was eluted at 350 mM NaCl in Buffer A. Fractions containing 6x FLAG-tagged Psf1p were pooled, supplemented with bovine serum albumin (BSA) to a final concentration of 5 mg/ml and with a mixture of protease inhibitors consisting of 4-(2-aminoethyle)-benzensulfonyl fluoride, aprotinin, benzamidine hydrochloride, leupeptin, and pepstatin A to final concentrations of 1 mM,2 µg/ml, 1 mM,10 µg/ml, and 1 µg/ml, respectively, and loaded to a 400 µl of anti-FLAG M2 agarose bead column. The column was washed three times with 10 bed volumes of buffer B (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10% glycerol, 0.05% (v/v) Tween20, 0.005% (v/v) Nonidet P-40, 2 mM -glycerophosphate, and 2 mM NaF) containing 300 mM NaCl and 0.1 mg/ml BSA, three times with 10 bed volumes of buffer B containing 300 mM NaCl, and once with 4 bed volumes of buffer B containing 150 mM NaCl and 10 µg/ml 1x FLAG peptide (Sigma). Bound proteins were eluted in 6 bed volumes of buffer B containing 300 mM NaCl and 100 µg/ml 3x FLAG peptide (Sigma). Eluted fractions were combined, applied to a Mono Q column (HR5/5, GE), the column was washed with 5 ml of 100 mM NaCl in Buffer A, and 6x FLAG-tagged Psf1p was eluted with 10 ml of a linear gradient from 100 to 700 mM NaCl in buffer A. Fractions containing 6x FLAG-Psf1p were pooled, dialyzed against buffer G (50% glycerol, 50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, and 3 mM 2-mercaptoethanol(50 µg/total 1 ml) and frozen in liquid nitrogen.

Pol and Other Replication Proteins—Pol holoenzyme was purified as described previously (8, 34). The Dpb3p·Dpb4p and Pol2p·Dpb2p complexes were kindly provided by Dr. S. Maki (Nara Institute of Science and Technology) (35). Purification of S. cerevisiae Pol , PCNA, and RPA was previously described (36). Pol -primase was the same as described (8). Recombinant S. cerevisiae RF-C-1DN (37) was a gift of Dr. P. Burgers (Washington University).

Pol -GINS Binding Assay—The purified Pol , the Pol2p·Dpb2p complex, or the Dpb3p·Dpb4p complex was incubated with purified GINS containing 6x FLAG-tagged Psf1p, or with FLAG-tagged BAP (Sigma) as a control in 50 µl of binding mixtures consisting of 50 mM HEPES-KOH (pH 7.6), 50 mM KOAc, 5 mM MgOAc, 1 mM EDTA, 10% glycerol, 0.01% Nonidet P-40, and 0.1 mg/ml BSA at 4 °C for 10 min. After incubation, BSA was added to a final concentration of 5 mg/ml, and the binding mixture was incubated with 10 µl of anti-FLAG M2-conjugated agarose beads at 4 °C for 1 h. Then, beads were washed three times with wash buffer (50 mM HEPES-KOH, pH 7.6, 50 mM KOAc, 5 mM MgOAc, 1 mM EDTA, 10% glycerol, 0.01% Nonidet P-40, 0.1 mg/ml BSA) and boiled in SDS sample buffer. Proteins associated with beads were analyzed by SDS-PAGE, followed by immunoblotting.

Immunoprecipitation of 6x FLAG-Psf1p from Yeast Cell Extracts—6x FLAG-tagged Psf1p was immunoprecipitated with anti-FLAG M2 agarose beads (Sigma) from the whole cell extracts prepared from asynchronously growing or hydroxyurea (HU)-treated yeast YYK61 cells as previously described (24). The precipitates were boiled in SDS-sample buffer at 95 °C for 30 min and subjected to SDS-PAGE, followed by immunoblotting.

Glycerol Gradient Sedimentation—Purified GINS and Pol (molar ratio was 1:1) were incubated in 100 µl of binding mixtures consisting of 50 mM HEPES-KOH pH 7.6, 5 mM MgOAc, 1 mM dithiothreitol, 0.01% Nonidet P-40, 0.1 mg/ml BSA at 4 °C for 1 h. For cross-linking experiments, after the incubation, 1 µl of 0.2 M dithiobis (succinimidylpropionate) (DSP) was added to a binding mixture, which was further incubated at 24 °C for 30 min. The reaction was quenched by addition of 5 µl of 1 M Tris-HCl, pH 7.5, and incubation at 24 °C for 5 min. The mixtures were layered onto a 5 ml of 15-40% glycerol density gradient in buffer containing 50 mM HEPES-KOH (pH 7.6), 5 mM MgCl₂, 1 mM dithiothreitol, 0.01% Nonidet P-40, and 50 mM KOAc and centrifuged in a Hitachi P55ST2 rotor at 45,000 rpm for 14 h at 2 °C. The sample was fractionated into 23 fractions from top of the gradient and proteins in the fractions were separated by SDS-PAGE and detected by silver staining or immunoblotting.

Immunoblotting—6x FLAG-tagged Psf1p was detected with anti-FLAG (Affinity BioReagents) or anti-Psf1p antibody. 9x Myc-tagged Sld3p was detected with anti-Myc antibody (28). Anti-Mcm2p antibodies were purchased from Santa Cruz Biotechnology. Sld5p, Psf2p, Dpb3p, and Dpb4p were detected with anti-Sld5p (30), anti-Psf2p-(30), anti-Psf3p-,³ and anti-Dpb4p rabbit polyclonal antibodies (22), respectively. Pol2p, Pol12p, PCNA, Rpa1p, and Mcm10p were detected with anti-yeast Pol II^* (Pol holoenzyme)-(38), Pol -primase-(8), -PCNA- (22), -RPA- (22), and -Mcm10 rabbit polyclonal antibodies (39), respectively. Anti-Psf1p-, -Dpb2p-, -Dpb11p-, -Cdc45p-, and -Sld2p rabbit antibodies were prepared using each protein, which was expressed in Escherichia coli and purified from E. coli.³

Gel Mobility Shift Assay without Cross-linking—The GINS complex, Pol and Cy5-labeled DNA fragments were incubated in reaction mixtures consisting of 20 mM HEPES-KOH (pH 7.6), 0.05% Nonidet P-40, 10% glycerol, and 0.1 mg/ml BSA on ice for 10 min. Ficoll was added to the samples at a final concentration of 15%, and the samples were run on a 4% polyacrylamide gel in TBE. For some experiments, MgOAc was added to the reaction mixture, and TBE at a final concentration of 5 mM. The sequences of the oligonucleotides used were shown in Table 1. Cy5-7 was used as single-stranded linear DNA (sslDNA). Cy5-7 and 71, Cy5-16, and 17, 21, and Cy5-22, Cy5-16 and 18, Cy5-19 and 20 were annealed for preparing a double-stranded linear DNA, 3'-overhang DNA, 5'-overhang DNA, Y-form DNA and bubble form DNA, respectively. The images were detected using a fluorescent image analyzer FLA-3000 (Fuji film).

Stimulation of Pol by GINS on a Replication Substrate Consisting of a 65-mer Template Annealed with 5'-³²P-labeled 34-Nucleotide Primer—The 10-µl reactions contained 50 mM Tris-HCl, pH 7.5, 5 mM MgCl₂, 100 mM potassium glutamate, 0.1 mg/ml BSA, 1 mM dithiothreitol, 50 µM each dNTPs, 600 fmol of 5'-³²P-labeled primer/template, 25-100 fmol of Pol , and 500 fmol of GINS. After incubation at 30 °C for 1-30 min, the reactions were stopped with 7 µl of 95% formamide-20 mM EDTA and electrophoresed on a 15% denaturing polyacrylamide gel containing 7 M urea.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
GINS Is Part of the Yeast Replisome—Protein-interacting partners of GINS were identified by immunoprecipitating 6x FLAG-tagged Psf1p from logarithmically growing yeast cells with anti-FLAG antibody beads. Immunoprecipitates were prepared in the presence or absence of a protein cross-linking agent, and the coprecipitating proteins were analyzed by SDS-PAGE, followed by Western blot. In the absence of cross-linking agent, a small fraction of soluble Dpb2p (the second subunit of Pol ) and Sld3p were detected in the immunoprecipitates (shown by arrows in Fig. 1A), but in the presence of a protein cross-linking reagent, significant amounts of Dpb2p, Dpb11p, phosphorylated Sld2p, Cdc45p (data not shown) and Sld3p coprecipitated with 6x FLAG-Psf1p (Fig. 1A). Other DNA replication factors also coprecipitated with Psf1p including Mcm2p (a member of Mcm2-7p), PCNA, and RPA. The latter two proteins are believed to be involved in lagging strand synthesis catalyzed by Pol -primase and Pol (2, 10). Yeast cells expressing 3x FLAG-tagged Psf2p were also treated with 0.2 M HU-containing YPD for 2 h, collected, and used to isolate stable S phase-specific protein complexes in the absence of protein cross-linking. Under these conditions, Dpb2p, Mcm10p, Cdc45p, and Mcm2p co-immunoprecipitated with 3x FLAG-tagged Psf2p (Fig. 1B). These results suggest that Pol , Cdc45p, Mcm2p, and Mcm10p form a stable complex with GINS in vivo during S phase. Thus, these results are consistent with the notion that GINS is a part of the yeast replisome.

Purification and Characterization of GINS—The above results suggest that GINS may interact directly with Pol and other replication proteins, although these interactions might be weak, transient, and/or salt-sensitive. To investigate this possibility directly, GINS was purified by affinity chromatography from yeast cells expressing 6x FLAG-tagged Psf1p as described under "Experimental Procedures." Purified GINS consisted of four proteins of 35, 30, 25, and 22 kDa, respectively (Fig. 2A), as previously reported (30). The purity of GINS was estimated to be >95%, and the protein subunits were approximately equimolar, although the third subunit of GINS (Psf2p) seemed to be a little more than other proteins (actual molar ratio between Sld5p, Psf1p, Psf2p, and Psf3p was 1:1:1.3:1). The identity of each GINS subunit was confirmed by immunoblotting purified GINS with anti-FLAG-, anti-Sld5p-, anti-Psf1p-, anti-Psf2p-, and anti-Psf3p antibodies (Fig. 2A) and by MALDI-TOF mass spectrometry analysis of gel-purified polypeptides from purified GINS (data not shown). The results confirmed that purified GINS is composed of Sld5p, Psf1p, Psf2p, and Psf3p. Purified GINS did not have any detectable DNA polymerase-, endonuclease-, exonuclease-, DNA helicase-, ATPase-, ssDNA-binding-, or dsDNA binding activity (data not shown).

View larger version (45K): [in this window] [in a new window]   FIGURE 1. GINS and its protein-interacting partners in yeast cell extracts. A, asynchronously growing cells expressing 6x FLAG-tagged Psf1p (30) were incubated with (+) or without (-) formaldehyde and whole cell extracts (WCS) were prepared as described previously (24). 6x FLAG-tagged Psf1p was immunoprecipitated from the whole cell extracts with anti-FLAG beads. Proteins bound to the beads were released by boiling in SDS, and analyzed by SDS-PAGE, followed by immunoblotting with the indicated antibodies. WCS were also analyzed by SDS-PAGE, followed by immunoblotting with the indicated antibodies. In the figure, arrows indicate Sld3p and Dpb2p (a representative subunit of Pol holoenzyme) bands that were immunoprecipitated with anti-FLAG beads. B, asynchronously growing cells expressing 3x FLAG-tagged Psf2p were treated with 0.2 M HU for 120 min at 30 °C, collected by centrifugation, and WCS were prepared without any cross-linking reagent. 3x FLAG-tagged Psf2p was immunoprecipitated with anti-FLAG antibody beads as above, and proteins associated with antibody were analyzed by immunoblotting as A. As a control (-), untagged yeast cells were grown, treated with 0.2 M HU for 120 min at 30 °C, whole cell extracts were prepared and used for immunoprecipitation. Asterisk indicates a protein band that interacts nonspecifically with anti-Dpb11p- or anti-Sld2p antibodies.

GINS Specifically Binds to Pol —The physical interaction between GINS and Pol was examined using FLAG-tagged Psf1p and anti-FLAG antibody pull-down assays. When GINS and Pol were incubated at 25 °C for 30 min, anti-FLAG antibody beads coprecipitated FLAG-tagged Psf1p (GINS) and the Pol including Pol2p, Dpb2p, Dpb3p, and Dpb4p (Fig. 3A). GINS appeared to bind to Pol2p·Dpb2p as efficiently as to Pol holoenzyme (Fig. 3B), but bound much less efficiently to Dpb3p·Dpb4p (35)(Fig. 3C) and to the145 kDa-degradation product of the full size Pol2p (265 kDa), which has a DNA polymerization activity as Pol (34)(data not shown). However, GINS did not bind to other replicative polymerases, Pol or Pol -primase complex (Fig. 3D and data not shown for Pol -primase). These results suggest that GINS interacts specifically with the C-terminal-half portion of Pol2p (a catalytic subunit of Pol ) and possibly with Dpb2p, which is the second subunit of S. cerevisiae Pol . The C-terminal-half portion of Pol2p, where other subunits (Dpb2p, Dpb3p, and Dpb4p) of Pol interact and form Pol holoenzyme (1), is known to be essential for yeast cell growth (20, 21).

The interaction between GINS and Pol was also examined by glycerol density gradient sedimentation in the presence or absence of a protein cross-linking agent. GINS and Pol co-sedimented through the gradient, when the samples were preincubated with a cross-linking agent, but the complex dissociated spontaneously during sedimentation in the absence of a protein cross-linker (Fig. 4A). These results suggest that the interaction between GINS and Pol is relatively weak and/or transient. In contrast, GINS and Pol are stable as independent complexes in the absence of protein cross-linking during glycerol density gradient sedimentation (Fig. 4A). The glycerol density gradient data of their apparent molecular weights also suggests that GINS and Pol exist as monomers in solution and that they form a 1:1 GINS·Pol complex (Fig. 4B).

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Purification of GINS and Pol holoenzyme. A, GINS complex was purified as described under "Experimental Procedures." Purified preparations were analyzed by 5-20% SDS-PAGE and stained with Coomassie Brilliant Blue (CBB) or immunoblotted with antibodies to FLAG, Psf1p, Sld5p, Psf2p, or Psf3p as indicated. To estimate molar ratio between each polypeptides in GINS complex, the CBB-stained bands were densitometry-scanned. B, purified Pol holoenzyme was analyzed by 5-20% SDS-PAGE and stained with CBB or immunoblotted with anti-Pol II*, anti-Dpb3p, or anti-Dpb4p as indicated. The anti-Pol II* antibody recognizes Pol2p, Dpb2p, Dpb3p, and Dpb4p subunits of Pol (34, 38).

View larger version (28K): [in this window] [in a new window]   FIGURE 3. The GINS complex interacts with Pol , but not with Pol in vitro. A, fixed amount of purified Pol was incubated with increasing amounts of purified GINS containing 6x FLAG-tagged Psf1p (FLAG-GINS) or with FLAG-tagged BAP (FLAG-BAP) at 25 °C for 30 min. The samples contained 2.0 pmol of Pol and 0, 0.3, 0.5, or 1.0 pmol of GINS or 0, 0.6, 1.2, or 2.4 pmol of FLAG-BAP. The GINS complex or BAP was immunoprecipitated with anti-FLAG beads, and the precipitated proteins were analyzed by SDS-PAGE, followed by immunoblotting with antibodies to FLAG and Pol holoenzyme (Pol II*). B and C, GINS complex interacts with the Pol2·Dpb2 subcomplex, but not with the Dpb3p·Dpb4p subcomplex of Pol in vitro. The samples contained 2 pmol of GINS and 2 pmol of Pol holoenzyme (Pol ), 0.7 or 1.8 pmol of Pol2-Dpb2 (Pol2·Dpb2) (B), or 0, 2, 4, or 10 pmol of Dpb3·Dpb4 (C). Binding reactions were carried out at 25 °C for 30 min. The GINS complex was immunoprecipitated with anti-FLAG beads and analyzed as in A using anti-Pol II*-, anti-Dpb3p-, and anti-Dpb4p antibodies. D, GINS complex was incubated with increasing amounts of purified Pol . The GINS complex was immunoprecipitated with anti-FLAG beads, and the precipitated proteins were probed with antibody to Pol31p (Hys2p), a subunit of Pol (4).

View larger version (41K): [in this window] [in a new window]   FIGURE 4. GINS forms a 1:1 complex with Pol holoenzyme. A, purified GINS, Pol holoenzyme or both were incubated with (cross-linked) or without 2 mM DSP (not cross-linked) and analyzed by sedimentation through a 15-40% glycerol gradient. The gradient was fractionated and samples were analyzed by SDS-PAGE, followed by immunoblotting or by silver staining. The positions of markers are indicated at the top. B, to quantify Pol2p, Dpb4p, Psf1p, and Psf2p in A, each band of the proteins was densitometry-scanned and estimated the relative intensity to the strongest band. Panel a, distribution of Pol2p and Psf1p of the Pol ·GINS complex with cross-linking; panel b, distribution of Pol 2p and Dpb4p of Pol alone without cross-linking; panel c, distribution of Psf1p and Psf2p of GINS without cross-linking.

View larger version (45K): [in this window] [in a new window]   FIGURE 5. Gel shift assay of DNA binding activity by Pol or GINS in the absence of Mg2+. Either Pol (A) or GINS (B) and various Cy5-labeled DNA fragments were incubated in reaction mixtures consisting of 20 mM HEPES-KOH (pH 7.6), 0.05% Nonidet P-40, 10% glycerol, 2 mM EDTA, and 0.1 mg/ml BSA on ice for 10 min. Ficoll was added to the samples at a final concentration of 15%, and the samples were run on a 4% polyacrylamide gel in TBE. The images were detected using a fluorescent image analyzer FLA-3000 (Fuji film). Asterisks show the position of Cy5 label on the DNA. C, gel-shift assay was carried out using GINS (0.6 pmol), Pol (0.8 pmol), GINS (0.6 pmol)-Pol (0.8 pmol), or GINS (0.6 pmol)-Pol (0.8 pmol)-FLAG antibody (10 pmol) and a 32P-labeled partially double-stranded 60-mer oligonucleotide DNA substrate (1.1 pmol) + and - indicate with or without indicated proteins.

View larger version (43K): [in this window] [in a new window]   FIGURE 6. GINS stimulates DNA synthesis by Pol . A, Pol holoenzyme (25 or 50 fmol) was incubated with 600 fmol of DNA substrate (a 32P-labeled 34-nucleotide primer annealed to a 65-mer oligonucleotide template) with or without GINS (200 fmol) at 30 °C. Reaction products were analyzed on a 15% denaturing polyacrylamide gel, followed by autoradiography for 60 min or 18 h. B, results shown in A were quantified by measuring radioactivity of the 65-mer product. C, reactions were carried out and analyzed as in A except that the ratio of GINS to Pol was varied as indicated, and reactions were incubated for 5 or 10 min at 30 °C. D, PCNA and RF-C also stimulates DNA synthesis catalyzed by Pol . 25 fmol of Pol holoenzyme was incubated with 600 fmol of DNA substrate and 600 fmol of RF-C with or without various amounts of PCNA (0.8, 1.6, and 8 pmol) for 1, 5, and 10 min at 30 °C. The products were analyzed as in A, except for autoradiography for 12 h.

View larger version (50K): [in this window] [in a new window]   FIGURE 7. GINS stimulates DNA synthesis catalyzed by Pol holoenzyme on singly primed X174 ssDNA. A, reaction mixtures contained 15 pmol of RPA, 100 fmol of a 30-mer singly primed X174 sscDNA, 25 fmol of Pol holoenzyme and 50 µM each dNTPs ([-32P]dTTP was included). Reactions were incubated at 30 °C with or without GINS (100 fmol) for 0.5, 1, 3, 5, and 10 min. The products were analyzed by 1.2% alkaline agarose gel electrophoresis, followed by autoradiography as published (41). B, reaction mixtures were the same as in A, except for 100 fmol of a 30-mer singly primed X174 sslDNA. C, reaction mixtures were the same as in A, except for with or without GINS (100 fmol), RF-C (250 fmol), and/or PCNA (1.6 pmol) for 20 min. + and - indicate addition of each protein into the reaction mixture. The bottom numbers in the figure indicate total nucleotides (pmol) synthesized in each reaction.

GINS Stimulates DNA Synthesis Catalyzed by Pol on a Singly Primed X174 Single-stranded DNA—Next, the effect of GINS on the rate of DNA synthesis catalyzed by Pol was examined using singly primed X174 single-stranded circular DNA (sscDNA) coated with yeast RPA as published (34). After incubation, the products of elongation were analyzed by electrophoresis on an alkaline agarose gel. As shown in Fig. 7A, during the first 3-5 min, DNA synthesis was almost linear in both reactions without and with GINS. Furthermore, total DNA synthesized by Pol with GINS was two to three times higher than without GINS and the DNA products produced by Pol were longer in the presence of GINS than in the absence of GINS at every time points (0.5, 1, 2, 3, 5, and 10 min). The product DNA at these early time points ran as rather distinct bands, and the size gradually increased to reach the full-length within 10 min incubation with GINS (Fig. 7A). Because the levels of incorporation during the incubation were less than 2% of the total incorporation obtained (only small portion of the input template primers were utilized), these results indicate that Pol starts elongation more or less uniformly and that the polymerase has a capacity to elongate DNA all the way around the viral DNA circle without dissociating from it in the presence or absence of GINS. These results suggest that GINS stimulates either the processivity or rate of DNA synthesis catalyzed by Pol or it stimulates both. Rates of DNA elongation by Pol and Pol with GINS, calculated from the maximum size of the elongation products at the first three time points, were about 15 and 30 nucleotides/s, respectively. After 5 min in the time course, the overall rate of DNA synthesis decreased gradually. This may be caused simply by a slowdown of the rate of DNA synthesis. And, these rates were estimated to be about 8 and 20 nucleotides/s, respectively. These are considerably slower than the estimated 35 nucleotides/s rate of replication fork movement in vivo in S. cerevisiae (22).

Although Pol holoenzyme itself is able to bind a singly primed X174 sscDNA and GINS does not bind either ssDNA or dsDNA, GINS·Pol holoenzyme complex would have some difficulty to bind it, due to the predicted structure of GINS (33). Thus, above experiments were repeated using a singly primed X174 sslDNA coated with yeast RPA. As shown in Fig. 7B, GINS also stimulated DNA synthesis by Pol as much as with X174 sscDNA. Therefore, it is concluded that GINS·Pol complex is able to bind sscDNA as efficiently as it binds sslDNA. The in vitro DNA synthesis reactions catalyzed by Pol or Pol ·GINS were not further stimulated by addition of PCNA and RF-C (Fig. 7C). These results suggest that GINS is an accessory protein for Pol and stimulates the Pol rate and possibly processivity of DNA synthesis in the absence of DNA polymerase clamp, PCNA, and its loader, RFC, in vitro.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
This study shows that purified GINS forms a 1:1 complex with Pol , but not other replicases, (such as Pol or Pol -primase), and that GINS stimulates Pol -catalyzed DNA synthesis in vitro. In the presence of GINS, the rate and possibly processivity of DNA synthesis catalyzed by Pol increase significantly (Figs. 6 and 7). This stimulation is dependent on the ratio of GINS to Pol and also on the ratio of GINS to the DNA substrate (Fig. 6C), unlike other nonspecific DNA-binding proteins. Thus, we concluded that GINS is an accessory protein of Pol and Pol ·GINS complex catalyzes much more rapid and processive DNA synthesis than Pol alone in vitro. Because it is known that Pol readily dissociates from a priming site by addition of excess ssDNA, which may be related to the other function of Pol , S-phase checkpoint regulation (34), we could not use well establishing protocols (such as chasing the reaction with a large excess of template primer or of ssDNA or use of a large excess template primer substrate) for measuring processivity of Pol or Pol ·GINS complex. However, total DNA synthesized by Pol and Pol ·GINS during incubation was less than 2% of total DNA template primer, whose molar concentration was 5-fold excess over that of Pol , and the products were rather uniform in size during the incubation (Fig. 7). Thus, it could be roughly estimated that the processivity of Pol ·GINS is 5.3 kb/binding event in vitro. Interestingly, Pol recycles many times during in vitro DNA synthesis on a linear small template primer in the presence of GINS, but not in its absence (Fig. 6 and supplementary Fig. S1). These reactions were partially substituted by PCNA and RF-C (Fig. 6D). These characteristics suggest that GINS may increase the suitability of Pol for catalyzing leading strand DNA synthesis in vivo. Thus, the data presented here are consistent with the proposal that GINS is a Pol accessory factor and that Pol ·GINS plays a significant role as a leading strand DNA polymerase.

Previous studies showed that GINS associates with origins of replication and adjacent DNA sequences in yeast DNA (30, 31) and that GINS facilitates association of Dpb11p and Cdc45p with chromatin (30). Genetic and physical interaction studies also suggested that GINS interacts with Sld3p and Dpb11p and that Sld3p and Dpb11p facilitate binding of GINS to origins of replication (30). This study provides evidence that GINS is a part of the yeast replisome in vivo (Fig. 1) and that GINS directly interacts with Pol in vitro (Figs. 3 and 4). Other groups also showed recently that GINS is one of many replication proteins found at the replication forks (40, 41). Particularly, it has been shown that all three Pols, , , and , Mcm2-7p, Cdc45p, GINS, and Mcm10p are found in the vertebrate replisome. Furthermore, in the presence of the DNA polymerase inhibitor aphidicolin, which causes uncoupling of a highly processive DNA helicase from the stalled replisome, only Cdc45p, GINS, and Mcm2-7p, but not any of Pols, are enriched at the pause site (41). These results are consistent with our finding of a weak and/or transient association of GINS to Pol holoenzyme. Therefore, we suggest that Dpb11p, Sld3p, Cdc45p, GINS, and Pol assemble in a coordinated manner on replication origins prior to initiation of DNA synthesis in vivo.

One of the functions of PCNA is to recruit Pol onto a primer site and to stimulate the processivity of Pol during Okazaki fragments synthesis on lagging strand DNA (reviewed in Ref. 11. Previous studies also suggest that PCNA and RF-C stimulate Pol -catalyzed DNA synthesis on a singly primed M13 viral sscDNA under certain conditions (42). In this study, we showed that GINS greatly stimulates Pol -catalyzed DNA synthesis on a 65-mer oligonucleotide template annealed with a 34-mer primer in vitro (Fig. 6 and supplementary Fig. S1) and PCNA also stimulates DNA synthesis by Pol in the presence of RF-C, but its stimulation is much less than with GINS (Fig. 6D). Using singly primed linear X174 (about 5.3 kb) as DNA substrates, Pol synthesized a full-length double-stranded DNA product without dissociating from the template and these reactions were stimulated by addition of GINS (Fig. 7, A and B), but not further stimulated by addition of PCNA and RF-C (Fig. 7C). However, these stimulations by addition of GINS were much less than those of a small primer template. This may be because of the unique properties of Pol . We showed previously that Pol , which is actively synthesizing DNA, rapidly dissociates from a priming site by addition of a large single-stranded DNA (34). Alternatively, a singly primed X174 ssc or sslDNA used in this study may not be a representative substrate for leading strand synthesis even in the presence of RPA, thus Pol may have a difficulty to elongate a primer on ssDNA coated with RPA in the presence of GINS. Consistent with this interpretation, other studies show that PCNA and RF-C greatly stimulate both Pol - and Pol -catalyzed elongation of a short oligonucleotide hybridized on X174 or M13 ssDNA in the presence of high salt (10, 42). Thus, we conclude that Pol holoenzyme primarily utilizes GINS as an accessory factor for DNA synthesis in vitro and in vivo.

Based on results from our previous studies (30) and the present article, we propose the following model to explain how Pol ·GINS complex binds a template-primer DNA during DNA synthesis and how both leading- and lagging strand synthesis are achieved by three different DNA polymerases at eukaryotic chromosomal replication forks. Recently, we observed that Xenopus GINS, which was reconstituted and purified from insect cells (33), stimulates DNA synthesis catalyzed by Xenopus Pol as Fig. 6 and supplemental Fig. S1.⁴ Thus, it is highly possible that the structure of S. cerevisiae GINS is similar to that of Xenopus GINS and that GINS acts as a clamp for Pol , although there is no evidence to date that GINS binds ds- or ssDNA in vitro (Fig. 5). In any case, if GINS were a clamp for Pol , then a few new questions arise as followings; how is GINS·Pol loaded onto a template primer in vitro and the replication origin in vivo (presumably RNA-DNA primer synthesized by Pol -primase on a leading strand at origin)? Is there a clamp loader for GINS? It is widely believed that each clamp has a specific clamp loader, proteins that usually belong to the AAA+ protein family (43). Several candidate GINS loaders should be mentioned here, including Mcm2-7p, Pol , or Pol·Dpb11p·Sld2p. Mcm2-7p is the most likely candidate, as it is a AAA+ protein family, is loaded onto replication origins, is a component of the pre-RC during M and G₁ phase, and is a helicase that is predicted to unwind DNA at the replication fork. However, this is unlikely at present time, as GINS stimulates DNA synthesis catalyzed by Pol on a singly primed X174 sscDNA (which has no end) as much as on a singly primed X174 sslDNA (which has ends) without Mcm2-7p (Fig. 7). Thus, as shown in Fig. 8A, we propose that a ringshaped GINS interacts with the C-terminal portion of Pol catalytic polypeptide and with the second subunit of Pol , Dpb2p, and forms a 1:1 complex. Then, the GINS·Pol subsequently undergoes a conformational transition to an "open structure" that binds to the junction between single-stranded and double-stranded DNA by using a single cleft in the Pol catalytic subunit that seems wide enough to accommodate double-stranded DNA (44) and the Dpb2p·GINS, which may interact with a single-stranded template DNA. In our model shown in Fig. 8B, GINS can be located on the leading strand of the replication fork between Cdc45p-Mcm2-7p (40, 41) and Pol during chromosomal DNA replication. Thus, this model differs from a previously proposed model (43). More importantly, the configuration of GINS on the leading strand of the replication fork is different from that of PCNA on the lagging strand, where PCNA is behind Pol (Fig. 8B). If this model were correct, it is still possible that PCNA is able to bind to the priming site of leading strand as to the priming sites of lagging strand with help of RF-C.

View larger version (25K): [in this window] [in a new window]   FIGURE 8. Proposed model for molecular dynamics at the eukaryotic replication fork. A, components of S. cerevisiae Pol holoenzyme. A multiprotein Pol holoenzyme, which is composed of Pol2p, Dpb2p, Dpb3p, and Dpb4p, interacts with a ring-shaped accessory factor, GINS, and forms "an open complex" that is then used by Pol , which has capability of both ds- and ssDNA binding (34, 35), as a tether to the DNA template, although Pol complex itself may accommodate direct interaction to primer template DNA (44). The red wavy line indicates a primer strand. B, in this model, hexameric Mcm2-7p encircles leading strand DNA and Pol is on the leading strand along with heterotetrameric GINS and Cdc45p. Pol is on the lagging strand with PCNA, RF-C, Fen1p, and Dna2p, along with Pol -primase and RPA bound to the looping single-stranded DNA. Although we could not rule out a possibility that PCNA also binds on the leading strand in this study, PCNA is omitted from the figure. Previous studies indicate that Dpb11p and Sld2p are loaded onto replication origin, but they do not travel with the replication fork (27). The purple wavy line indicates newly synthesized DNA, and the red line indicates the RNA primer synthesized by Pol -primase.

	FOOTNOTES

 
^* This work was supported by grants from the Ministry of Education, Science, Sports, Culture, and Technology of Japan (to H. A. and A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental figures.

¹ To whom correspondence should be addressed: Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan. Tel.: 81-6-6879-4661; Fax: 81-6-6879-4663; E-mail: asugino{at}fbs.osaka-u.ac.jp.

² The abbreviations used are: Pol, DNA polymerase; ssDNA, single-stranded DNA; dsDNA, double-stranded DNA; ssc, single-stranded circular; ssl, single-stranded linear; RPA, replicative protein A; RF-C, replication factor C; PCNA, proliferating cell nuclear antigen; Mcm, mini-chromosome maintenance protein; BSA, bovine serum a