- Email: [email protected]
Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication
FEBS Letters 581 (2007) 3973–3978
Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication Poonam Tanejaa,1, Irene Bochea,2, Hella Hartmannb,3, Heinz-Peter Nasheuerc, Frank Grosseb, Ellen Fanninga, Klaus Weisshartb,* b
a Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA Leibniz Institute for Age Research–Fritz Lipmann Institute (former Institute for Molecular Biotechnology), Beutenbergstrasse 11, 07745 Jena, Federal Republic of Germany c Department of Biochemistry, National University of Ireland, Galway, Ireland
Received 25 April 2007; revised 6 July 2007; accepted 16 July 2007 Available online 25 July 2007 Edited by Angel Nebreda
Abstract Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase a-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein–protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: SV40 DNA replication; Replication protein A; Protein–protein interaction
1. Introduction Replication protein A (RPA), a hetero-trimeric protein involved in various aspects of DNA metabolism, is highly conserved in its subunit structure, DNA binding activity and posttranslational modifications in all examined eukaryotes [1,2]. The requirement of all three genes coding for the p70, p32 and p14 subunits for cell viability in yeast argues that RPA functions in vital reactions as a complex [3]. The integrity of the human trimer is also needed for Simian virus 40 (SV40) cell-free in vitro DNA replication, since neither the p70 subunit alone nor a sub-complex of p32 and p14 were able to support the reaction and antibodies directed against any one of the subunits were able to inhibit the process [4,5]. * Corresponding author. Present address: Carl Zeiss MicroImaging GmbH, Carl-Zeiss-Promenade 10, 07745 Jena, Germany. Fax: +49 3641 643144. E-mail address: [email protected] (K. Weisshart). 1 Present address: Molecular Devices Corporation, 1311 Orleans Drive, Sunnyvale, CA 94089, USA. 2 Present address: Xantos Biomedicine AG, Max-Lebsche-Platz 31, 81377 Muenchen, Germany. 3 Present address: Biotechnology Center, TU Dresden, Tatzberg 47-51, 01307, Germany.
Human RPA’s tight association to single-stranded DNA (ssDNA) is mediated primarily by p70 [4,6]. The subunit is oriented on the DNA in a polar fashion and positions the p32 subunit in the vicinity of the 3 0 -end of a primer-template junction [7]. Binding occurs in three modes, whereby the protein occludes 8–10, 12–23 and 28–30 nucleotides, respectively [8– 10]. The crystal structure of the ssDNA binding domain of p70 bound to DNA comprises two structurally homologous sub-domains, the so-called oligosaccharide/oligonucleotide binding (OB)-folds, which form the DNA binding domains A and B (DBD-A and DBD-B) [8]. The ssDNA lies in a cleft extending from one domain to the other [11]. OB-folds corresponding to other putative DNA binding domains have been mapped to the N- and C-termini of p70 (DBD-C and DBDF) and both the p32 (DBD-D) and p14 (DBD-E) subunits [12–16]. a-helices C-terminally located to OB-fold motifs of DBD-C, -D, and -E are arranged in parallel and mediate trimerization [9]. The orchestration of SV40 in vitro DNA replication requires the interaction of RPA with the viral initiator protein T-antigen (Tag) and the cellular DNA polymerase a-primase (Pol) and these interactions were mediated mainly by p70 and p32 [17–20]. Interaction sites of different strengths have been mapped to DBD-A and N-terminal sequences of p70 and to a winged helix–loop–helix motif in the C-terminus of p32 [18,21,22]. To probe the importance of p70’s interaction with the other two subunits, DNA and its replicative partner proteins we have expressed specific regions corresponding to formerly defined structural domains and sub-sequences of those [23]. These peptides displayed properties expected from previous deletion analysis. Peptide inhibition studies revealed that the cooperation of all activities of the p70 subunit is essential for the initiation process and that a stable primosome is assembled on a primer, which acts in a processive manner.
2. Materials and methods 2.1. Cloning and expression of RPA subunits RPA subunit sequences were amplified from a cDNA library obtained with the 5 0 -RACE kit of GibcoBRL from human 293S cells using primer pairs that allowed for directional cloning in amplification and expression vectors. Subunits were either expressed alone or linked together in various combinations. Likewise sub-regions of p70 were expressed alone or co-transcriptionally with p14 and p32.
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.07.038
3974
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978
2.2. Expression and purification of proteins Baculovirus expressed Tag and Pol were immunoaffinity purified. Bacterially expressed RPA, his-tagged RPA (H6-RPA) as well as topoisomerase I from calf thymus were purified by a combination of affinity, adsorption and ion-exchange chromatography steps. Maltose binding protein (MBP)-fusion proteins were affinity purified on amylose resin according to the manufacturer’s instructions (New England Biolabs). All p70 derived fusions were soluble and concentrations of 1 mg/ml and higher were obtained. If required, fusion peptides were concentrated to 5–10 mg/ml using Centricon P-20 centrifugal filter units (Millipore).
nucleotides. After removal of proteins from the rest of the sample by phenol/chloroform extraction DNA was ethanol precipitated and double restricted with EcoRI to linearize the DNA (form II) and DpnI to remove any un-replicated template DNA. Products were separated by agarose gel electrophoresis and analyzed by autoradiography. Peptides were added at different time points to the reaction at a 5–100-fold molar excess over Tag. Alternatively, the replication reaction was stalled at the unwinding, primer synthesis and primer extension steps by omitting ATP, other rNTPs and dNTPs, respectively. Peptides added to these reactions were pre-incubated with the mix for 30 min prior to the addition of the missing nucleotides to relieve the replication block.
2.3. Protein–protein interactions 17.5 pmol RPA (0.116 lg/pmol) were incubated with an equal amount of Tag or Pol, which were coupled to beads via specific antibodies, in the absence or presence of a 100-fold molar excess of peptides. The amounts of co-precipitated RPA were quantified by densitometric analysis of western blots developed with enhanced chemoluminescence (ECL, GE Healthcare). Alternatively, Tag and Pol were passed over amylose columns to which MBP-fusion proteins were coupled at a ratio of 1 mg/ml. Bound proteins were eluted and detected by western blotting using ECL.
2.7. DNA unwinding For bidirectional unwinding reactions 6.25 pmol Tag (0.096 lg/ pmol) were incubated with 0.1 pmol supercoiled SV40-origin containing pUC-HS DNA in the presence of 1.2 pmol toposiomerase I (0.1 lg/ pmol) and 4 pmol RPA (0.116 lg/pmol) Where indicated, RPA was replaced by the same amounts of MBP-fusion peptides. Alternatively peptides were added in addition to RPA. Under-wound DNA (form U) was detected by agarose gel electrophoresis and ethidium bromide staining and quantified by densitometric scanning. Detailed protocols can be obtained in the methods section of the Supplementary data.
2.4. Protein–DNA interactions 100 pmol of RPA (0.116 lg/pmol) or MBP-fusion peptides were incubated with 0.083 pmol circular single-stranded M13mp18 DNA (2.4 lg/pmol). Free DNA was separated from protein–DNA complexes by agarose gel electrophoresis. DNA was visualized by ethidium bromide staining. 2.5. Subunit complex formation Various p70 MBP-fusion proteins were co-transcriptionally expressed with p14, p32 or both. Extracts were passed over amylose resin to capture the MBP fusion and co-eluted subunits were analyzed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting. To test for soluble proteins, crude extracts were analyzed in parallel. 2.6. SV40 in vitro DNA replication SV40 in vitro DNA replication was carried out with S100 extracts prepared from human 239S cells using 0.1 pmol of pUC-HS DNA (2 lg/pmol), which contains the complete SV40 origin. The assay was supplemented with ATP to drive the unwinding reaction catalyzed by Tag, as well as other ribonucleotides (rNTPs) and (radioactive labeled) deoxyribonucleotides (dNTPs) to support the primer synthesis and primer extension reactions catalyzed by Pol. The mix was assembled on ice and the reaction was started by the addition of 6.25 pmol Tag (0.096 lg/pmol). Incubation lasted for 120 min at 37 C. Five ll of the reaction were spotted on DE81 paper to quantify incorporated
3. Results 3.1. Activities of p70 peptides To probe the importance of different parts of the p70 subunit of replication protein A (RPA) for the replication process we have expressed different domains and sub-regions thereof as soluble maltose binding protein (MBP) fusion proteins. Amino acids 174–330 were sufficient for DNA binding, 1–173 for binding DNA polymerase a-primase (Pol), 174–250 for binding both SV 40 large T-antigen (Tag) and Pol, and 421–525 as well as 525–616 for complex formation with the other two smaller subunits (for a detailed characterization see results section and Figs. 1 and 2 of the Supplementary data). All fragments containing amino acids 174–250 reduced the amounts of RPA co-precipitated with Pol and Tag, respectively (Fig. 1). Peptide 1–173 reduced the co-precipitated RPA only to a minor extent if tested with Pol, but showed no effect on RPA’s interaction with Tag. C-terminal derived peptides did not reduce the amounts of co-precipitated RPA. Peptide 174–250 was chosen to probe the importance of protein–protein interactions between replicative factors during cell
Fig. 1. Interference of p70 peptides with protein interactions. Co-precipitations of 17.5 pmol soluble H6-RPA were performed with the same amounts of immobilized Tag (Panel A) and Pol (Panel B) in the presence of a 100-fold molar excess of the indicated p70 competitor polypeptides. Bound H6-RPA was detected with his-tag specific antibodies by western blotting and ECL detection. The numbers below the lanes refer to the fractions of H6-RPA bound in relation to the amount obtained in the presence of MBP. The position of the p70 subunit bearing the histidin-tag is indicated. Lane 1 represents a loading control of 1/10th of the input material, whereas in lane 2 no RPA was added.
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978
3975
Fig. 2. SV40 DNA replication in the presence of peptide 174–250. (Panel A) Inhibition of SV40 DNA replication by peptide 174–250. The peptide was titrated into the assay in increasing quantities defined as a molar excess over the 6.25 pmol of Tag present in the assay (lanes 3 and 4). Lane 2 (0·) contained no peptide and a control reaction MBP in a 100-fold molar excess over Tag (lane 7). Tag was back titrated with the indicated amounts where indicated (lanes 5 and 6). Lane 1 (Tag) contained no Tag to determine background replication. (Panel B) Kinetics of inhibition by peptide 174–250. The peptide was added to the mix at the indicated times after start of the replication reaction with a 100-fold molar excess over the 6.25 pmol of Tag (lanes 3–8). Incubation continued for a total of 120 min. Reactions in the presence (+Tag, lane 1) and absence (Tag, lane 2) of Tag without peptide served as positive and negative controls, respectively. (Panel C) Kinetics of inhibition relief by extra Tag. Reactions were supplied with peptide 174–250 at the onset of the reaction in a 100-fold molar excess over the 6.25 pmol of Tag. After the indicated times an additional 6 pmol of Tag was added (lanes 3–8). Incubation continued for a total of 120 min. Reactions in the presence (+Tag, lane 1) and absence (Tag, lane 2) of Tag without peptide served as positive and negative controls, respectively. The position of the linearized plasmid DNA (form II) is indicated. The numbers below each lane indicate the pmol of dNMPs incorporated.
free SV40 DNA replication because it interfered reasonable well with RPA’s ability to bind to both Tag and Pol, but had on its own no DNA binding activity. 3.2. Influence of peptide 174–250 on cell-free SV40 DNA replication If peptide 174–250 was titrated into the replication assays in the presence of Tag, incorporation rates decreased in a dose dependent manner (Fig. 2A, lanes 2–4). Adding back extra molecules of Tag restored incorporation levels (lanes 4–6). MBP alone had no adverse effect on the replication efficiency (lane 7). Even at a 100-fold molar excess of the peptide over Tag, replication rates did not drop to zero as exemplified for a reaction without Tag (compare lane 4 to lane 1). The peptide interfered with the replication reaction only when it was titrated into the assay within a maximum time delay of 15 min after start of the reaction (Fig. 2B, lanes 4–7). In contrast, incorporation levels comparable to the control reaction without peptide were obtained when the peptide was added after 30 or 60 min (compare lanes 7 and 8 to lane 1). Back titration of Tag at various times in the presence of the inhibitory peptide could more effectively restore the replication activity if Tag was added within 15 min after start (Fig. 2C, lanes 3–6). In these cases, incorporation rates that closely matched the one obtained in the control reaction were obtained (lane 2). If Tag was added after 30 or 60 min had elapsed, its positive influence on DNA synthesis was substantially decreased (lanes 7 and 8). 3.3. Influence of peptide 174–250 on different steps during cellfree SV40 DNA replication The initiation of SV40 DNA replication can be divided into different steps including the assembly of the pre-initiation complex, bidirectional unwinding catalyzed by Tag in the presence
of a single-stranded DNA binding protein, as well as primer synthesis and primer extension carried out by Pol (Fig. 3A). Complex formation and unwinding reaction comprise the pre-initiation phase that lasted for about 15 min (see supplementary Fig. 3). Reactions were set up with certain nucleotides omitted to stall the reaction before some of these steps. Peptide 174–250 was added to these stalled reactions and incubated with the mix before release of the block by providing the missing nucleotides. The peptide did not inhibit the unwinding reaction per se, if it was supplemented prior to the addition of ATP (Fig. 3B, compare lanes 1 and 2). If peptide 174–250 was present in the replication reaction before the unwinding step, it inhibited nucleotide incorporation to a great extent (Fig. 3C, lane 1). Intermediate and no inhibition were observed if the peptide was added prior to the primer synthesis (lane 2) and primer extension (lane 3) reactions, respectively. 3.4. Influence of other p70 peptides on cell-free SV40 DNA replication We also investigated other p70 regions for their inhibitory influence on SV40 DNA replication (Fig. 4). Of these, all peptides comprising the DNA binding domain (p70, 1–440, 174– 440, 174–330) showed strong inhibition (Fig. 4B). A near complete stop of nucleotide incorporation were obtained at a 10-, 25- and 50-fold excess of p70, 1–440 and 174–330, respectively (Fig. 4A). As a comparison, peptide 174–250 showed intermediate inhibition capabilities. A slight decrease in incorporation levels was seen with peptides 1–173 and those derived from the C-terminal part (441–616, 441–525, 525–616). No or very low interference was observed for p14, p32, 250–330 and 330–440. All inhibitory peptides other than 174–250 show inhibition regardless when they were added into the reaction (Fig. 4B). Particularly peptides with DNA binding capacity were strongly inhibitory even when added at late times into the reac-
3976
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978
Fig. 3. Effect of peptide 174–250 at different steps during SV40 DNA replication. (Panel A) Enzymatic events during initiation of cell-free SV40 DNA replication. The sketch shows mechanistic views of the single steps: assembly of Tag, Pol and RPA on origin sequences and unmelting of duplex DNA (step 1); recruitment of Topoisomerase I (Topo), extensive unwinding by Tag and coating of ssDNA by RPA (step 2); RNA primer synthesis by the primase subunit of Pol (step 3); and primer extension with DNA by the polymerase subunit of Pol (step 4). Steps 1 and 2 belong to the pre-initiation or lag phase and are ATP dependent, steps 3 and 4 comprise the initiation phase and depend on rNTPs and dNTPs, respectively. (Panel B) Effect of peptide 174–250 at the unwinding step. The peptide was added before the addition of ATP, which is needed for the bidirectional unwinding reaction (lane 2). Lane 1 is a positive control without peptide. In lane 3 Tag was omitted. The position of supercoiled template DNA (form I), topoisomers (form I 0 ) and underwound DNA (form U) are indicated. The amount of form U is given below each lane. (Panel C) Effect of peptide 174–250 at different replication steps. The peptide was added to the assay before unwinding (U, lane 1), primer synthesis (I, lane 2) and primer extension (E, lane 3). The respective template DNAs present at the time point of peptide addition are supercoiled, underwound and primed underwound plasmid DNA. All are represented by symbols below each lane. Reactions with (lane 4) and without (lane 5) Tag in the absence of the peptide served as positive and negative controls. The pmol of incorporated dNMPs are indicated.
Fig. 4. Inhibition of SV40 DNA replication by specific regions of p70. (Panel A) Titration of peptides. Peptides were added in increasing amounts expressed as fold molar excess over the 6.25 pmol of Tag present at the onset of the reaction. After a total of 120 min incubation time incorporated nucleotides were determined and plotted versus the molar excess of the respective peptides. (Panel B) Influence of peptides on different replication steps. Peptides were added at a 50-fold molar excess over Tag before unwinding, primer synthesis and primer extension as indicated (>). The pmol of incorporated nucleotides were determined for each case.
tion. The Escherichia coli single-stranded DNA binding protein (SSB) had similar inhibition capabilities (data not shown). With the exception of peptide 174–250 addition of extra Tag molecules did not relieve the inhibition caused by other inhibitory peptides (data not shown).
4. Discussion In this report we have expressed various sub-regions of the p70 subunit of replication protein A as soluble maltose binding protein (MBP)-fusions to study their influence on SV40 DNA
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978
replication. Specifically, we were able to express the protein interaction-, complex formation-, and DNA binding-sites of p70 and demonstrate their importance for the replication process (see the discussion section of the Supplementary data for more details on the activities of the peptides). SV40 in vitro DNA replication exhibited a characteristic initial lag time of about 10–15 min preceding the linear DNA synthesis step, which lasted for 60–120 min (supplementary Fig. 3) [24,25]. This lag time is referred to as the pre-synthesis or pre-initiation phase, in which it is assumed that Tag, Pol and RPA form a productive complex on the template DNA (Fig. 3A) [26]. Since the initial delay can be overcome by preincubation of cellular extracts with Tag and SV40 DNA prior to the addition of nucleotides to start RNA/DNA synthesis, the assembly of a pre-initiation complex consisting of the three protein players on the origin DNA is considered to be the rate limiting step [27]. It was exactly during this period that p70 peptide 174–250 proved to be inhibitory when titrated into replication assays (Fig. 2A and B), suggesting that it interferes with protein assembly. Indeed, the presence of the peptide reduced the association of RPA with both Tag and Pol (Fig. 1) and it was most potent when added prior to the unwinding step (Fig. 3C). Consistent with this interpretation were back titration experiments in which additional Tag restored activity only during the pre-initiation phase (Fig. 2C). Binding of the peptide to Tag and Pol during the lag period might lead to unproductive complexes at the transition to the initiation stage. Once the initiation stage comprising primer-synthesis and extension was reached, interference capability was lost, implying that the assembled initiation complex is stable enough to prevent the peptide from gaining access to the bona fide interacting surfaces and that any re-arrangements of contacts between RPA and the other replication factors during the later stages of initiation occur within tight complexes. On the other hand, the pre-initiation complex seemed to be meta-stable, because only intermediate inhibition was achieved if peptides were added before the primer synthesis step. The assembly of the pre-initiation complex therefore seems to be reversible during the pre-initiation period, but once the initiation complex (primosome) is formed, it acts in a processive way during primer synthesis and extension. Several other peptides reduced incorporation rates as well. In these cases inhibition was not due solely to a disruption of RPA’s interaction with Tag, since back titrating Tag did not overcome the negative effects (supplementary Fig. 4). Hence interference with other replicative activities must have caused or augmented inhibition, implying the importance of various p70 domains for the replication process. For peptide 1–173 inhibition was due to sequestering Pol (Fig. 1 and supplementary Fig. 1). Peptides 1–440, 174–440 and 174–330, in addition to bind Pol and Tag, interacted unproductively with single-stranded DNA akin to Escherichia coli SSB [19] and can therefore prevent the formation of a productive initiation complex, albeit they support bi-directional unwinding (see supplementary Fig. 1). Finally, C-terminal peptides 441–616, 441– 525 and 525–616 could potentially interfere with processes that require rearrangements between the DBDs of all RPA subunits. Such reorganization of the trimerization core is likely to occur before the primer synthesis step, and results in a switch in the DNA binding mode of RPA [8,9,28] (see discussion section of the Supplementary data for more details). Understanding the timed formation and significance of protein–protein interactions during the different steps of the initi-
3977
ation reaction remains an active field of research [22,29]. The peptides with their marked specificities should be helpful in future studies to delineate more precisely the importance of mutual interactions between RPA, Tag and Pol in this process. Acknowledgements: This work was supported by NIH (GM52948 to EF, C00004284-1 to HPN), Vanderbilt University (to EF), Deutsche Forschungsgemeinschaft (SFB604 to FG), Health Research Board Ireland (RP/2004/154 to HPN) and Science Foundation Ireland (05/RFP/ BIC0022 to HPN). The Leibniz Institute for Age research is financially supported by the federal government and the Land Thueringen.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet. 2007.07.038. References [1] Wold, M.S. (1997) Replication protein A: a heterotrimeric, singlestranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61–92. [2] Fanning, E., Klimovich, V. and Nager, A.R. (2006) A dynamic model for replication protein A (RPA) function in DNA processing pathways. Nucleic Acids Res. 34, 4126–4137, Epub 2006 Aug 25. [3] Brill, S.J. and Stillman, B. (1991) Replication factor-A from Saccharomyces cervisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev. 5, 1589–1600. [4] Kenny, M.K., Schlegel, U., Furneaux, H. and Hurwitz, J. (1990) The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J. Biol. Chem. 265, 7693–7700. [5] Umbricht, C.B., Erdile, L.F., Jabs, E.W. and Kelly, T.J. (1993) Cloning, overexpression, and genomic mapping of the 14-kDa subunit of human replication protein A. J. Biol. Chem. 268, 6131– 6138. [6] Erdile, L.F., Wold, M.S. and Kelly, T.J. (1990) The primary structure of the 32-kDa subunit of human replication protein A. J. Biol. Chem. 265, 3177–3182. [7] Kolpashchikov, D.M., Khodyreva, S.N., Khlimankov, D.Y., Wold, M.S., Favre, A. and Lavrik, O.I. (2001) Polarity of human replication protein A binding to DNA. Nucleic Acids Res. 29, 373–379. [8] Bochkareva, E., Belegu, V., Korolev, S. and Bochkarev, A. (2001) Structure of the major single-stranded DNA-binding domain of replication protein A suggests a dynamic mechanism for DNA binding. EMBO J. 20, 612–618. [9] Bochkareva, E., Korolev, S., Lees-Miller, S.P. and Bochkarev, A. (2002) Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA. EMBO J. 21, 1855– 1863. [10] Bastin-Shanower, S.A. and Brill, S.J. (2001) Functional analysis of the four DNA binding domains of replication protein A. The role of RPA2 in ssDNA binding. J. Biol. Chem. 276, 36446– 36453. [11] Bochkarev, A., Pfuetzner, R.A., Edwards, A.M. and Frappier, L. (1997) Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA. Nature 385, 176–181. [12] Philipova, D., Mullen, J., Maniar, H., Lu, J., Gu, C. and Brill, S. (1996) A hierarchy of SSB protomers in replication protein A. Genes Dev. 10, 2222–2233. [13] Bochkareva, E., Frappier, L., Edwards, A.M. and Bochkarev, A. (1998) The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain. J. Biol. Chem. 273, 3932–3936. [14] Bochkarev, A., Bochkareva, E., Frappier, L. and Edwards, A.M. (1999) The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for singlestranded DNA binding. EMBO J. 18, 4498–4504.
3978 [15] Brill, S.J. and Bastin-Shanower, S. (1998) Identification and characterization of the fourth single-stranded-DNA binding domain of replication protein A. Mol. Cell. Biol. 18, 7225–7234. [16] Binz, S.K., Sheehan, A.M. and Wold, M.S. (2004) Replication protein A phosphorylation and the cellular response to DNA damage. DNA Repair 3, 1015–1024. [17] Dornreiter, I., Erdile, L.F., Gilbert, I.U., von Winkler, D., Kelly, T.J. and Fanning, E. (1992) Interaction of DNA polymerase aprimase with cellular replication protein A and SV40 T antigen. EMBO J. 11, 769–776. [18] Braun, K.A., Lao, Y., He, Z., Ingles, C.J. and Wold, M.S. (1997) Role of protein–protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase a by multiple mechanisms. Biochemistry 36, 8443–8454. [19] Han, Y., Loo, Y.M., Militello, K.T. and Melendy, T. (1999) Interactions of the papovavirus DNA replication initiator proteins, bovine papillomavirus type 1 E1 and simian virus 40 large T antigen, with human replication protein A. J. Virol. 73, 4899– 4907. [20] Weisshart, K., Forster, H., Kremmer, E., Schlott, B., Grosse, F. and Nasheuer, H.P. (2000) Protein–protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system. J. Biol. Chem. 275, 17328–17337. [21] Gomes, X.V. and Wold, M.S. (1995) Structural analysis of human replication protein A. Mapping functional domains of the 70-kDa subunit. J. Biol. Chem. 270, 4534–4543.
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978 [22] Arunkumar, A.I., Klimovich, V., Jiang, X., Ott, R.D., Mizoue, L., Fanning, E. and Chazin, W.J. (2005) Insights into hRPA32 Cterminal domain-mediated assembly of the simian virus 40 replisome. Nat. Struct. Mol. Biol. 12, 332–339. [23] Gomes, X.V., Henricksen, L.A. and Wold, M.S. (1996) Proteolytic mapping of human replication protein A: evidence for multiple structural domains and a conformational change upon interaction with single-stranded DNA. Biochemistry 35, 5586–5595. [24] Wobbe, C.R., Weissbach, L., Borowiec, J.A., Dean, F.B. and Hurwitz, J. (1985) In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc. Natl. Acad. Sci. USA 82, 5710–5714. [25] Fairman, M.P. and Stillman, B. (1988) Cellular factors required for multiple stages of SV40 replication in vitro. EMBO J. 7, 1211–1218. [26] Dornreiter, I., Copeland, W.C. and Wang, T.S. (1993) Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen. Mol. Cell. Biol. 13, 809–820. [27] Tsurimoto, T., Fairman, M.S. and Stillman, B. (1989) Simian Virus 40 DNA replication in vitro: identification of multiple stages of initiation. Mol. Cell. Biol. 9, 3839–3849. [28] Blackwell, L.J. and Borowiec, J.A. (1994) Human replication protein A binds single-stranded DNA in two distinct complexes. Mol. Cell. Biol. 14, 3993–4001. [29] Simmons, D.T., Gai, D., Parsons, R., Debes, A. and Roy, R. (2004) Assembly of the replication initiation complex on SV40 origin DNA. Nucleic Acids Res. 32, 1103–1112.
Different activities of the largest subunit of replication protein A cooperate during SV40 DNA replication Poonam Tanejaa,1, Irene Bochea,2, Hella Hartmannb,3, Heinz-Peter Nasheuerc, Frank Grosseb, Ellen Fanninga, Klaus Weisshartb,* b
a Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA Leibniz Institute for Age Research–Fritz Lipmann Institute (former Institute for Molecular Biotechnology), Beutenbergstrasse 11, 07745 Jena, Federal Republic of Germany c Department of Biochemistry, National University of Ireland, Galway, Ireland
Received 25 April 2007; revised 6 July 2007; accepted 16 July 2007 Available online 25 July 2007 Edited by Angel Nebreda
Abstract Replication protein A (RPA) is a stable heterotrimeric complex consisting of p70, p32 and p14 subunits. The protein plays a crucial role in SV40 minichromosome replication. Peptides of p70 representing interaction sites for the smaller two subunits, DNA as well as the viral initiator protein large T-antigen (Tag) and the cellular DNA polymerase a-primase (Pol) all interfered with the replication process indicating the importance of the different p70 activities in this process. Inhibition by the peptide disrupting protein–protein interactions was observed only during the pre-initiation stage prior to primer synthesis, suggesting the formation of a stable initiation complex between RPA, Tag and Pol at the primer end. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: SV40 DNA replication; Replication protein A; Protein–protein interaction
1. Introduction Replication protein A (RPA), a hetero-trimeric protein involved in various aspects of DNA metabolism, is highly conserved in its subunit structure, DNA binding activity and posttranslational modifications in all examined eukaryotes [1,2]. The requirement of all three genes coding for the p70, p32 and p14 subunits for cell viability in yeast argues that RPA functions in vital reactions as a complex [3]. The integrity of the human trimer is also needed for Simian virus 40 (SV40) cell-free in vitro DNA replication, since neither the p70 subunit alone nor a sub-complex of p32 and p14 were able to support the reaction and antibodies directed against any one of the subunits were able to inhibit the process [4,5]. * Corresponding author. Present address: Carl Zeiss MicroImaging GmbH, Carl-Zeiss-Promenade 10, 07745 Jena, Germany. Fax: +49 3641 643144. E-mail address: [email protected] (K. Weisshart). 1 Present address: Molecular Devices Corporation, 1311 Orleans Drive, Sunnyvale, CA 94089, USA. 2 Present address: Xantos Biomedicine AG, Max-Lebsche-Platz 31, 81377 Muenchen, Germany. 3 Present address: Biotechnology Center, TU Dresden, Tatzberg 47-51, 01307, Germany.
Human RPA’s tight association to single-stranded DNA (ssDNA) is mediated primarily by p70 [4,6]. The subunit is oriented on the DNA in a polar fashion and positions the p32 subunit in the vicinity of the 3 0 -end of a primer-template junction [7]. Binding occurs in three modes, whereby the protein occludes 8–10, 12–23 and 28–30 nucleotides, respectively [8– 10]. The crystal structure of the ssDNA binding domain of p70 bound to DNA comprises two structurally homologous sub-domains, the so-called oligosaccharide/oligonucleotide binding (OB)-folds, which form the DNA binding domains A and B (DBD-A and DBD-B) [8]. The ssDNA lies in a cleft extending from one domain to the other [11]. OB-folds corresponding to other putative DNA binding domains have been mapped to the N- and C-termini of p70 (DBD-C and DBDF) and both the p32 (DBD-D) and p14 (DBD-E) subunits [12–16]. a-helices C-terminally located to OB-fold motifs of DBD-C, -D, and -E are arranged in parallel and mediate trimerization [9]. The orchestration of SV40 in vitro DNA replication requires the interaction of RPA with the viral initiator protein T-antigen (Tag) and the cellular DNA polymerase a-primase (Pol) and these interactions were mediated mainly by p70 and p32 [17–20]. Interaction sites of different strengths have been mapped to DBD-A and N-terminal sequences of p70 and to a winged helix–loop–helix motif in the C-terminus of p32 [18,21,22]. To probe the importance of p70’s interaction with the other two subunits, DNA and its replicative partner proteins we have expressed specific regions corresponding to formerly defined structural domains and sub-sequences of those [23]. These peptides displayed properties expected from previous deletion analysis. Peptide inhibition studies revealed that the cooperation of all activities of the p70 subunit is essential for the initiation process and that a stable primosome is assembled on a primer, which acts in a processive manner.
2. Materials and methods 2.1. Cloning and expression of RPA subunits RPA subunit sequences were amplified from a cDNA library obtained with the 5 0 -RACE kit of GibcoBRL from human 293S cells using primer pairs that allowed for directional cloning in amplification and expression vectors. Subunits were either expressed alone or linked together in various combinations. Likewise sub-regions of p70 were expressed alone or co-transcriptionally with p14 and p32.
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.07.038
3974
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978
2.2. Expression and purification of proteins Baculovirus expressed Tag and Pol were immunoaffinity purified. Bacterially expressed RPA, his-tagged RPA (H6-RPA) as well as topoisomerase I from calf thymus were purified by a combination of affinity, adsorption and ion-exchange chromatography steps. Maltose binding protein (MBP)-fusion proteins were affinity purified on amylose resin according to the manufacturer’s instructions (New England Biolabs). All p70 derived fusions were soluble and concentrations of 1 mg/ml and higher were obtained. If required, fusion peptides were concentrated to 5–10 mg/ml using Centricon P-20 centrifugal filter units (Millipore).
nucleotides. After removal of proteins from the rest of the sample by phenol/chloroform extraction DNA was ethanol precipitated and double restricted with EcoRI to linearize the DNA (form II) and DpnI to remove any un-replicated template DNA. Products were separated by agarose gel electrophoresis and analyzed by autoradiography. Peptides were added at different time points to the reaction at a 5–100-fold molar excess over Tag. Alternatively, the replication reaction was stalled at the unwinding, primer synthesis and primer extension steps by omitting ATP, other rNTPs and dNTPs, respectively. Peptides added to these reactions were pre-incubated with the mix for 30 min prior to the addition of the missing nucleotides to relieve the replication block.
2.3. Protein–protein interactions 17.5 pmol RPA (0.116 lg/pmol) were incubated with an equal amount of Tag or Pol, which were coupled to beads via specific antibodies, in the absence or presence of a 100-fold molar excess of peptides. The amounts of co-precipitated RPA were quantified by densitometric analysis of western blots developed with enhanced chemoluminescence (ECL, GE Healthcare). Alternatively, Tag and Pol were passed over amylose columns to which MBP-fusion proteins were coupled at a ratio of 1 mg/ml. Bound proteins were eluted and detected by western blotting using ECL.
2.7. DNA unwinding For bidirectional unwinding reactions 6.25 pmol Tag (0.096 lg/ pmol) were incubated with 0.1 pmol supercoiled SV40-origin containing pUC-HS DNA in the presence of 1.2 pmol toposiomerase I (0.1 lg/ pmol) and 4 pmol RPA (0.116 lg/pmol) Where indicated, RPA was replaced by the same amounts of MBP-fusion peptides. Alternatively peptides were added in addition to RPA. Under-wound DNA (form U) was detected by agarose gel electrophoresis and ethidium bromide staining and quantified by densitometric scanning. Detailed protocols can be obtained in the methods section of the Supplementary data.
2.4. Protein–DNA interactions 100 pmol of RPA (0.116 lg/pmol) or MBP-fusion peptides were incubated with 0.083 pmol circular single-stranded M13mp18 DNA (2.4 lg/pmol). Free DNA was separated from protein–DNA complexes by agarose gel electrophoresis. DNA was visualized by ethidium bromide staining. 2.5. Subunit complex formation Various p70 MBP-fusion proteins were co-transcriptionally expressed with p14, p32 or both. Extracts were passed over amylose resin to capture the MBP fusion and co-eluted subunits were analyzed by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and western blotting. To test for soluble proteins, crude extracts were analyzed in parallel. 2.6. SV40 in vitro DNA replication SV40 in vitro DNA replication was carried out with S100 extracts prepared from human 239S cells using 0.1 pmol of pUC-HS DNA (2 lg/pmol), which contains the complete SV40 origin. The assay was supplemented with ATP to drive the unwinding reaction catalyzed by Tag, as well as other ribonucleotides (rNTPs) and (radioactive labeled) deoxyribonucleotides (dNTPs) to support the primer synthesis and primer extension reactions catalyzed by Pol. The mix was assembled on ice and the reaction was started by the addition of 6.25 pmol Tag (0.096 lg/pmol). Incubation lasted for 120 min at 37 C. Five ll of the reaction were spotted on DE81 paper to quantify incorporated
3. Results 3.1. Activities of p70 peptides To probe the importance of different parts of the p70 subunit of replication protein A (RPA) for the replication process we have expressed different domains and sub-regions thereof as soluble maltose binding protein (MBP) fusion proteins. Amino acids 174–330 were sufficient for DNA binding, 1–173 for binding DNA polymerase a-primase (Pol), 174–250 for binding both SV 40 large T-antigen (Tag) and Pol, and 421–525 as well as 525–616 for complex formation with the other two smaller subunits (for a detailed characterization see results section and Figs. 1 and 2 of the Supplementary data). All fragments containing amino acids 174–250 reduced the amounts of RPA co-precipitated with Pol and Tag, respectively (Fig. 1). Peptide 1–173 reduced the co-precipitated RPA only to a minor extent if tested with Pol, but showed no effect on RPA’s interaction with Tag. C-terminal derived peptides did not reduce the amounts of co-precipitated RPA. Peptide 174–250 was chosen to probe the importance of protein–protein interactions between replicative factors during cell
Fig. 1. Interference of p70 peptides with protein interactions. Co-precipitations of 17.5 pmol soluble H6-RPA were performed with the same amounts of immobilized Tag (Panel A) and Pol (Panel B) in the presence of a 100-fold molar excess of the indicated p70 competitor polypeptides. Bound H6-RPA was detected with his-tag specific antibodies by western blotting and ECL detection. The numbers below the lanes refer to the fractions of H6-RPA bound in relation to the amount obtained in the presence of MBP. The position of the p70 subunit bearing the histidin-tag is indicated. Lane 1 represents a loading control of 1/10th of the input material, whereas in lane 2 no RPA was added.
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978
3975
Fig. 2. SV40 DNA replication in the presence of peptide 174–250. (Panel A) Inhibition of SV40 DNA replication by peptide 174–250. The peptide was titrated into the assay in increasing quantities defined as a molar excess over the 6.25 pmol of Tag present in the assay (lanes 3 and 4). Lane 2 (0·) contained no peptide and a control reaction MBP in a 100-fold molar excess over Tag (lane 7). Tag was back titrated with the indicated amounts where indicated (lanes 5 and 6). Lane 1 (Tag) contained no Tag to determine background replication. (Panel B) Kinetics of inhibition by peptide 174–250. The peptide was added to the mix at the indicated times after start of the replication reaction with a 100-fold molar excess over the 6.25 pmol of Tag (lanes 3–8). Incubation continued for a total of 120 min. Reactions in the presence (+Tag, lane 1) and absence (Tag, lane 2) of Tag without peptide served as positive and negative controls, respectively. (Panel C) Kinetics of inhibition relief by extra Tag. Reactions were supplied with peptide 174–250 at the onset of the reaction in a 100-fold molar excess over the 6.25 pmol of Tag. After the indicated times an additional 6 pmol of Tag was added (lanes 3–8). Incubation continued for a total of 120 min. Reactions in the presence (+Tag, lane 1) and absence (Tag, lane 2) of Tag without peptide served as positive and negative controls, respectively. The position of the linearized plasmid DNA (form II) is indicated. The numbers below each lane indicate the pmol of dNMPs incorporated.
free SV40 DNA replication because it interfered reasonable well with RPA’s ability to bind to both Tag and Pol, but had on its own no DNA binding activity. 3.2. Influence of peptide 174–250 on cell-free SV40 DNA replication If peptide 174–250 was titrated into the replication assays in the presence of Tag, incorporation rates decreased in a dose dependent manner (Fig. 2A, lanes 2–4). Adding back extra molecules of Tag restored incorporation levels (lanes 4–6). MBP alone had no adverse effect on the replication efficiency (lane 7). Even at a 100-fold molar excess of the peptide over Tag, replication rates did not drop to zero as exemplified for a reaction without Tag (compare lane 4 to lane 1). The peptide interfered with the replication reaction only when it was titrated into the assay within a maximum time delay of 15 min after start of the reaction (Fig. 2B, lanes 4–7). In contrast, incorporation levels comparable to the control reaction without peptide were obtained when the peptide was added after 30 or 60 min (compare lanes 7 and 8 to lane 1). Back titration of Tag at various times in the presence of the inhibitory peptide could more effectively restore the replication activity if Tag was added within 15 min after start (Fig. 2C, lanes 3–6). In these cases, incorporation rates that closely matched the one obtained in the control reaction were obtained (lane 2). If Tag was added after 30 or 60 min had elapsed, its positive influence on DNA synthesis was substantially decreased (lanes 7 and 8). 3.3. Influence of peptide 174–250 on different steps during cellfree SV40 DNA replication The initiation of SV40 DNA replication can be divided into different steps including the assembly of the pre-initiation complex, bidirectional unwinding catalyzed by Tag in the presence
of a single-stranded DNA binding protein, as well as primer synthesis and primer extension carried out by Pol (Fig. 3A). Complex formation and unwinding reaction comprise the pre-initiation phase that lasted for about 15 min (see supplementary Fig. 3). Reactions were set up with certain nucleotides omitted to stall the reaction before some of these steps. Peptide 174–250 was added to these stalled reactions and incubated with the mix before release of the block by providing the missing nucleotides. The peptide did not inhibit the unwinding reaction per se, if it was supplemented prior to the addition of ATP (Fig. 3B, compare lanes 1 and 2). If peptide 174–250 was present in the replication reaction before the unwinding step, it inhibited nucleotide incorporation to a great extent (Fig. 3C, lane 1). Intermediate and no inhibition were observed if the peptide was added prior to the primer synthesis (lane 2) and primer extension (lane 3) reactions, respectively. 3.4. Influence of other p70 peptides on cell-free SV40 DNA replication We also investigated other p70 regions for their inhibitory influence on SV40 DNA replication (Fig. 4). Of these, all peptides comprising the DNA binding domain (p70, 1–440, 174– 440, 174–330) showed strong inhibition (Fig. 4B). A near complete stop of nucleotide incorporation were obtained at a 10-, 25- and 50-fold excess of p70, 1–440 and 174–330, respectively (Fig. 4A). As a comparison, peptide 174–250 showed intermediate inhibition capabilities. A slight decrease in incorporation levels was seen with peptides 1–173 and those derived from the C-terminal part (441–616, 441–525, 525–616). No or very low interference was observed for p14, p32, 250–330 and 330–440. All inhibitory peptides other than 174–250 show inhibition regardless when they were added into the reaction (Fig. 4B). Particularly peptides with DNA binding capacity were strongly inhibitory even when added at late times into the reac-
3976
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978
Fig. 3. Effect of peptide 174–250 at different steps during SV40 DNA replication. (Panel A) Enzymatic events during initiation of cell-free SV40 DNA replication. The sketch shows mechanistic views of the single steps: assembly of Tag, Pol and RPA on origin sequences and unmelting of duplex DNA (step 1); recruitment of Topoisomerase I (Topo), extensive unwinding by Tag and coating of ssDNA by RPA (step 2); RNA primer synthesis by the primase subunit of Pol (step 3); and primer extension with DNA by the polymerase subunit of Pol (step 4). Steps 1 and 2 belong to the pre-initiation or lag phase and are ATP dependent, steps 3 and 4 comprise the initiation phase and depend on rNTPs and dNTPs, respectively. (Panel B) Effect of peptide 174–250 at the unwinding step. The peptide was added before the addition of ATP, which is needed for the bidirectional unwinding reaction (lane 2). Lane 1 is a positive control without peptide. In lane 3 Tag was omitted. The position of supercoiled template DNA (form I), topoisomers (form I 0 ) and underwound DNA (form U) are indicated. The amount of form U is given below each lane. (Panel C) Effect of peptide 174–250 at different replication steps. The peptide was added to the assay before unwinding (U, lane 1), primer synthesis (I, lane 2) and primer extension (E, lane 3). The respective template DNAs present at the time point of peptide addition are supercoiled, underwound and primed underwound plasmid DNA. All are represented by symbols below each lane. Reactions with (lane 4) and without (lane 5) Tag in the absence of the peptide served as positive and negative controls. The pmol of incorporated dNMPs are indicated.
Fig. 4. Inhibition of SV40 DNA replication by specific regions of p70. (Panel A) Titration of peptides. Peptides were added in increasing amounts expressed as fold molar excess over the 6.25 pmol of Tag present at the onset of the reaction. After a total of 120 min incubation time incorporated nucleotides were determined and plotted versus the molar excess of the respective peptides. (Panel B) Influence of peptides on different replication steps. Peptides were added at a 50-fold molar excess over Tag before unwinding, primer synthesis and primer extension as indicated (>). The pmol of incorporated nucleotides were determined for each case.
tion. The Escherichia coli single-stranded DNA binding protein (SSB) had similar inhibition capabilities (data not shown). With the exception of peptide 174–250 addition of extra Tag molecules did not relieve the inhibition caused by other inhibitory peptides (data not shown).
4. Discussion In this report we have expressed various sub-regions of the p70 subunit of replication protein A as soluble maltose binding protein (MBP)-fusions to study their influence on SV40 DNA
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978
replication. Specifically, we were able to express the protein interaction-, complex formation-, and DNA binding-sites of p70 and demonstrate their importance for the replication process (see the discussion section of the Supplementary data for more details on the activities of the peptides). SV40 in vitro DNA replication exhibited a characteristic initial lag time of about 10–15 min preceding the linear DNA synthesis step, which lasted for 60–120 min (supplementary Fig. 3) [24,25]. This lag time is referred to as the pre-synthesis or pre-initiation phase, in which it is assumed that Tag, Pol and RPA form a productive complex on the template DNA (Fig. 3A) [26]. Since the initial delay can be overcome by preincubation of cellular extracts with Tag and SV40 DNA prior to the addition of nucleotides to start RNA/DNA synthesis, the assembly of a pre-initiation complex consisting of the three protein players on the origin DNA is considered to be the rate limiting step [27]. It was exactly during this period that p70 peptide 174–250 proved to be inhibitory when titrated into replication assays (Fig. 2A and B), suggesting that it interferes with protein assembly. Indeed, the presence of the peptide reduced the association of RPA with both Tag and Pol (Fig. 1) and it was most potent when added prior to the unwinding step (Fig. 3C). Consistent with this interpretation were back titration experiments in which additional Tag restored activity only during the pre-initiation phase (Fig. 2C). Binding of the peptide to Tag and Pol during the lag period might lead to unproductive complexes at the transition to the initiation stage. Once the initiation stage comprising primer-synthesis and extension was reached, interference capability was lost, implying that the assembled initiation complex is stable enough to prevent the peptide from gaining access to the bona fide interacting surfaces and that any re-arrangements of contacts between RPA and the other replication factors during the later stages of initiation occur within tight complexes. On the other hand, the pre-initiation complex seemed to be meta-stable, because only intermediate inhibition was achieved if peptides were added before the primer synthesis step. The assembly of the pre-initiation complex therefore seems to be reversible during the pre-initiation period, but once the initiation complex (primosome) is formed, it acts in a processive way during primer synthesis and extension. Several other peptides reduced incorporation rates as well. In these cases inhibition was not due solely to a disruption of RPA’s interaction with Tag, since back titrating Tag did not overcome the negative effects (supplementary Fig. 4). Hence interference with other replicative activities must have caused or augmented inhibition, implying the importance of various p70 domains for the replication process. For peptide 1–173 inhibition was due to sequestering Pol (Fig. 1 and supplementary Fig. 1). Peptides 1–440, 174–440 and 174–330, in addition to bind Pol and Tag, interacted unproductively with single-stranded DNA akin to Escherichia coli SSB [19] and can therefore prevent the formation of a productive initiation complex, albeit they support bi-directional unwinding (see supplementary Fig. 1). Finally, C-terminal peptides 441–616, 441– 525 and 525–616 could potentially interfere with processes that require rearrangements between the DBDs of all RPA subunits. Such reorganization of the trimerization core is likely to occur before the primer synthesis step, and results in a switch in the DNA binding mode of RPA [8,9,28] (see discussion section of the Supplementary data for more details). Understanding the timed formation and significance of protein–protein interactions during the different steps of the initi-
3977
ation reaction remains an active field of research [22,29]. The peptides with their marked specificities should be helpful in future studies to delineate more precisely the importance of mutual interactions between RPA, Tag and Pol in this process. Acknowledgements: This work was supported by NIH (GM52948 to EF, C00004284-1 to HPN), Vanderbilt University (to EF), Deutsche Forschungsgemeinschaft (SFB604 to FG), Health Research Board Ireland (RP/2004/154 to HPN) and Science Foundation Ireland (05/RFP/ BIC0022 to HPN). The Leibniz Institute for Age research is financially supported by the federal government and the Land Thueringen.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet. 2007.07.038. References [1] Wold, M.S. (1997) Replication protein A: a heterotrimeric, singlestranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 66, 61–92. [2] Fanning, E., Klimovich, V. and Nager, A.R. (2006) A dynamic model for replication protein A (RPA) function in DNA processing pathways. Nucleic Acids Res. 34, 4126–4137, Epub 2006 Aug 25. [3] Brill, S.J. and Stillman, B. (1991) Replication factor-A from Saccharomyces cervisiae is encoded by three essential genes coordinately expressed at S phase. Genes Dev. 5, 1589–1600. [4] Kenny, M.K., Schlegel, U., Furneaux, H. and Hurwitz, J. (1990) The role of human single-stranded DNA binding protein and its individual subunits in simian virus 40 DNA replication. J. Biol. Chem. 265, 7693–7700. [5] Umbricht, C.B., Erdile, L.F., Jabs, E.W. and Kelly, T.J. (1993) Cloning, overexpression, and genomic mapping of the 14-kDa subunit of human replication protein A. J. Biol. Chem. 268, 6131– 6138. [6] Erdile, L.F., Wold, M.S. and Kelly, T.J. (1990) The primary structure of the 32-kDa subunit of human replication protein A. J. Biol. Chem. 265, 3177–3182. [7] Kolpashchikov, D.M., Khodyreva, S.N., Khlimankov, D.Y., Wold, M.S., Favre, A. and Lavrik, O.I. (2001) Polarity of human replication protein A binding to DNA. Nucleic Acids Res. 29, 373–379. [8] Bochkareva, E., Belegu, V., Korolev, S. and Bochkarev, A. (2001) Structure of the major single-stranded DNA-binding domain of replication protein A suggests a dynamic mechanism for DNA binding. EMBO J. 20, 612–618. [9] Bochkareva, E., Korolev, S., Lees-Miller, S.P. and Bochkarev, A. (2002) Structure of the RPA trimerization core and its role in the multistep DNA-binding mechanism of RPA. EMBO J. 21, 1855– 1863. [10] Bastin-Shanower, S.A. and Brill, S.J. (2001) Functional analysis of the four DNA binding domains of replication protein A. The role of RPA2 in ssDNA binding. J. Biol. Chem. 276, 36446– 36453. [11] Bochkarev, A., Pfuetzner, R.A., Edwards, A.M. and Frappier, L. (1997) Structure of the single-stranded-DNA-binding domain of replication protein A bound to DNA. Nature 385, 176–181. [12] Philipova, D., Mullen, J., Maniar, H., Lu, J., Gu, C. and Brill, S. (1996) A hierarchy of SSB protomers in replication protein A. Genes Dev. 10, 2222–2233. [13] Bochkareva, E., Frappier, L., Edwards, A.M. and Bochkarev, A. (1998) The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain. J. Biol. Chem. 273, 3932–3936. [14] Bochkarev, A., Bochkareva, E., Frappier, L. and Edwards, A.M. (1999) The crystal structure of the complex of replication protein A subunits RPA32 and RPA14 reveals a mechanism for singlestranded DNA binding. EMBO J. 18, 4498–4504.
3978 [15] Brill, S.J. and Bastin-Shanower, S. (1998) Identification and characterization of the fourth single-stranded-DNA binding domain of replication protein A. Mol. Cell. Biol. 18, 7225–7234. [16] Binz, S.K., Sheehan, A.M. and Wold, M.S. (2004) Replication protein A phosphorylation and the cellular response to DNA damage. DNA Repair 3, 1015–1024. [17] Dornreiter, I., Erdile, L.F., Gilbert, I.U., von Winkler, D., Kelly, T.J. and Fanning, E. (1992) Interaction of DNA polymerase aprimase with cellular replication protein A and SV40 T antigen. EMBO J. 11, 769–776. [18] Braun, K.A., Lao, Y., He, Z., Ingles, C.J. and Wold, M.S. (1997) Role of protein–protein interactions in the function of replication protein A (RPA): RPA modulates the activity of DNA polymerase a by multiple mechanisms. Biochemistry 36, 8443–8454. [19] Han, Y., Loo, Y.M., Militello, K.T. and Melendy, T. (1999) Interactions of the papovavirus DNA replication initiator proteins, bovine papillomavirus type 1 E1 and simian virus 40 large T antigen, with human replication protein A. J. Virol. 73, 4899– 4907. [20] Weisshart, K., Forster, H., Kremmer, E., Schlott, B., Grosse, F. and Nasheuer, H.P. (2000) Protein–protein interactions of the primase subunits p58 and p48 with simian virus 40 T antigen are required for efficient primer synthesis in a cell-free system. J. Biol. Chem. 275, 17328–17337. [21] Gomes, X.V. and Wold, M.S. (1995) Structural analysis of human replication protein A. Mapping functional domains of the 70-kDa subunit. J. Biol. Chem. 270, 4534–4543.
P. Taneja et al. / FEBS Letters 581 (2007) 3973–3978 [22] Arunkumar, A.I., Klimovich, V., Jiang, X., Ott, R.D., Mizoue, L., Fanning, E. and Chazin, W.J. (2005) Insights into hRPA32 Cterminal domain-mediated assembly of the simian virus 40 replisome. Nat. Struct. Mol. Biol. 12, 332–339. [23] Gomes, X.V., Henricksen, L.A. and Wold, M.S. (1996) Proteolytic mapping of human replication protein A: evidence for multiple structural domains and a conformational change upon interaction with single-stranded DNA. Biochemistry 35, 5586–5595. [24] Wobbe, C.R., Weissbach, L., Borowiec, J.A., Dean, F.B. and Hurwitz, J. (1985) In vitro replication of duplex circular DNA containing the simian virus 40 DNA origin site. Proc. Natl. Acad. Sci. USA 82, 5710–5714. [25] Fairman, M.P. and Stillman, B. (1988) Cellular factors required for multiple stages of SV40 replication in vitro. EMBO J. 7, 1211–1218. [26] Dornreiter, I., Copeland, W.C. and Wang, T.S. (1993) Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen. Mol. Cell. Biol. 13, 809–820. [27] Tsurimoto, T., Fairman, M.S. and Stillman, B. (1989) Simian Virus 40 DNA replication in vitro: identification of multiple stages of initiation. Mol. Cell. Biol. 9, 3839–3849. [28] Blackwell, L.J. and Borowiec, J.A. (1994) Human replication protein A binds single-stranded DNA in two distinct complexes. Mol. Cell. Biol. 14, 3993–4001. [29] Simmons, D.T., Gai, D., Parsons, R., Debes, A. and Roy, R. (2004) Assembly of the replication initiation complex on SV40 origin DNA. Nucleic Acids Res. 32, 1103–1112.