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Nucleic Acids Research Pages 5636-5643  

Unwinding of origin-specific structures by human replication protein A occurs in a two-step process
Introduction
Materials And Methods
   DNA substrate and SSBs
   Binding and unwinding reactions
   Determination of hRPA-DNA dissociation constants
Results
   Pseudo-origin binding and unwinding activities of recombinant hRPA similar to HeLa hRPA
   Binding and unwinding activities of hRPA increase proportionally with the protein amount
   Pseudo-origin binding and denaturation occur at elevated salt concentrations
   Binding and unwinding by hRPA do not occur simultaneously
   The binding and unwinding activities of hRPA are temperature dependent
   Some SSBs that bind the PO-8 substrate cannot support its unwinding
Discussion
Acknowledgements
References

Unwinding of origin-specific structures by human replication protein A occurs in a two-step process

Unwinding of origin-specific structures by human replication protein A occurs in a two-step process

Cristina Iftode and James A. Borowiec*

Department of Biochemistry and Kaplan Comprehensive Cancer Center, New York University Medical Center, 550 First Avenue, New York, NY 10016, USA

Received August 26, 1998; Revised and Accepted November 3, 1998

ABSTRACT

The simian virus 40 (SV40) large tumor antigen(T antigen) has been shown to induce the melting of 8 bp within the SV40 origin of replication. We found previously that a `pseudo-origin' DNA molecule (PO-8) containing a central 8 nt single-stranded DNA (ssDNA) bubble was efficiently bound and denatured by human replication protein A (hRPA). To understand the mechanism by which hRPA denatures these pseudo-origin molecules, as well as the role that hRPA plays during the initiation of SV40 DNA replication, we characterized the key parameters for the pseudo-origin binding and denaturation reactions. The dissociation constant of hRPA binding to PO-8 was observed to be 7.7 × 10-7 M, compared to 9.0 × 10-8 M for binding to an identical length ssDNA under the same reaction conditions. The binding and denaturation of PO-8 occurred with different kinetics with the rate of binding determined to be ~4-fold greater than the rate of denaturation. Although hRPA binding to PO-8 was relatively temperature independent, an increase in incubation temperature from 4 to 37°C stimulated denaturation nearly 4-fold. At 37°C, denaturation occurred on ~1/3 of those substrate molecules bound by hRPA, showing that hRPA can bind the pseudo-origin substrate without causing its complete denaturation. Tests of other single-stranded DNA-binding proteins (SSBs) over a range of SSB concentrations revealed that the ability of the SSBs to bind the pseudo-origin substrate, rather than denature the substrate, correlated best with the known ability of these SSBs to support the T antigen-dependent SV40 origin-unwinding activity. Our data indicate that hRPA first binds the DNA substrate using a combination of contacts with the ssDNA bubble and duplex DNA flanks and then, on only a fraction of the bound substrate molecules, denatures the DNA substrate.

INTRODUCTION

The most abundant single-stranded DNA-binding protein (SSB) in eukaryotic cells, replication protein A (RPA), plays critical roles in chromosomal DNA replication and repair and is involved in DNA recombination and the regulation of transcription (1). Human RPA (hRPA), first isolated as an essential component in cell-free reactions supporting simian virus 40 (SV40) DNA replication (2-4), is perhaps the best characterized member of this family. During the initiation of SV40 DNA replication, hRPA is a required factor together with the viral T antigen for origin denaturation (5,6). Subsequently, hRPA stabilizes the single-stranded DNA (ssDNA) that is generated by the DNA helicase activity of T antigen during viral DNA unwinding and facilitates nascent strand synthesis by the replicative DNA polymerases.

Similar to other RPA homologs, hRPA is a heterotrimeric protein composed of 70, 29 and 14 kDa subunits (3,4). Although all three protein subunits appear to be essential for SV40 DNA replication, only the 70 kDa subunit is known to contain high-affinity ssDNA-binding activity (7,8). Studies of ssDNA binding suggest that hRPA first binds 8 nt of DNA unstably (9,10), and then reorients on the DNA to stably bind 30 nt in a process involving conformational changes within hRPA (9-13). Although the affinity of RPA binding to double-stranded DNA substrates is generally three to four orders of magnitude lower than that for ssDNA (11,12), the Saccharomyces cerevisiae RPA (scRPA) and hRPA can bind specific sequences found within the promoter regions of various genes, including those expressing proteins involved in DNA repair (14-16).

hRPA binds with high affinity to an SV40 pseudo-origin substrate, a partially duplex molecule containing a central 8 nt bubble (17). As the substrate resembles a structure found within the T antigen-origin complex (18,19), the interaction of hRPA with the pseudo-origin can potentially shed light on the events taking place during the initiation of SV40 DNA replication. The pseudo-origin can be unwound by hRPA (17), suggesting that hRPA plays an active role in the denaturation of the SV40 origin and the release of the T antigen DNA helicase from strong contacts with recognition elements within the origin. The pseudo-origin unwinding reaction of hRPA resembles helix-destabilizing activities found previously for hRPA and RPA from calf thymus (ctRPA) (20,21). In apparent contrast to pseudo-origin unwinding which can occur using physiological salt levels (17), the helix-destabilizing activities are highly salt sensitive.

To understand the mechanism by which hRPA denatures pseudo-origin substrates, we examined the importance of various reaction parameters on binding and unwinding of the pseudo-origin substrate by hRPA. We found that only a fraction (~1/3) of the bound substrates were denatured under standard conditions (incubation at 37°C in the presence of 20 mM NaCl and 7 mM MgCl2), indicating that hRPA can bind the substrate without causing its complete denaturation. Identification of conditions at which the two reactions can be separated strongly suggests that hRPA-mediated denaturation of these substrates is a two-step process in which hRPA first binds the partially duplex fragment and then induces its unwinding.

MATERIALS AND METHODS

DNA substrate and SSBs

The SV40 pseudo-origin substrate (PO-8) was prepared by annealing two partially complementary oligonucleotides to generate a 48 bp DNA molecule with central 8 nt ssDNA region. The top and bottom strand sequences were as follows (ssDNA regions underlined): top, 5[prime]-AGG CCT CCA AAA AAG CCT CCT CAC TAC TTC TGG AAT AGC TCA GAG GCC; bottom, 5[prime]-GGC CTC TGA GCT ATT CCA GAT CAT CAC TGG AGG CTT TTT TGG AGG CCT. The annealing and 32P-labeling conditions were as described previously (17).

Recombinant hRPA was expressed in and purified from Escherichia coli using a modification of the protocol described by Henricksen et al. (22). The p11d-tRNA expression vector (generously provided by Marc Wold) was transformed into E.coli strain BL21. Following induction and cell lysis, the supernatant from a 2 l culture was loaded onto a 10-ml Affi-Gel blue column (2 × 5 cm) equilibrated with HI buffer (30 mM HEPES, 1 mM dithiothreitol, 0.25 mM EDTA, 0.25% inositol and 0.01% Nonidet P-40) containing 100 mM KCl. The protein was eluted with HI buffer containing 1.5 M sodium thiocyanate. After dialysis against HI buffer containing 100 mM KCl, the peak fraction was applied to a Mono Q Sepharose column (HR 5/5; Pharmacia-LKB). The column was eluted with a 100-400 mM KCl gradient in HI buffer, and hRPA eluted at ~300 mM KCl. The purity of hRPA, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver-staining, was found to be ~98%. Other SSBs were kindly provided by Kenneth Marians (EcoSSB), I. Robert Lehman (HSV ICP8), Ronald T. Hay (AdDBP) and Charles Richardson (T7 gp2.5). Bacteriophage T4 gene 32 protein (T4 gp32) was purchased from Boehringer Mannheim Biochemical.

Binding and unwinding reactions

Binding reactions between hRPA (0.01-6 pmol) and the PO-8 substrate (0.15 pmol; 5[prime]-32P-labeled on the top strand) were performed as previously described (17). Standard reaction mixtures contained 20 mM NaCl and 7 mM MgCl2 and were incubated for 20 min at 37°C. Where indicated, the concentrations of MgCl2 (0.5-7 mM) and NaCl (3-100 mM) in the binding buffer were varied, as was the incubation time and temperature. hRPA binding to the pseudo-origin substrate was detected by a nitrocellulose filter binding assay. Reaction mixtures (20 µl) were applied to nitrocellulose filters pretreated with alkali as described by McEntee et al. (23). Glutaraldehyde crosslinking was not employed in these reactions. The radioactivity retained on the membranes was detected in a scintillation counter. For the electrophoretic mobility shift experiments, binding reactions were performed and crosslinked with glutaraldehyde as previously described (17). To detect the ssDNA generated as a result of unwinding by hRPA, the binding reaction products were extracted with phenol:chloroform (1:1, v/v) in the presence of a 10-fold molar excess of unlabeled top strand (to reduce reannealing of the labeled top strand; see Results). The DNA samples were separated on 8% non-denaturing polyacrylamide gels (29:1; acrylamide:bisacrylamide) and subjected to autoradiography. Quantitation of unwound versus total DNA was carried out by densitometric analysis with NIH Image (version 1.61) software.

Determination of hRPA-DNA dissociation constants

Binding reactions between recombinant hRPA (0.01-300 pmol) and the 32P-labeled PO-8, PO-0 (a 48 bp completely duplex DNA) (17) or 48 nt ssDNA (top strand of PO-8) substrates (0.15 pmol) were performed as indicated (17), except that the incubation temperature was 15°C instead of 37°C. The amount of bound DNA (quantitated by the nitrocellulose filter binding assay as described above) was represented as a function of the active hRPA concentration. The percentage of active hRPA, measured as described by Kim et al. (12), was determined to be 80%. In order to calculate the binding constants, the titration curves of hRPA binding to the PO-0, PO-8 and ssDNA substrates were fitted using SigmaPlot Software. The binding constants were expressed as the recombinant hRPA concentration at which 50% saturation is achieved (24).

RESULTS

Pseudo-origin binding and unwinding activities of recombinant hRPA similar to HeLa hRPA

Our previous examination of pseudo-origin denaturation employed hRPA that was purified from HeLa cell lysates (17). Because this procedure yields a relatively low amount of protein, we tested recombinant hRPA expressed in E.coli (22). The recombinant protein can be produced in relatively large amounts (in our hands, ~0.5 mg/l of infected cells) and has been reported to be functionally indistinguishable from hRPA isolated from human cells (22).

We compared the ability of the HeLa and recombinant hRPA (1.5 pmol) to bind and unwind the PO-8 substrate (0.15 pmol). The PO-8 molecule contains an 8 nt bubble flanked on each side by 20 bp of duplex DNA (17), and resembles the structure induced in the SV40 origin upon the ATP-dependent binding of T antigen (18,19). Using an electrophoretic mobility shift assay, we found that the recombinant hRPA and HeLa hRPA bound PO-8 with similar affinity, although the recombinant protein bound in a higher oligomeric state (Fig. 1A, lanes 1-4). HeLa hRPA gave rise to two primary bands (lanes 2 and 3) that corresponded to hRPA dimers and trimers (previously identified by Blackwell et al.; 10), while the recombinant protein bound PO-8 in the form of trimers and tetramers. To detect DNA unwinding, similar reactions were prepared in the absence of glutaraldehyde crosslinking and then subjected to native gel electrophoresis (Fig. 1B) (17). For each protein, the incubation with PO-8 caused the appearance of a band that migrated faster than the free PO-8 substrate and corresponded to the denatured substrate (Fig. 1B; lanes 3 and 6). The recombinant protein was slightly more efficient in the generation of ssDNA product compared to HeLa hRPA. Thus, pseudo-origin binding and denaturation are not activities limited to hRPA purified from HeLa cells but are intrinsic properties of hRPA.


Figure 1. Comparison of the PO-8 binding and denaturation activities of HeLa hRPA or recombinant hRPA purified from E.coli. To examine PO-8 binding (A), the 32P-labeled PO-8 substrate (0.15 pmol) was incubated in the absence or presence of either HeLa (lane 2) or recombinant (lane 4) hRPA (1.5 pmol). The reaction mixtures were crosslinked with glutaraldehyde and subjected to native gel electrophoresis. The positions of the PO-8 substrate bound by hRPA monomers, dimers, trimers and tetramers are indicated by numerals 1-4, as is the position of unbound PO-8. The band migrating slightly below the monomer band in the HeLa hRPA lane, detected previously (17), was not observed in the recombinant hRPA preparations or in all preparations of HeLa hRPA. To examine PO-8 denaturation (B), the 32P-labeled PO-8 substrate was similarly incubated in the absence (lanes 2 and 5) or presence of either HeLa (lane 3) or recombinant (lane 6) hRPA except that no glutaraldehyde was utilized in the reaction. The marker lane (M; lanes 1 and 4) contains the 32P-labeled top strand of the PO-8 substrate. The location of the unbound PO-8 substrate and the denatured product (labeled `ssDNA') are indicated. The product molecules in lanes 4-6 were electrophoresed a greater distance than those in lanes 1-3, causing the apparent difference in separation of the PO-8 and the ssDNA molecules.

To more accurately quantitate hRPA binding and denaturation of the PO-8 substrate, we modified our assay procedures that were used previously (17) and are described above (Fig. 1). First, to measure PO-8 binding, we used a nitrocellulose filter binding assay in place of the electrophoretic mobility shift assay. In control experiments, we observed that the two methods yielded similar values of hRPA binding (data not shown), but the filter binding assay was more simple and easier to quantitate. Second, the initial electrophoretic mobility shift assay used to detect PO-8 denaturation likely underestimated the amount of ssDNA produced because additional ssDNA was likely present in the hRPA-bound complexes. To modify this assay system, we used a pseudo-origin substrate of which the top strand was 32P-labeled. Following formation of the hRPA-DNA complexes, the reaction mixtures were extracted with phenol:chloroform in the presence of a 10-fold molar excess (over substrate) of unlabeled single-stranded top strand, to prevent reannealing of the labeled top strand product with the available bottom strand ssDNA. The reaction products were then subjected to native gel electrophoresis to separate the ssDNA product from the pseudo-origin substrate. Following autoradiography, the unwound products were quantitated by densitometric analysis. As shown in a representative experiment, addition of the unlabeled top strand led to a 2-fold increase in the amount of ssDNA generated (Fig. 2, lanes 3 and 4). Note that extraction of the PO-8 substrate in the absence of hRPA and in the presence of unlabeled ssDNA competitor did not cause substrate denaturation (lane 2). In addition, boiling the PO-8 substrate prior to extraction showed that the recovered product was completely ssDNA, with little, if any, duplex substrate observed (lane 6). Thus, the extraction procedure allows accurate determination of the amount of PO-8 unwinding caused by hRPA.


Figure 2. Denaturation of the PO-8 substrate by hRPA. The PO-8 substrate (0.15 pmol; 32P-labeled on the top strand) was incubated at 37°C for 20 min in the absence (lanes 1 and 2) or presence (lanes 3 and 4) of recombinant hRPA (1.5 pmol). Reaction mixtures were then extracted with phenol:chloroform in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of a 10-fold molar excess of unlabeled top strand to prevent reannealing of the labeled top strand. To verify that the extraction procedure accurately recovers the denatured ssDNA, the PO-8 substrate (lane 5; labeled `M' for marker) was boiled for 15 min, and then extracted as indicated above (lane 6; labeled `[Delta]'). Reaction products were subjected to native gel electrophoresis through an 8% polyacrylamide gel, and autoradiographed.

Binding and unwinding activities of hRPA increase proportionally with the protein amount

The effect of hRPA concentration on both the binding and unwinding activities of hRPA was investigated. Increasing levels of hRPA (0.01-6 pmol) were titrated against 0.15 pmol of 5[prime]-32P-labeled PO-8 substrate at 37°C (Fig. 3A). Pseudo-origin binding increased linearly with hRPA concentration up to 0.5 pmol of hRPA when 1/2 of the DNA substrate was bound. In parallel hRPA titrations (0.01-300 pmol) at 15°C, we determined the dissociation constant for hRPA binding to the PO-8 substrate to be 7.7 × 10-7 M, compared to a value of 9.0 × 10-8 M for a 48 nt ssDNA molecule (Fig. 3B). Under these conditions, the dissociation constant of hRPA binding to a completely duplex 48 bp DNA molecule (PO-0) (17) was of insufficient strength to be accurately determined (>1.5 × 10-4 M). Thus, although hRPA binds the PO-8 substrate with ~10-fold lower affinity compared to a ssDNA substrate, the binding is still >100-fold stronger than to a duplex DNA substrate, consistent with the results obtained previously using the electrophoretic mobility shift assay (17).


Figure 3. Effect of hRPA protein concentration on PO-8 binding and denaturation. (A) Increasing amounts of recombinant hRPA were incubated with the PO-8 substrate (0.15 pmol; 32P-labeled on the top strand) for 20 min at 37°C. The amount of bound PO-8 (filled circles) was measured by a nitrocellulose filter binding assay as described in Materials and Methods. PO-8 denaturation (open circles) was determined by phenol:chloroform extraction of the reaction mixtures in the presence of a 10-fold molar excess of unlabeled top strand, followed by native gel electrophoresis, as indicated in Materials and Methods. Autoradiographs were subjected to densitometric analysis to quantify the amount of unwound DNA. (B) Determination of dissociation constants for recombinant hRPA binding to different DNA substrates. Each 32P-labeled DNA substrate [0.15 pmol; PO-0 (duplex DNA), triangles; PO-8, squares; ssDNA (top strand of PO-8); circles)] was incubated with a wide range of hRPA concentrations (0.01-300 pmol) for 20 min at 15°C. The amount of bound DNA was quantitated as indicated for (A). The fraction of bound DNA was plotted as a function of total concentration of active hRPA, and the best fitting theoretical curves are shown (Materials and Methods). Each curve is based upon one representative experiment of two to three performed for each DNA substrate.

The effect of hRPA concentration on substrate unwinding was also examined. Although DNA unwinding increased as the hRPA level was raised, substrate unwinding only occurred on a fraction of the substrate that was bound by hRPA (Fig. 3A). Pseudo-origin unwinding was observed at levels ~1/4-1/3 of those observed for binding, with the average unwinding for all levels of hRPA found to be 37.5% of the bound substrate. Thus, we conclude that efficient binding of hRPA to the PO-8 substrate can occur on the partially duplex substrate. In other words, these results show that hRPA binding does not require complete unwinding of the substrate.

Pseudo-origin binding and denaturation occur at elevated salt concentrations

It has been previously shown that the general DNA unwinding reaction of ctRPA is inhibited 90% by the presence of 1 mM Mg2+ or 50 mM NaCl (21) and somewhat similar observations were noted for hRPA (20). To determine if the PO-8 unwinding reaction has a similar salt response, we examined the effect of salt concentration on PO-8 binding and unwinding by hRPA, choosing reaction conditions similar to those used previously (20,21,25). Thus, we varied the concentration of either MgCl2 (from 0.5 to 7 mM) or NaCl (from 3 to 100 mM) in the reaction buffer, while keeping the protein-DNA ratio constant (1.5 pmol-0.15 pmol). Increasing the MgCl2 concentration from 0.5 to 7 mM (in the presence of 20 mM NaCl) caused both PO-8 binding and unwinding to decrease only modestly (32 and 33%, respectively; Fig. 4). As significant unwinding activity was observed even at 7 mM, the effect of an increase in Mg2+ concentration on the pseudo-origin unwinding reaction is different from that previously reported for the general DNA unwinding reactions mediated by RPA (20,21,25).


Figure 4. Effect of increasing MgCl2 concentration on PO-8 binding and denaturation by hRPA. Recombinant hRPA (1.5 pmol) was incubated at 37°C for 20 min with the PO-8 substrate (0.15 pmol; 32P-labeled on the top strand) in the presence of increasing concentrations of MgCl2. Following incubation, the efficiency of PO-8 binding (filled circles) was established by a nitrocellulose filter binding assay as described in Materials and Methods. In separate reactions, PO-8 denaturation (open circles) was determined by phenol:chloroform extraction in the presence of a 10-fold molar excess of unlabeled ssDNA competitor and subsequent native gel electrophoresis, as indicated in Materials and Methods. In order to quantify the amount of single-stranded DNA product, autoradiographs were subjected to densitometric analysis.

The binding of hRPA to the pseudo-origin substrate was tested in the presence of increasing levels of NaCl (3-100 mM; Fig. 5), and in the presence of either 0.5 mM (filled symbols) or 7 mM (open symbols) MgCl2. Somewhat surprisingly, hRPA binding to the pseudo-origin was stimulated ~2-3-fold as the salt concentration was raised from 3 to 20 mM NaCl (Fig. 5, square symbols), regardless of the Mg2+ concentration used. As the NaCl concentration was raised further, hRPA binding decreased gradually to levels slightly above those observed at the lowest salt concentration. The increase in the apparent binding affinity of hRPA for PO-8 with increasing salt was unexpected because the equilibrium constants of most protein-nucleic acid complexes strongly decrease with increases in salt concentration because of the disruption of electrostatic contacts (e.g. 26). A few exceptions to this rule have been noted such as the interaction of E.coli ribosomal S1 protein with RNA, although this effect was observed at higher salt concentrations (between 0.1 and 1 M NaCl) (27). As postulated for such cases (26,27), salt may induce favorable changes in nucleic acid conformation or drive cooperative hRPA binding. Alternatively, low salt concentrations may reduce the stability of hRPA, although previous studies did not show any apparent diminution of hRPA activity under such conditions (20,21).


Figure 5. Effect of increasing NaCl concentration on PO-8 binding and denaturation by hRPA. Recombinant hRPA (1.5 pmol) was incubated at 37°C for 20 min with the PO-8 substrate (0.15 pmol; 32P-labeled on the top strand) in the presence of increasing concentrations of NaCl. Reaction mixtures contained either 0.5 mM MgCl2 (squares) or 7 mM MgCl2 (circles). The amount of bound PO-8 (filled symbols) was measured by a nitrocellulose filter binding assay as described in Materials and Methods. In parallel reactions, PO-8 denaturation (open symbols) was determined by extracting the reaction mixtures with phenol:chloroform in the presence of a 10-fold molar excess of unlabeled top strand, and then separating the reaction products by native gel electrophoresis as indicated in Materials and Methods. Densitometric analysis of the autoradiographs was performed to quantify the amount of ssDNA produced.

Pseudo-origin unwinding had a similar although relatively smaller response to NaCl. Like the effect on hRPA binding, increases in the salt concentration from 3 to 20 or 30 mM stimulated DNA unwinding 2.5-fold in the presence of 0.5 mM MgCl2, and 1.7-fold with 7 mM MgCl2 (Fig. 5). Increasing the NaCl concentration to 100 mM reduced PO-8 unwinding to levels similar to those observed at 3 mM. We therefore find that both PO-8 binding and unwinding are initially stimulated and then inhibited in response to increasing salt levels. Furthermore, our data indicate that elevated salt concentrations (i.e., 100 mM NaCl and 7 mM MgCl2) reduce both the binding and unwinding activities of hRPA from peak levels, but does not completely inhibit them.

Binding and unwinding by hRPA do not occur simultaneously

We investigated the time dependence of the PO-8 binding and unwinding reactions by hRPA. Initial binding studies performed at 37°C showed that binding was essentially complete at the earliest time point tested (10 s). In an attempt to determine rate constants for the interaction of hRPA with the PO-8 substrate, the incubation temperature was lowered to 15°C. The time courses of the PO-8 binding and unwinding reactions were then tested over a 60-fold range of hRPA concentrations using these conditions (Fig. 6). Unfortunately, we found that the binding and unwinding data did not follow first-order kinetics over the earliest time points tested, preventing accurate determination of the rate constants for these reactions. Furthermore, our data suggest that the PO-8 binding and unwinding reactions cannot be modeled using a simple kinetic scheme, and will require a more sophisticated analysis. With these caveats, estimates of the initial rate of hRPA binding and unwinding were determined (Fig. 6, legend). Over the range of hRPA concentrations tested, we calculated that the initial rate of PO-8 binding was, on average, 3.8-fold faster than the rate of PO-8 unwinding. Because DNA unwinding occurs with a significantly slower rate compared to DNA binding, these data indicate that these reactions do not take place simultaneously.


Figure 6. Kinetics of PO-8 binding and denaturation by hRPA. A constant amount (0.15 pmol) of PO-8 substrate (32P-labeled on the top strand) was incubated at 15°C for various times with variable amounts of recombinant hRPA (0.5 pmol, squares; 1 pmol, circles; 3 pmol, inverted triangles; 5 pmol, diamonds; 30 pmol, triangles). Following incubation, a nitrocellulose filter binding assay was performed to determine the amount of PO-8 binding [(A), filled symbols] as described in Materials and Methods. Separately, PO-8 denaturation [(B), open symbols] was ascertained by phenol:chloroform extraction of the reaction mixtures in the presence of a 10-fold molar excess of unlabeled top strand and subsequent native gel electrophoresis. Autoradiographs were subjected to densitometric analysis to quantify the amount of ssDNA. The initial rates of binding and unwinding were obtained from the slopes of semi-logarithmic plots showing the loss of the unbound or unwound PO-8 substrate, respectively, versus time of incubation (41). The calculated rates for each amount of hRPA tested were as follows: (i) binding-0.5 pmol, 0.79/min; 1.0 pmol, 0.80/min; 3 pmol, 1.7/min; 5 pmol, 2.4/min; 30 pmol, 3.3/min; (ii) unwinding-0.5 pmol, 0.09/min; 1 pmol, 0.48/min; 3 pmol, 0.65/min; 5 pmol, 0.77/min; 30 pmol, 1.1/min.

The binding and unwinding activities of hRPA are temperature dependent

We tested the effect of temperature on the ability of hRPA to bind and unwind the PO-8 substrate. We found that considerable hRPA binding (44%) could occur at low temperatures (0 and 4°C; Fig. 7) and increased 1.6-fold as the temperature was raised to 37°C (to 70% of the available substrate). As expected for a DNA unwinding event, PO-8 denaturation was more sensitive to temperature, with only 8% of the substrate pool denatured at 4°C. Substrate denaturation increased 3.7-fold to 30% of the pool as the temperature was raised to 37°C. In the absence of hRPA, we detected no significant denaturation of the substrate at any of the tested temperatures (data not shown), confirming that ssDNA formed only as a result of the hRPA unwinding activity. Thus, we find that substrate denaturation is significantly more sensitive than binding to reaction temperature.


Figure 7. Effect of temperature on the binding and denaturation of PO-8 by hRPA. Recombinant hRPA (1.5 pmol) was incubated at various temperatures with the PO-8 substrate (0.15 pmol; 32P-labeled on the top strand). Subsequent to incubation, the efficiency of PO-8 binding (filled circles) was measured by a nitrocellulose filter binding assay as described in Materials and Methods. To determine PO-8 denaturation (open circles), the reaction mixtures were extracted by phenol:chloroform in the presence of a 10-fold molar excess of competitor (unlabeled top strand), and then subjected to native gel electrophoresis as indicated in Materials and Methods. Autoradiographs were analyzed by densitometry to quantitate the amount of ssDNA obtained.

Some SSBs that bind the PO-8 substrate cannot support its unwinding

We previously reported that hRPA and E.coli SSB (EcoSSB) bound the PO-8 substrate with high affinity, while moderate binding was detected for the adenovirus DNA-binding protein (AdDBP) and herpes simplex virus ICP8 protein (ICP8), and poor, if any, binding was observed for the bacteriophage T4 gene 32 (T4 gp32) and bacteriophage T7 gene 2.5 (T7 gp2.5) proteins (17). Unfortunately, these electrophoretic mobility shift experiments were unable to provide information concerning the ability of the various SSB proteins to denature the pseudo-origin substrate. We re-examined this issue using our improved detection system for substrate unwinding.

Increasing amounts of each SSB (from 0.5 to 6 pmol of their active oligomeric form) were incubated with 0.15 pmol of the PO-8 substrate. Similar to our previous results, hRPA and EcoSSB were found to bind the PO-8 substrate most efficiently (Fig. 8A). The ICP8 and AdDBP proteins bound the substrate at lower though detectable levels, and T4 gp32 and T7 gp2.5 bound most weakly.


Figure 8. Binding and denaturation of PO-8 by different SSBs. The PO-8 substrate (0.15 pmol; 32P-labeled on the top strand) was incubated with increasing amounts (0.5-6 pmol of the active oligomer) of each SSB (recombinant hRPA, filled squares; EcoSSB, open circles; AdDBP, filled circles; HSV ICP8, open triangles; T4 gp32, open squares; T7 gp2.5, filled triangles). After incubation, the amount of PO-8 binding (A) was determined by a nitrocellulose filter binding assay as described in Materials and Methods. In separate reactions, PO-8 denaturation (B) was determined by phenol:chloroform extraction of the reaction mixtures in the presence of a 10-fold molar excess of unlabeled top strand, followed by native gel electrophoresis as indicated in Materials and Methods. The amount of ssDNA was then measured by densitometric analysis of the autoradiographs.

Of all the SSB proteins tested, only hRPA and EcoSSB were able to unwind a significant fraction of the PO-8 substrate pool (Fig. 8B). Although the binding activity of EcoSSB is somewhat less than that of hRPA using [le]1 pmol of the active oligomeric form of each protein (i.e., an hRPA heterotrimer and an EcoSSB tetramer), the unwinding activities of these proteins were comparable at these and higher SSB levels. ICP8, AdDBP, T4 gp32 and T7 gp2.5 displayed no significant substrate unwinding activity. The unwinding activity of EcoSSB is particularly interesting because a helix-destabilizing activity for this protein has not been reproducibly detected (28). Each of these SSB proteins has been tested previously for its ability to replace hRPA in the T antigen-mediated SV40 origin denaturation reaction and it was found that hRPA and EcoSSB were most active, ICP8 and AdDBP had intermediate activity, and T4 gp32 and T7 gp2.5 were not functional in this reaction (29,30). As ICP8 and AdDBP did not cause pseudo-origin denaturation, our data indicate that the ability of an SSB to function in the T antigen-mediated SV40 origin denaturation reaction correlates with the ability to bind the PO-8 substrate. However, those SSBs most active in the SV40 origin denaturation reaction were also most active in the PO-8 DNA unwinding reaction.

DISCUSSION

A critical role for hRPA in the denaturation of the SV40 origin was previously suggested by the ability of hRPA to bind and denature a DNA structure resembling that induced within the origin by T antigen (17). In combination with our previous observations, our data suggest that hRPA interacts with the duplex DNA flanking the central 8 nt of ssDNA, inducing duplex destabilization and subsequent denaturation on a fraction of the bound molecules. These data indicate that denaturation of the pseudo-origin molecule occurs in a pathway in which binding and unwinding are sequential events.

The pseudo-origin binding and unwinding activities of hRPA are clearly separated by various criteria. First, kinetic examination of each reaction indicates that DNA unwinding occurs with an ~4-fold slower rate relative to DNA binding. Second, the dissociation constant of hRPA binding to the PO-8 substrate was ~10-fold lower than to a comparable ssDNA molecule, indicating that hRPA was not merely binding to transiently-formed ssDNA regions on the PO-8 substrate. Third, at low temperatures (i.e. 4°C), only 18% of the bound substrates were unwound, and this fraction increased to 43% as the temperature was raised to 37°C. These data indicate that hRPA initially binds to the non-denatured pseudo-origin substrate and these complexes have sufficient stability to be retained on nitrocellulose filters (this work) or during electrophoretic mobility shift assays (17). On a fraction of the bound templates (~1/3 under our standard conditions), the reaction proceeds to complete denaturation of the DNA molecule.

hRPA was previously found to induce structural distortion within the duplex DNA flanking the melted region (17). Because we find that hRPA can bind the PO-8 substrate in lieu of its complete denaturation, our data therefore suggest that the distortion is a result of hRPA binding the duplex DNA. Previous copper-phenanthroline footprinting of hRPA-pseudo-origin complexes did not reveal significant protection of the duplex DNA (17) suggesting that this interaction is likely to be transient in nature. We previously proposed that the binding of hRPA to ssDNA initially involves an unstable recognition of 8 nt, and then stabilization of complex formation through extension of the bound ssDNA to 30 nt (10). Biochemical and physical examination of hRPA indicate that the primary high-affinity DNA binding site, sufficient to interact with 8 nt of ssDNA, is contained in the middle region of the 70 kDa subunit (31-34). More recent studies have revealed the presence of two additional, albeit weaker, DNA-binding sites that are located on the C-terminal portion of the 70 kDa subunit and within the 29 kDa subunit (35,36). Thus, it is possible that these other hRPA elements interact with the duplex flanks of the PO-8 molecule. Alternatively, as we find that three to four hRPA molecules can bind to the PO-8 substrate (Fig. 1), multiple hRPA molecules may interact with the substrate, each using the primary 70 kDa DNA-binding domain, such that one hRPA molecule binds the 8 nt ssDNA bubble and an adjacent molecule binds duplex DNA on the flank. In either case, binding of double-stranded DNA causes the duplex to become structurally deformed.

In all experiments examining the unwinding of the PO-8 molecule, we observed that hRPA was only able to unwind a fraction of the bound substrate. Although various causes are possible, one likely explanation is that the hRPA pool contains a fraction of inactive molecules which are unable to catalyze the DNA unwinding reaction. As PO-8 becomes bound by hRPA oligomers (e.g., trimers and tetramers), these inactive molecules could act to inhibit PO-8 unwinding by other bound molecules.

The general DNA unwinding activity previously described for ctRPA or hRPA (20,21) differs from the pseudo-origin unwinding activity in various aspects. The primary difference is the response to elevated salt concentrations. General unwinding is unable to proceed in the presence of >5 mM Mg2+ while the pseudo-origin unwinding progresses with 2/3 of the efficiency observed in Mg2+-free reactions. Moreover, NaCl concentrations >50 mM prevented general DNA unwinding, while pseudo-origin unwinding in 100 mM NaCl was ~1/2 that of peak unwinding levels (at 20-30 mM NaCl). Surprisingly, we also found that unwinding was stimulated when the salt concentration was increased from 3 to 20 mM. Although different substrates were used to test these unwinding reactions and modest differences in reaction conditions were employed, the pseudo-origin and general DNA unwinding reactions are apparently distinct. It may be that the pseudo-origin denaturation reaction is a subset of more general DNA unwinding reactions that can take place under physiological salt concentrations.

Tests of the ability of other SSB proteins to interact with the pseudo-origin substrate over a wide range of protein concentrations showed that PO-8 binding rather than PO-8 denaturation correlated with the ability of these proteins to support the T antigen-mediated origin-unwinding reaction. However, it is notable that all the proteins that support the origin unwinding reaction (hRPA, EcoSSB, AdDBP, ICP8) also have helix destabilizing activity (17,20,21,37-39; EcoSSB, this work). We conclude that origin denaturation is a process mediated both by hRPA and T antigen. The ATP-dependent binding of T antigen destabilizes the flanking DNA elements (40) and provides an ssDNA binding site that can be utilized by hRPA (in the normal context of infected cells) or by other SSBs (using in vitro systems). Binding of the SSB to the destabilized DNA, in combination with its intrinsic unwinding activity, leads to further denaturation of the origin. T antigen is thus released from sequence-specific contacts to the origin allowing the T antigen DNA helicase to unwind the remainder of the viral DNA molecule and provide a template for the DNA replication machinery.

ACKNOWLEDGEMENTS

We thank Marc Wold for his kind gift of the p11d-tRNA vector overexpressing hRPA, and Kenneth Marians (Memorial Sloan-Kettering Cancer Center), I. Robert Lehman (Stanford University School of Medicine), Ronald T. Hay (University of St Andrews, UK) and Charles Richardson (Harvard Medical School) for generously supplying EcoSSB, ICP8, AdDBP and T7 gp2.5, respectively. We also thank Natalia Smelkova, Jennifer Garner, Thomas Gillette, Yaron Daniely and Mehboob Shivji for constructive comments during the course of this project and for critical reading of the manuscript. This research was supported by NIH grant AI29963 and Kaplan Cancer Center Developmental Funding and Kaplan Cancer Center Support Core Grant (NCI P30CA16087).

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*To whom correspondence should be addressed. Tel: +1 212 263 8453; Fax: +1 212 263 8166; Email: borowj01@mcrcr.med.nyu.edu

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