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Home , Protein A, Replication factor C, Replication protein A

1993 Oxford University Press Nucleic Acids Research, 1993, Vol. 21, No. 16 3659-3665

DNA unwinding by replication A is a property of the 70 kDa subunit and is facilitated by phosphorylation of the 32 kDa subunit

Anthi Georgaki and Ulrich Hubscher* Department of Veterinary Biochemistry, University of Zurich-lrchel, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland

Received May 28, 1993; Revised and Accepted July 1, 1993

ABSTRACT (RP-A) is a heterotrimeric single- three subunits of 70 kDa, 32-34 kDa and 11-14 kDa and binds stranded DNA binding protein with important functions preferentially to ssDNA through its 70 kDa subunit. RP-A in DNA replication, DNA repair and DNA recombination. appears to have an important role in the T dependent We have found that RP-A from calf thymus can unwind generation of a ss region at the SV40 origin prior to initiation DNA in the absence of ATP and MgCI2, two essential of DNA synthesis. This protein is highly conserved since it has cofactors for bona fide DNA (Georgaki, A., been found to have a similar subunit structure and function in Strack, B., Podust, V. and Hubscher, U. FEBS Lett. 308, Saccharomyces cerevisiae [3]. RP-A has a preference to bind 240 - 244, 1992). DNA unwinding by RP-A was found to ss pyrimidine stretches in a stoichiometry of 1 molecule to to be sensitive to MgCI2, ATP, heating and freezing/ 30 bases [2]. In addition, it has direct effects on DNA thawing. Escherichia coil single stranded DNA binding a and a [4]. RP-A can specifically stimulate DNA protein at concentrations that coat the single stranded ca under conditions where heterologous SSB's cannot regions had no influence on DNA unwinding by RP-A [5]. The 70 kDa subunit is responsible for the interaction with suggesting that RP-A binds fast and tightly to single- DNA polymerase ca/ [6]. The stimulatory effect on DNA stranded DNA. DNA unwinding by RP-A did not show polymerase 6 is more complex. First, DNA polymerase 6 requires directionality. Experiments with monoclonal the presence of at least the two other auxiliary being strongly suggested that the 7OkDa subunit is proliferating cell nuclear antigen (PCNA) and replication factor responsible for DNA unwinding. Phosphorylation of the C (RF-C) for optimal stimulation by RP-A [4, 5]. DNA 32kDa subunit of RP-A by chicken cdc2 kinase polymerase 6 is less dependent on RP-A originating from the facilitated DNA unwinding indicating that this post- same organism, since bacteriophage T4 gene 32 protein and translational modification might be important for SSB's from and adenovirus could substitute for modulating this activity of RP-A. Finally, DNA the RP-A derived from the same tissue. RP-A concentration in unwinding of a primer recognition complex for DNA the SV40 replication system is critical for the specificity for DNA polymerase 6 which is composed of proliferating cell polymerase a [7]. The individual RP-A subunits have different nuclear antigen, and ATP bound to roles in DNA replication, in the sence that DNA polymerase ca a singly-primed M13DNA slightly inhibited DNA is stimulated by the 70 and 32 kDa subunits while DNA unwinding. An important role for DNA unwinding by polymerase 6 on the other hand only by the 70 kDa subunit [8]. RP-A in processes such as initiation of DNA replication, Furthermore, an interaction between RP-A and SV40 T antigen fork propagation, DNA repair and DNA recombination appears to be essential for primosome assembly during SV40 is discussed. DNA replication [9]. The 32-34 kDa subunits of yeast and human RP-A are phosphorylated in a cell-cycle dependent manner INTRODUCTION [10], RP-A-32 appears to be phosphorylated within the replication complex [11] and its phosphorylation is carried out in part by Fractionation of cellular proteins involved in Simian Virus 40 the cdc2 family of protein kinases [12]. Our recent observation (SV40) DNA replication in vitro identified a single-stranded (ss) that RP-A alone is able perform unwinding of DNA might be binding protein (reviewed in [1] and see also references in [2] physiologically relevant to its role in initiation of DNA replication for further citations). Depending on the laboratory this [13]. An early role of RP-A in replication appears to be heterotrimeric protein was called replication protein A (RP-A), suppression of unspecific priming events through its interaction replication factor A (RF-A) or human single-stranded DNA with DNA primase [14], SV40 T antigen [6] and DNA binding protein (HSSB). The protein complex is composed of polymerase a [6]. RP-A from calf thymus could replace the

* To whom correspondence should be addressed 3660 Nucleic Acids Research, 1993, Vol. 21, No. 16 human counterpart in the SV40 replication system [15]. Finally, sodiumbisulfite, lmM PMSF, 0.5 mg/ml TLCK and lytg/ml each RP-A also seems to be involved in cellular DNA repair [16] and of pepstatin, leupeptin, aprotinin and chymostatin. Buffer B: 30 DNA recombination events [17]. mM Tris-HCI (pH 8.0), 0.02%(v/v) Nonidet P-40, We have recently observed that RP-A can unwind DNA 0.125 %(w/v) myo-inositol, 2 mM EDTA, 1 mM dithiothreitol independent of ATP and MgCl2. The length of the DNA and all the protease inhibitors mentioned in buffer A. Buffer C: unwound is more than 350 nucleotides and stoichiometric 20 mM Tris-HCl (pH 8.0), 10%(v/v) glycerol, 0.02%(v/v) amounts of RP-A are required [13]. In this communication we Nonidet P-40, 2 mM EDTA, 1 mM dithiothreitol and all the have further characterized this activity and show that DNA protease inhibitors mentioned in buffer A. unwinding is due to the 70 kDa subunit and that the 500 g of fetal calf thymus (the tissues were stored at -70°C phosphorylation of the 32 kDa subunit in vitro stimulates DNA until use) were homogenized in 1500 ml buffer A in a Waring unwinding while dephoshyphorylation by alkaline phosphatase blender for 5 min. The suspension was centrifuged at 10,000 inhibits the same activity. g for 30 min. After passage through several layers of cheesecloth the cleared supernatant was loaded onto a 350 ml DEAE-Sephacel column equilibrated with buffer B containing 100 mM KCl. The MATERIALS AND METHODS column was washed with 3500 ml buffer B containing 100 mM Materials, proteins and nucleic acids KCI. RP-A was eluted in one step with buffer B, containing 350 Nucleotides, column supports, nucleic acids and other chemicals mM KC1 and the fractions containing RP-A were dialysed and were exactly as descibed [18]. The protease inhibitors pepstatin, loaded onto a 160 ml hydroxylapatite column equilibrated in leupeptin, PMSF, sodium bisulfite, aprotinin, chymostatin, buffer B. RP-A was eluted in one step with buffer B, containing 1-chloro-3-tosylamido-7-amino-2-heptane (TLCK) were from 150 mM potassium phosphate (pH 7.5). The fractions containing Sigma. DNA polymerase 6, Escherichia coli SSB, PCNA, and RP-A were dialyzed against buffer B. The dialyzed RP-A were RF-C were donated by V.Podust (this Institute) and had been brought to 800 mM NaCl. This solution was then loaded onto purified as described earlier [19, 20]. E.Nigg (Lausanne) a 10 ml single-stranded DNA cellulose column equilibrated in provided chicken cdc2 kinase and M.Kenny (Dundee) the four buffer C containing 800 mM NaCl. The column was washed with monoclonal antibodies against RP-A (70A, 70B, 70C and 34A). 100 ml buffer C containing 900 mM NaCl and RP-A eluted with Polyclonal antibodies against PCNA were raised in chickens as 30 ml buffer C containing 2.5 M NaCl. The fractions containing described [21] and a polyclonal against the 40 kDa RP-A were pooled, dialyzed against buffer C and loaded onto subunit ofRF-(; was a gift ofJ.Hurwitz (New York). Calfthymus a 1 ml FPLC Mono Q column equilibrated with the same buffer. histone HI was donated by Ph. Panceter (Zurich), aLkaline Elution of RP-A was performed with a linear gradient of 20 ml phosphatase and the restriction EcoRI, Hind III, Sma from 50 to 500 mM NaCl in the above mentioned buffer. RP-A I, BamHl, were from Boehringer. eluted at 250 mM NaCl and the active fractions in all three assays as describe above were stored in small aliquots in liquid nitrogen Preparation of DNA substrates until further use. A DNA was finally separated on the A short substrate (24mer) hybridized onto a M13mp8 DNA was last column from RP-A and eluted at 300-350 mM NaCl (see prepared as outlined for DNA helicases [18]. To determine the Results, Figure 1). direction of unwinding two substrates were constructed from a 30mer XbaI-EcoRI polylinker oligonucleotide hybridized to Inactivation by heating and freezing/thawing ssM13mpl 1 DNA and restricted with SmaI according to Heat inactivation was performed by heating 300 ng RP-A for Thommes et al.(1992). 3 min at different temperatures (0, 24, 37, 60, 80°C) and the samples were subsequently added to the reaction mixtures as Assays for RP-A described for the DNA unwinding assay. For freezing/thawing: RP-A were frozen in thawed on ice and The fate of RP-A during isolation was followed with three assays: 300 ng liquid nitrogen, added to the reaction mixtures as described for the (i) by testing individual fractions with a polyclonal antibody raised subsequently in chicken; (ii) by in vitro complementation of DNA polymerase DNA unwinding assay. 6, PCNA and RF-C on a singly-DNA primed M13 DNA template Experiments with monoclonal antibodies against RP-A as described below and (iii) with an DNA unwinding assay. Unwinding of the short 24 mer substrate was carried out in a In preliminary experiments we have found that the four final volume of 25 ,d containing: 20 mM Tris-HCI (pH 7.5), monoclonal antibodies against human RP-A (ceRP-A 70A, oa RP- 4%(w/v) sucrose, 8 mM dithiotreitol, 80 yg/ml bovine serum A 70B, aRP-A 70C and aRP-A 34A) [8] reacted in immunoblots albumin, 10 ng DNA substrate (3000 cpm/pmol) and protein to with calf thymus RP-A (data not shown). The interactions with be tested. Incubation was for 60 min at 37°C unless otherwise the monoclonal antibodies were carried out in a final volume of mentioned. The reaction was stopped, the product electro- 22.5 1l containing: 20 mM Tris-HCI (pH 7.5), 4% (w/v) phoresed, autoradiographed and quantified as described [18]. sucrose, 8 mM dithiotheitrol, 80 tg/ml bovine serum albumin, 150 ng RP-A (to give about 20% unwinding) and antibodies to Purification of RP-A be tested. The reactions were incubated under gentle mixing for The purification of RP-A was essentiallly as described [13]. 60 min at 4°C. After addition of 2.5 Al (10 ng) of DNA substrate Briefly, the purification procedure included the four steps DEAE- DNA unwinding was measured as decribed above. Sephacel, hydroxylapatite, ssDNA cellulose and FPLC Mono Q. Buffers: buffer A: 20 mM Tris-HCI (pH 8.0), 10 mM KCI, In vitro phosphorylation of calf thymus RP-A 250 mM sucrose, 10% (v/v) glycerol, 1 mM dithiothreitol, 2 The in vitro phosphorylation of RP-A was carried out in a final mM EDTA, 0.5 mM spermidin, 10 mM benzamidine, 10 mM volume of25 Al containing: 50 mM Tris-HCI (pH 7.5), 10 mM Nucleic Acids Research, 1993, Vol. 21, No. 16 3661

MgCl2, 1 mM dithiothreitol, 4 mM ATP, 0.2 yd chicken cdc2 A. S 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 cd ..I i kinase (200 pmole/s/4l) and 5 itg of RP-A. The reaction was _lo,400 incubated for 30 min at 24°C. As a positive control for cdc2 kinase 5 Ag calf thymus histone HI were phosphorylated in the same way. In vitro dephosphorylation of calf thymus RP-A a' The in vitro dephosphorylation of RP-A was carried out in a final volume of 25 ,tl containing: 50 mM Tris-HCI (pH 8.5), 0.1 mM EDTA, 5 U alkaline phosphatase and 5 itg RP-A. The reaction was incubated for 3 min at 37°C followed by 30 min at 240C. B s 31 33 35 37 39 41 43 45 47 49 51 53 55 57 59 cd _ ..______In vitro replication of singly primed M13 DNA This replication assay was carried out as described [19]. A final volume of 25 tl contained the following components: 40 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 10 mM MgCl2, dATP, dGTP, dCTP each at 50 ,uM, 15 AM of [3H]dTTP (1500 cpm/pmol), 100 ng of singly-DNA primed M13 DNA, 100 ng of PCNA, 10 ng RF-C, 350 ng ofEscherichia coli SSB, and 0.25 U of DNA polymerase C. 37 39 41 43 45 47 | a. The reactions were incubated for 60 min at 37°C and DNA ..! . .... synthesis measured as described [19]. 70 kDa 4m -a "I=IS 55 Isolation of the primer recognition complex with gel filtration .. ...- _ _ A final volume of 100 ,ul contained: 40 mM Tris-HCI (pH 7.5), 34 assi_-- . 1 mM dithiothreitol, 0.2 mg/ml bovine serum albumin, 10 mM 'I MgCl2, 600 ng singly-DNA primed M13DNA, 2 jg 11 __ Escherichia coli SSB, 600 ng PCNA, 15 ng RF-C, and 1 mM ATP. After incubation for 2 min at 37°C, the mixture was x __...1 immediately loaded on a 1.8 ml (0.4 14 cm) Bio-Gel A-Sm Figure 1. FPLC Mono Q separates calf thymus RP-A from a DNA helicase. column equilibrated in 30 mM Tris-HCI (pH 7.5), 20 mM Chromatography on FPLC Mono Q was performed as described in Materials NaCl, 5% (v/v) glycerol, 2 mM dithiothreitol, 8 mM MgCl2 and Methods. 1 pd of every other fraction tested for DNA unwinding in the absence and 0.1 mg/ml bovine serum albumin. Gel filtration was carried of ATP and MgCl2 (A) and in the presence of lmM each of ATP and MgCl2 out at 4°C with a flow rate of 2 ml/h. Two-drop fractions (B) C: 2 isg of the peak fractions of the DNA unwinding activity dependent on ATP and MgCl2 were loaded onto a 12.5% SDS polyacrylamide gel, separated corresponding to about 90 Al were collected. Under these and silver stained. conditions DNA mainly eluted in fractions 5 and 6 and free nucleotides in fractions 12-16. The fractions were assayed for DNA synthesis activity by incubating 22,l with 3,tl of a solution which yielded finally 50 ltM each of dATP, dCTP, dGTP and MgCl2 [13]. Figure 1 shows that on FPLC Mono Q an 15 ,uM [3H]dTTP (1500 cpm/pmol) and 0.25 units DNA unwinding activity independent of ATP and MgCl2 separated pollymerase 6. Incubation was for 30 min at 37°C followed by from an unwinding activity that was evident only in the presence the determination of acid-precipitable material. of the two cofactors mentioned and thus being a bonafide DNA helicase activity (compare Figure IA and 1B). The peak of ATP Other methods and MgCl2 independent DNA unwinding activity contained the Protein concentrations were determined according to Bradford subunits of RP-A, being 70 kDa, 55 kDa (a proteolytic [22], immunoblots performed according to Towbin et al. [23] degradation product of the 7OkDa subunit always seen in calf and for silver staining the protocol of Arezzo and Rose [24] was thymus, even in the presence of eight different protease inhibitors, employed. [13]), 32 kDa and I1 kDa sububits (Figure 1C). The subunits reacted with antibodies raised against the individual subunits of RP-A (data not shown). The DNA helicase separated from the RESULTS RP-A will be described elsewhere. Separation of the calf thymus RP-A unwinding activity from Effect of MgCl2, ATP, heat and freezing/thawing on the a DNA helicase unwinding activity of calf thymus RP-A The purification procedure for RP-A includes the four steps MgCl2 at 0.750 mM inhibited unwinding to 50% in the absence DEAE-Sephacel, hydroxylapatite, ssDNA cellulose and FPLC of ATP (Figure 2A), while 1.5 mM were required to observe Mono Q [13] and the procedure was followed by using the three the same effect if 1mM ATP was included in the reaction assays of (i) immunoblotting for RP-A, (ii) complementation of (Figure 2B). ATP on the other hand in the absence of MgCl2 DNA polymerase 6 in a holoenzyme assay [19] and (iii) by DNA stimulated unwinding 2-fold at 500 ,uM, while 1 mM and more unwinding [13]. RP-A after the FPLC Mono Q column was abolished the activity (Figure 2C). Complex formation of Mg- shown to have unwinding activity independent of ATP and ATP is a likely explanation for this result. In the presence of 3662 Nucleic Acids Research, 1993, Vol. 21, No. 16 A.

c EI

a

MgCi2 (mM) MgC12 (mM)

C. B. i 4

60-

2. 40 aI U9

20

2 3 4 5 6 0 1 2 3 4 0 ATP (mM) ATP (mM) 0 2 4 6 8 10 Freezing / thawing (x)

Figure 2. Effect of ATP and MgC12 on the DNA unwinding by calf thymus RP- A. The DNA unwinding experiments were performed as described in Materials FIgure 3. Inactivation of calf thymus RP-A by heating (A) and by fizing/thawing and Methods by using 150 ng RP-A and by varying the ATP and MgCl2 (B). Heating and freezing/thawing experiments were performed by using 300 concentrations either alone (A, C) or in the presence of 1mM ATP (B) or MgC12 ng of RP-A with subsequent testing for DNA unwinding as described in Materials (D). and Methods.

1 mM MgCl2 and absence of ATP unwinding is inhibited more than 50% (Figure 2A). This inhibition of unwinding at 0 mM ATP is overcome by the addition of 2 mM ATP (Figure 2D) suggesting that a Mg-ATP complex did not interfere anymore with the unwinding by RP-A. The unwinding activity is very labile since heating for 10 min at 25 -37°C reduced the activity by more than 40% (Figure 3A). Upon freezing/thawing twice RP-A unwinding was reduced more than 70% and after five times a complete loss of the activity was seen (Figure 3B). These data 0 100 300 indicated that the unwinding activity can easily go undetected RP-A(ng in the presence of MgCl2 or ATP and by the usual handling of the protein. Figure 4. DNA unwinding by calfthymus RP-A on an Escherichia coli covered primed DNA substrate. DNA unwinding was performed with 10 ng substrate Eschenchia coli SSB does not affect unwinding by calf thymus by testing 0, 25, 50, 100, 200 and 300 ng RP-A. 10 ng of the substrate (for RP-A details see Materials and Methods) were preincubated for 1 min on ice with none (open circles), 30ng (closed circles) or 100 ng (open squares) Escherichia coli a Under optimal conditions for DNA unwinding of 24mer SSB. Data represent a mean value of three experiments carried out in triplicate. substrate annealed to ssM13DNA no unwinding was observed with Escherichia coli SSB alone [13]. Titration of RP-A in the presence of increasing amounts of the bacterial SSB showed no nucleotide hybridized to ssM13mpl DNA and restricted with stimulation of unwinding by RP-A (Figure 4). This observation SmaI [18]. RP-A can unwind DNA with the 5'- 3' and 3'-.5' suggested that RP-A appears to bind more efficiently or more directionality substrate indicating that RP- A has no preference tightly to ssDNA than the bacterial counterpart since at all for the direction of unwinding (Figure 5). This result was not concentrations of SSB tested stoichiometric amounts of RP-A, unexpected since the herpes simplex virus type 1 ICP8 protein, enough to cover the ss region ofthe DNA were required to detect which is an analogous protein of RP-A has been shown to unwind DNA unwinding. SSB alone did not show unwinding under these in both directions [25]. conditions (Figure 4, data points at 0 ng RP-A). The 7OkDA subunit of calf thymus RP-A appears to be Unwinding by calf thymus RP-A shows no directionality responsible for DNA unwinding To determine the direction of unwinding two substrates were Polyclonal antibodies raised in chickens against the heterotrimeric constructed from a 30mer XbaI-EcoRl polylinker oligo- protein as well as against the three subunits individually did not Nucleic Acids Research, 1993, Vol. 21, No. 16 3663

A. s@A3

SI 4Smai

1j i 35'-:3' direction S.

3JIII 3'-Sdirectiond 3.

B. Antibodies (pg)

Figure 6. The 70kDa subunit of calf thymus RP-A appears to be responsible for DNA unwinding. 150 ng RP-A (enough to give 20% unwinding) were incubated with vanous amounts of monoclonal antibodies (0-1 zg) dircted against

80. the 70 kDa (aRP-A70A, ciRP-A70B and aRP-A70C) and the 34 kDa subunit (cxRP-A34A). After 60 min at 0°C the fractions were tested for DNA unwinding 8 as described in Materials and Methods. Data represent a mean value of three experiments carried out in triplicate. ai

A. RP-A ne.control Hi 94 kDa -67 3P4L32 RPA -43 -30 RP-A (ng) -20.1 -14.4

Figure5. Calf thymus RP-A has no directionality ofunwinding. The directionality B. substrate was prepared as outlined by Th6mmes et al. (1992). Different amounts (0-200 ng) of calf thymus RP-A were titrated with the 5'-3' (open circles) so. e: lsoited RP-A and the 3'-5' (closed circles) directionality substrate as described in Materials b: phoPIyt RP-A and Methods. Data represent a mean value of three experiments carried out in c: dp bo d RP-A triplicate. 60.

40 affect the DNA unwinding of RP-A (data not shown). When, however, the well characterized monoclonal antibodies caRP- 20. A70A, aRP-A70B and aRP-A70C [8] were tested, the results shown in 6 Figure indicated that only the (xRP-A70C abolished ¢ unwinding, while the oaRP-A70A, caRP-A70B as well as the aRP- A34A had no inhibitoy effect. These results nicely support an earlier observation by Kenny et al.[8] in the SV40 in vitro C. replication system where the aRP-A70C antibody was able to (0) beolatd RP-A abolish the SV40 origin specific unwinding in the presence of (0) phoephoWrted RP-A (A) deposphorylted RP-A the large T antigen, RP-A and DNA I. SV40 origin dependent unwinding on the other hand was not affected by the aRP-A70A, aiRP-A70B and aRP-A34A antibodies [8] a result which is consistent with the data in Figure 6. From these we experiments conclude that the 70kDa subunit of RP-A is likely 0 160 200 responsible for DNA unwinding by RP-A. Finally, the aRP- RP-A (ng) A34A antibody stimulated unwinding by RP-A four fold. Figure 7. In vitro phosphorylation of the 32 kDa subunit facilitates unwinding In vitro phosphorylation of the 32kDa subunit facilitate DNA by calf thymus RP-A. In vitro phosphorylation of calf thymus RP-A and unwinding by calf thymus RP-A dephosphorylation were performed as described in Materials and Methods. A: RP-A (250 ng) was phosphorylated with cdc2 kinase in the presence of a The 32kDa subunit of RP-A is phosphorylated in cell-cycle [.y32P]ATP, separated on a 12% SDS polyacrylamide gel and the radioactive dependent manner [10] and cdc2 family kinases appear to be labeled protein detected by autoradiography as described in Materials and Medtods. responsible for phosphorylation leading to activation of DNA As a control the same labeling was performed with calf thymus histone Hi (5 replication [12]. In addition it had been shown that jLg). B: DNA unwinding by RP-A was carried opit with isolated RP-A (a), after in vitro phosphorylation (b) or after in vitro dephosphorylation (c). C: Titration phosphorylation appears to occur witiin the replication initiation of RP-A unwinding included the same amounts of isolated (open circles), complex [11]. We therefore tested the effect of RP-A phosphorylated (closed circles) and dephosphorylated (closed triangles) forms of phosphorylation/dephosphorylation on DNA unwinding. the protein. 3664 Nucleic Acids Research, 1993, Vol. 21, No. 16

A. 81 DNA and this complex can be isolated on a Bio-gel 5A column 0~~~~~~ [19]. We therefore isolated two substrates, namely (i) an .2 iO X~~~no, Escherichia coli SSB-coated sinlgy-primed M13DNA in the o mple absence ofthe primer recognition complex and ii) an Escherichia S O coplex coli SSB-coated sinlgy-primed ssM13 DNA containing a primer a. recognition complex. The second substrate contained PCNA and a 0 RF-C after gel filtration on a Bio-gel 5A column as tested in two Ij immunoblots and was able to support DNA replication upon O- . . . 0 20 40 60 8U addition of DNA polymerase 6 (data not shown). From Figure 8A RP-A (ng) and 8B it is evident that rate and extent of DNA unwinding was only slightly affected by the primer recognition complex B. indicating that RP-A could also unwind a substrate that contains no the auxiliary proteins for the DNA polymerase 6 action but less 160 complex 0-S0c efficienly than in their absence. E ---- C,complex 040- a. DISCUSSION 20. RP-A from calf thymus copurifies with a DNA helicase and can to yield an apparently 0 10 20 30 40 finally be separated from the DNA helicase Time (min) homogeneous preparation of of RP-A. The unwinding activity does not require ATP and MgC12 two essential cofactors for DNA helicases [27] and is extremely labile to heat as well as Figure 8. Unwinding of calfthymus primer recognition complex by calf thymus very sensitive to freezing/thawing. Escherichia coli SSB does RP-A. A: DNA unwinding was measured with increasing amounts ofcalf thymus RP-A by using Escherichia coli SSB-covered singly-primed M13 DNA (open not affect DNA unwinding by RP-A. Since the same amount of circles) or Escherichia coli SSB-covered singly-primed Ml3DNA as a substrate RP-A was required for unwinding ifEscherichia coli SSB covered in the presence of PCNA, RF-C and ATP (closed circles), representing a primer the entire ss region of the unwinding substrate, suggesting that recognition complex [19]. B: Time course of DNA unwinding with 50 ng RP-A RP-A might displace Escherichia coli SSB from the ss DNA. in the absence (open circles) or presence (closed circles) of the primer recognition The protein showed no directionality while unwinding since it complex. acted on two directionality substrates to determine 3'-5' and 5'- 3' direction [18]. experiments Figure 7A shows that the 32kDa subunit was preferentially suggested that the 70 kDa subunit of RP-A is responsible for phosphorylated in vitro by chicken cdc2 kinase. Phosphorylation unwinding. by cdc2 kinase stimulated DNA unwinding (Figure 7B and C). Phosphorylation of RP-A32 stimulated and dephosphorylation Under conditions where isolated RP-A (which represent a mixture inhibited unwinding suggesting an important role of of phoshporylated and non-phosphorylated RP-A, see [13] and phosphorylation for DNA unwinding. It has been demonstrated Figure 1 therein) exhibited 15% unwinding (140 ng) phosphoryl- that the 32kDa subunit of RP-A is phosphorylated in a cell cycle ation facilitated unwinding 5-6 fold, while dephosphorylation dependent manner [10], that phosphorylation appears to occur by aLkaline phophatase reduced DNA unwinding to less than within the replication initiation complex [11] and that this 10%. Titration of these three forms of RP-A confirmed that modification is carried out by the cdc2 family protein kinases phosphorylation reduced the amount of RP-A required for [12]. Thus after phosphorylation DNA unwinding could facilitate unwinding, since complete unwinding (>80%) was achieved origin unwinding to occur and thus DNA synthesis to start. The with the phosphorylated form while the non-phosphorylated fact that RP-A interacts with the origin binding protein SV40 protein at the highest concentration tested (250 ng) did only T antigen [6], with DNA polymerase a/primase [6] and with unwind to 60%. Extrapolating the curve to saturation predicts several cellular DNA helicase in a species specific way [18, 28] that 600-700 ng of unphosphorylated RP-A would be required suggests that the process of DNA unwinding by RP-A might be to unwind more than 80% under these conditions. Thus tightly coupled through protein-protein interaction with phosphorylation stimulated DNA unwinding by RP-A up to 5-fold replication proteins and that phosphorylation may influence these under these DNA unwinding conditions. The data from Figures interactions. Indeed an interaction of RP-A with SV40 T antigen 1-6 were influenced in an analogous manner as documented in might be essential for the correct assembly of a primosome on Figure 7. Two to three times less RP-A was required to give a ssDNA coated with RP-A [9]. Furthermore, in the presence the same effect ofunwinding and a filter binding assay experiment of RP-A, the SV40 T antigen, that was complexed to the SV40 with ssDNA indicated that the phosphorylation status of RP-A started unwinding of the DNA [29]. At low had no influence on the ssDNA binding activity of RP-A (data T antigen concentrations and in the presence of ATP, DNA not shown). polymerase at stimulated the binding of T antigen to the core origin, while RP-A did not, but this stimulation finally lead to Unwinding of a primer recognition complex by calf thymus an increase in the subsequent unwinding in the presence of RP- RP-A A [29]. Efficient replication of primed M13 DNA by DNA polymerase DNA unwinding has been observed with ICP8 of Herpes 6, requires PCNA, RF- C, RP-A (or Escherichia coli SSB) and Simplex Virus type 1 which also promotes strand displacement ATP [19, 26]. PCNA and RF-C in the presence of ATP can form independent of ATP and MgCl2 and exhibits no directionality a primer recognition complex on SSB-coated primed ss M13 [30]. The in vitro unwinding activity of RP-A and ICP 8 might Nucleic Acids Research, 1993, Vol. 21, No. 16 3665 favour functions for these protein in DNA replication to 23. Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. destabilize DNA during origin unwinding and possibly also during USA 76, 4350-4354. replication fork movement. 24. Arezzo, F. and Rose, K. M. (1987) Anal. Biochem. 167, 387-393. 25. Boehmer, P. E. and Lehman, I. R. (1993) J. Virol. 67, 711-715. What might the function of DNA unwinding by RP-A at the 26. Podust, V. N., Mikhailov, V., Georgald, A. and Huibscher, U. (1992) replication fork itself and in processes such as DNA repair and Chromosoma 102, 133-141. DNA replication? As suggested by the data in Figure 8 unwinding 27. Th6mmes, P. and Hubscher, U. (1992) Chromosoma 101, 467-473. occurs even in the presence of an primer recognition complex 28. Seo, Y.-S., Lee, S.-H. and Hurwitz, J. (1991) J. BioL Chem. 266, and ATP on an Escherichia SSB 13161-13170. (PCNA, RF-C coli coated 29. Murakami, Y. and Hurwitz, J. (1993) J. Biol. Chem. 268, 11008-11017. singly-primed M13DNA) that is tightly bound to a primer [19]. 30. Boelmer, P. E., Dodson, M. S. and Lehman, I. R. (1993) J. Biol. Chem. However less unwinding was observed in comparison to a primer 268, 1220-1225. free of the primer recognition complex. One function of RP-A could be to remove unproductive (= free) or abortive (primer recognition complexes not or already used by DNA polymerases 6 or e) primers thus allowing the assemly of a primosome [9]. Removal of primers might not only be important at the replication fork but also for processes such as DNA repair (e.g. nucleotide excision) or .

ACKNOWLEDGEMENTS We thank R.Fotedar (Grenoble) for stimulating discussions and for critical reading of the manusrcipt, V.Podust (this Institute) for the components of DNA polymerase 6 holoenzyme and for critical reading of the manuscript, B.Sturzenegger for her excellent technical assistance, E.Nigg (Lausanne) for chicken cdc2 kinase and M.Kenny (Dundee) for monoclonal antibodies against RP-A. This work was supported by the Swiss Cancer Society, by the Swiss National Science Foundation (grant 31.28692.90) and by the Kanton of Zurich.

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