mimic the nucleotide-bound state bovine papillomavirus e1 initiator

JOURNAL OF VIROLOGY, Feb. 2010, p. 1912–1919 Vol. 84, No. 40022-538X/10/$12.00 doi:10.1128/JVI.01756-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Mutations in Sensor 1 and Walker B in the Bovine Papillomavirus E1Initiator Protein Mimic the Nucleotide-Bound State�

Xiaofei Liu1,2 and Arne Stenlund1*Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724,1 and Graduate Program in Molecular and

Cellular Biology, State University of New York at Stony Brook, Stony Brook, New York 117942

Received 20 August 2009/Accepted 18 November 2009

Viral replication initiator proteins are multifunctional proteins that utilize ATP binding and hydrolysis bytheir AAA� modules for multiple functions in the replication of their viral genomes. These proteins aretherefore of particular interest for understanding how AAA� proteins carry out multiple ATP driven functions.We have performed a comprehensive mutational analysis of the residues involved in ATP binding andhydrolysis in the papillomavirus E1 initiator protein based on the recent structural data. Ten of the elevenresidues that were targeted were defective for ATP hydrolysis, and seven of these were also defective for ATPbinding. The three mutants that could still bind nucleotide represent the Walker B motif (D478 and D479) andSensor 1 (N523), three residues that are in close proximity to each other and generally are considered to beinvolved in ATP hydrolysis. Surprisingly, however, two of these mutants, D478A and N523A, mimicked thenucleotide bound state and were capable of binding DNA in the absence of nucleotide. However, these mutantscould not form the E1 double trimer in the absence of nucleotide, demonstrating that there are two qualitativelydifferent consequences of ATP binding by E1, one that can be mimicked by D478A and N523A and one whichcannot.

Viral initiator proteins from DNA viruses belong to thesuperfamily 3 (SF3) helicases (5, 9). Well-studied members ofthis group include the T-antigens from the polyomaviruses, theE1 proteins from the papillomaviruses, and the Rep proteinsfrom the adeno-associated viruses. These proteins are multi-functional proteins that utilize ATP binding and hydrolysis bytheir AAA� (ATPases associated with various cellular activi-ties) modules for multiple functions in the replication of theirviral genomes. AAA� modules are �250-amino-acid ATPbinding domains that carry out numerous ATP driven func-tions (for reviews, see references 6 and 7). For example, the E1protein, which plays an essential role in papillomavirus DNAreplication, has multiple functions that are affected by bindingor hydrolysis of ATP (14, 18, 21, 23, 24, 26). E1 is a DNA-binding protein, which binds specifically to E1 binding sites (E1BS) in the origin of DNA replication (2, 8, 15, 22, 25). DNA-binding activity requires nucleotide binding by E1 (15). In thepresence of ATP or ADP, E1 can form a specific double-trimer(DT) complex on the ori and, through ATP hydrolysis, thiscomplex can melt the ori DNA (13, 15, 16). In a process thatrequires ATP hydrolysis, the DT is then converted into a dou-ble hexamer (DH), which has ATP-dependent DNA helicaseactivity and is the replicative DNA helicase (15, 26). Conse-quently, the E1 AAA� module is utilized for ATP binding andhydrolysis in at least two different E1 complexes with differentfunctions. An interesting question is how the same motif forATP binding and hydrolysis is used in these different com-plexes to achieve their differing functions.

Structural studies of representatives from all threegroups—E1 proteins, T antigens, and Rep proteins—have pro-vided important information about how ATP is bound andhydrolyzed by these proteins and the structural consequencesthat result (1, 4, 7, 10–12). For example, in the recent crystalstructure of a hexamer of the E1 oligomerization and helicasedomains formed on single-stranded DNA, an ATP bindingpocket is formed by 11 residues from two adjacent monomersof the E1 helicase domain (Fig. 1A) (3). Because most of theresidues thought to be involved in ATP binding and hydrolysisin these AAA� proteins are highly conserved and form par-ticular substructures, the specific function of the individualresidues have been predicted for these proteins (6) (see Fig.1A). It is well established that the conserved residues in theWalker A and Walker B motifs are involved in both bindingand hydrolysis of ATP. The Sensor 1 residues are generallyinvolved in contacting Walker B and the �-phosphate of ATP.The Sensor 2 motif also participates in nucleotide binding andinteracts directly with the �-phosphate of ATP.

To gain a more precise understanding of the role of theseparticular residues in ATP binding and hydrolysis and becausea systematic analysis of such residues has not been performedfor the E1 initiator proteins, we performed a mutational anal-ysis of these 11 residues. Based on the behavior of mutants inthese residues, the residues can be classified into three groups.Seven of the mutations result in a protein that fails to bindnucleotide and consequently also fail to hydrolyze nucleotide.Three mutants can still bind nucleotide but fail to hydrolyzeATP. Surprisingly, two of these mutants mimic the ATP-boundstate and can bind DNA in the absence of nucleotide, demon-strating that E1 utilizes ATP binding for two different modes,only one of which can be mimicked by the mutations in theATP binding pocket.

* Corresponding author. Mailing address: Cold Spring Harbor Lab-oratory, P.O. Box 100, 1 Bungtown Road, Cold Spring Harbor, NY11724. Phone: (516) 367-8407. Fax: (516) 367-8454. E-mail: [email protected].

� Published ahead of print on 25 November 2009.

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MATERIALS AND METHODS

Recombinant proteins. Expression and purification of E1 and E2 proteinswere carried out as described previously (19).

EMSA. 4% acrylamide gels (39:1, acrylamide-bis) containing 0.5� Tris-borate-EDTA were used for all electrophoretic mobility shift assay (EMSA) experi-ments. E1 was added to the probe (5,000 cpm) in a solution containing 20 mMHEPES (pH 7.5), 100 mM NaCl, 0.7 mg of bovine serum albumin (BSA)/ml,0.1% NP-40, 5% glycerol, 5 mM dithiothreitol (DTT), 5 mM MgCl2, and 2 mMATP or ADP. After incubation at room temperature for 60 min, the sampleswere loaded and run for 2 h at 9 V/cm. For the E12E22-ori complex formation,EMSA was performed using the conditions described above except that nonucleotide was present. The 84-bp probe, which contains the E1 BS and theadjacent low-affinity E2 BS12, was used. The E12E22-ori complex formationrequires both the E1 and the E2 binding sites and is independent of nucleotide.

Pulldown experiments. The E1 oligomerization and helicase domain fragment(amino acids [aa] 308 to 605) was expressed in Escherichia coli as a glutathioneS-transferase (GST) fusion, purified as previously described (19), separated fromthe GST tag by thrombin cleavage and further purified by ion-exchange chro-matography on a Mono S column. A portion (0.5 �g) of the protein was labeledat residue S584 by incubation with 1 U of the protein kinase CK2 (New EnglandBiolabs) in the presence of [�-32P]ATP in a volume of 10 �l. The labeled proteinwas diluted 200-fold in a solution containing 20 mM HEPES (pH 7.5), 100 mMNaCl, 0.7 mg of BSA/ml, 0.1% NP-40, 5% glycerol, 5 mM DTT, 5 mM MgCl2,and 10 �l of the 32P-labeled 308-605 protein was incubated with the GST fusionproteins (�1 �g) in the absence of nucleotide or in the presence of 2 mM ADPor 2 mM ATP. After incubation at room temperature for 1 h, 5 �l of glutathioneagarose gel slurry was added, and incubation was continued for 30 min. Thebeads were washed in 4 � 500 �l of Tris-buffered saline with 0.1% NP-40, andthe bound material was analyzed by SDS-PAGE and quantitated by using a FujiBAS 5000 imager.

ATPase assays. ATPase assays were performed in a 20-�l reaction containing30 mM HEPES (pH 7.5), 30 mM NaCl, 1 mM DTT, 7 mM MgCl2, 100 �g ofbovine serum albumin/ml, 100 �M ATP, and 40,000 cpm of [�-32P]ATP and E1.Reactions were incubated for 1 h at room temperature and stopped by theaddition of EDTA to a final concentration of 10 mM. Then, 2-�l portions of thereactions were spotted onto a polyethyleneimine-cellulose plate, and the plateswere then developed in 1 M formic acid and 0.5 M LiCl2 for 40 min. After drying,each plate was exposed to a Fuji imaging plate, and the level of free phosphatewas determined by scanning the plate using a Fuji BAS imager.

RESULTS AND DISCUSSION

Based on the structure of the hexamer of the E1 oligomer-ization and helicase domains (Fig. 1A), we mutated all of theresidues predicted to contact ATP in a BPV1 E1 hexamer (3).These 11 substitutions were generated in the E. coli expressionvector pETGST-E1 by site-directed mutagenesis. Ten of theeleven residues (K425, K439, S440, D478, D479, D497, Y499,N523, Y534, and R538) were changed into alanine. Becausethe alanine substitution at R493 was not expressed, this residuewas mutated into Leu, Met, and Glu. After expression andpurification of the mutant proteins they were first tested for theability to bind to the origin of DNA replication, together withthe BPV E2 protein. The E1 and E2 proteins bind coopera-tively to adjacent sites in the origin of DNA replication (17, 20,25, 27). The resulting complex, E12E22-ori, does not rely onnucleotide binding or hydrolysis for formation and thereforeserves as a convenient control for the structural integrity of theE1 mutants and for the intrinsic ability of the mutant proteinsto bind to the E1 BS in the ori.

The alanine substitutions were tested for E12E22 complexformation to ascertain that they had no structural defects dueto the mutations (Fig. 1B). As is well established, E2 alonebinds to the E2 binding site present in the ori and forms an E2dimer complex (Fig. 1B, lane 23). In the presence of wild-type(wt) E1 a larger complex E12E22 forms through cooperative

FIG. 1. (A) Residues in E1 involved in nucleotide binding andhydrolysis. A schematic image is shown of the interface between twoE1 monomers that constitute the ATP binding pocket of BPV E1 withthe residues that are predicted to be involved in ATP binding andhydrolysis (adapted from reference 3). The Walker A, Walker B, andSensor 1 and Sensor 2 motifs and the arginine finger are indicated.(B) Formation of the E12E22-ori complex. EMSA was performed usingan 84-bp ori probe. Two quantities (1.5 and 3 ng) of wt E1 and of eachE1 substitution, as indicated at the top of the gels, were used in thepresence of 0.1 ng of full-length E2. In lane 23, E2 alone was added.The mobility of the E22 and E12E22 complexes are indicated.(C) ATPase activity of E1 substitution mutations of residues involvedin ATP binding and hydrolysis. Portions (80 ng) of wt E1 or of eachrespective E1 substitution mutant as indicated were tested for ATPaseactivity using 32P-labeled �-ATP. After the reaction the free phosphatewas separated from ATP by thin-layer chromatography and quanti-tated by using a Fuji imager. Lane 12 contained [�-32P]ATP only.

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binding of E1 and E2 to adjacent E1 and E2 binding sites (Fig.1B, lanes 1 and 2). All of the mutants, with one exception,could form this complex, as well as wt E1 (Fig. 1B, lanes 3 to20). The exception, R538A, showed a slight defect in formationof the E12E22 complex (Fig. 1B, lanes 21 and 22). R493E, -M,and -L, also formed wt levels of E12E22-ori complex formation(data not shown).

We next tested the mutants in an ATPase assay (Fig. 1C). Inthis assay, ATP with a radioactively labeled �-phosphate wasincubated with either the wt or the mutant E1 proteins. Hy-drolysis of the ATP would result in the appearance of radio-actively labeled free phosphate, which can be separated fromradioactively labeled ATP by thin-layer chromatography (Fig.1C). The 10 alanine substitutions were, with one exception,devoid of detectable ATPase activity, showing a �20-fold re-duction compared to wt E1. The exception (Y534, lane 10) had�25% of the ATPase activity of the wt E1 (compare lanes 1and 13). This result demonstrated that all of the 10 residuesplay a role in ATP hydrolysis. As expected, the mutantsR493E, M, and L was also devoid of ATPase activity (data notshown).

Measurement of nucleotide binding for proteins such as E1are complicated by the fact that ATP binds between two sub-units, and the level of ATP binding would therefore dependgreatly on whether the protein is monomeric or oligomeric. Wehave demonstrated that E1 in the absence of DNA is mono-meric, and methods such as filter-binding assays to measureATP binding are therefore not practical (18). Instead, we usedan indirect method to measure nucleotide binding. In the ab-sence of nucleotide, E1 binds to DNA weakly or not at all,depending on the conditions (15). The basis for this depen-dence on nucleotide binding for DNA binding is unknown butis likely the result of conformational changes in E1 that ex-poses the E1 DNA-binding domain.

To determine whether the 10 alanine substitutions were alsodefective for nucleotide binding, we utilized EMSA. E1 can, inthe presence of ADP or ATP, form a trimer complex on anyshort DNA sequence (15). We incubated the wt and mutant E1proteins with a 39-bp ori probe that lacks E1 BS. In the ab-sence of nucleotide, no complex is observed (Fig. 2A, lanes 1 to3), while in the presence of ADP (lanes 4 to 6) the trimer bandis observed. Two of the mutants, D479A and Y534A similar towt E1, bound as a trimer in the presence of ADP (Fig. 2A,lanes 10 to 12 and lanes 16 to 18, respectively) but not in itsabsence (Fig. 2A, lanes 7 to 9 and lanes 13 to 15, respectively).Two other mutants, D478A and N523A, showed a differentbehavior. These two mutants showed significant trimer forma-tion both in the absence (Fig. 2B, lanes 7 to 9 and lanes 13 to15, respectively) and in the presence of ADP (Fig. 2B, lanes 10to 12 and lanes 16 to 18, respectively). The remaining sixalanine substitutions—K425A, K439A, S440A, D497A,Y499A, and R538A—failed to form the trimer complex(Fig. 2C), as did the three R493 substitutions (Fig. 2D, lanes2 to 12).

These results placed the mutants in three different catego-ries. The largest group, seven mutants, failed to form a trimerin the absence and presence of ADP, indicating that thesemutants are defective for nucleotide binding. The secondgroup with the two mutants D479A and Y534A behaved as wtE1, i.e., these mutants formed the trimer, but only in the

presence of ADP. Since D479A was defective for ATP hydro-lysis, this demonstrates that this mutant can bind but not hy-drolyze ATP. Y534A can also clearly bind ATP but is onlyslightly defective for ATP hydrolysis (Fig. 1B). The thirdgroup, consisting of the mutants D478A and N523A, are themost interesting mutants, since they behaved as if they hadnucleotide bound even in the absence of nucleotide.

We focused on the four mutants that were capable of DNAbinding in the E1 trimer assay above and tested these mutantsfor the ability to form the functional double trimer (DT) anddouble hexamer (DH) (15) (Fig. 3). As expected, wt E1 failedto form a complex in the absence of nucleotide (Fig. 3A, lanes1 and 2) formed a DT (E16) in the presence of ADP (lanes 3to 4) and a DH (E112) in the presence of ATP (lanes 5 and 6).The mutant D478A (Fig. 3A, lanes 7 to 12) formed ladders inthe absence of nucleotide (lanes 7 and 8). In the presence ofADP and ATP a DT was formed (Fig. 3A, lanes 9 and 10 andlanes 11 and 12, respectively). N523A (Fig. 3B, lanes 10 to 18)behaved similarly. These results clearly indicate that D478Aand N523A are incapable of hydrolyzing ATP, a finding con-sistent with the ATPase assays but, because a qualitative effectof ADP addition is observed (a ladder versus a DT), theseproteins can both bind nucleotide. However, since DNA bind-ing is observed even in the absence of nucleotide these muta-tions clearly have an effect on the DNA binding properties ofthe protein in the absence of nucleotide, mimicking some as-pects of nucleotide binding, as observed above.

The mutants D479A (Fig. 3A, lanes 13 and 14) and Y534A(Fig. 3A, lanes 19 and 20) behaved as predicted from thetrimer formation and failed to form DT without nucleotide.D479A also formed the DT very weakly in the presence ofADP (Fig. 3A, lanes 15 to 18) and instead formed a largercomplex of unknown composition. This indicates that this mu-tant may have a defect in ADP binding; however, the DT wasformed more efficiently in the presence of ATP (lanes 17 to18). Y534A (Fig. 3A, lanes 21 to 24), however, formed DT inthe presence of both ADP and ATP, showing no indicationthat the residual ATPase activity of this mutant allows theformation of the DH.

The DNA binding properties of the two mutants D478A andN523A clearly indicate that they are defective for ATP hydro-lysis since the DH does not form in the presence of ATP. Thisresult is consistent with the ATPase assays, which also dem-onstrated a lack of ATPase activity for these mutants (Fig. 1C).Furthermore, these two mutants have substantial DNA-bind-ing activity in the absence of nucleotide, indicating that themutations may mimic the nucleotide bound state. Interestingly,however, both of these mutants are also affected by addition ofnucleotide, indicating that they can still bind nucleotide.

The behavior of D478A and N523A indicated that theseparticular mutations mimic a conformational change that nor-mally is induced by nucleotide binding, giving rise to DNA-binding activity in the absence of nucleotide. Another possi-bility is that these particular mutant proteins already havenucleotide stably bound that survives the purification proce-dure. To distinguish between these two possibilities, we per-formed the experiment shown in Fig. 4. We first incubated theE1 protein under EMSA conditions with 2 mM ADP at roomtemperature for 10 min. The sample was then diluted 20-foldto provide a final ADP concentration of 0.1 mM. Probe was

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then added to the reaction mixture, and the sample was dividedin two. To one half no ADP was added (lane 2), and to theother half 2 mM ADP was added (lane 3). wt E1 failed to formtrimer without nucleotide (lane 1) and also failed to formtrimer in the sample diluted without nucleotide (lane 2) butformed a robust complex in the presence of added ADP (lane3). This result demonstrates that 0.1 mM ADP is not sufficientfor trimer formation. It also demonstrates that wt E1 does notbind ADP sufficiently well to allow dilution and therefore thatthe half-life of nucleotide bound to wt E1 is very short since theduration of the dilution is less than 1 min.

We performed the same experiment with N523A andD478A (Fig. 4, lanes 4 to 9). These mutants, which bind DNA

without nucleotide, showed no difference in trimer formationwithout ADP and with the diluted ADP (Fig. 4, lanes 4 and 5and lanes 7 and 8, respectively), demonstrating either that theylike wt E1 bind nucleotide with a very short half-life or thatthey cannot bind nucleotide. Since we have already shown thatboth of these proteins can bind nucleotide in Fig. 3, this resultdemonstrates that the half-life for bound nucleotide is veryshort. That N523A is capable of binding ADP is also clearlydemonstrated by the incubation with 2 mM ADP, which re-sulted in a substantial increase in binding (Fig. 4, lane 6). TheD478A mutant behaved similarly except that addition of ADPdid not stimulate trimer formation (Fig. 4, lanes 7 to 9), afinding consistent with the results in Fig. 2.

FIG. 2. Trimer formation of wt E1 and E1 substitution mutants. wt E1 and the E1 point mutants were tested for complex formation by EMSAon a 39-bp ori probe on which the wt E1 forms a trimer. Three quantities (6, 12, and 24 ng) of wt E1 and the respective E1 mutants were usedin the absence or presence of 2 mM ADP as indicated at the top of the panels. (A) D479A and Y534A were tested. (B) D478A and N523A weretested. (C) K425A, K439A, S440A, D497A, Y499A, and R538A were tested in the presence of ADP. (D) R493L, R493M, and R493E were testedin the presence of ADP.


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These results demonstrate that N523A clearly can bind ADPbut that the resulting E1-ADP complex has a very short half-life, since after dilution, which takes less than 1 min, the effectof the ADP is completely lost (compare lanes 4 and 5 in Fig. 4).Since the E1 purification procedure does not include ADP andtakes about 48 h, it is very unlikely that ADP could still bebound to E1 after this procedure. This result demonstrates thatthese mutations induce conformational changes, which is sim-ilar to those induced by nucleotide binding.

Our expectation was that the majority of the residues that wemutated in E1 would affect both ATP binding and hydrolysis,and it is extremely gratifying to note the exceptional predictivevalue of the E1 hexamer structure. Of the 11 residues that werepredicted to be involved in ATP binding and hydrolysis in thestructure, when mutated 10 were devoid of ATPase activity,and one mutant (Y534A) showed a fourfold reduction inATPase activity. Seven of the residues (K425A, K439A,

S440A, D497A, Y499A, R538A, and R493M, -L, and -E) whenmutated, resulted in a protein that was defective for both ATPbinding and hydrolysis. These include the expected Walker Amutations (K439A and S440A), the Sensor 2 mutation(K425A), and the arginine finger (R538A), plus an additionalthree residues (R493A, D497A, and Y499A). The only sur-prises in this group were the Sensor 2 and the arginine fingermutations, which generally affect ATP hydrolysis but not ATPbinding.

We also hoped that some mutations would affect ATP hy-drolysis only, since these mutant proteins would be usefulreagents. Three of the mutants—D478A, D479A, andN523A—are completely defective for ATP hydrolysis (Fig. 1B)but still are affected by the addition of ATP, indicating thatthey can bind nucleotide (Fig. 3). Interestingly, these mutantsrepresent the Walker B motif (D478 and D479) and the Sensor1 (N523), three residues that are in close proximity to each

FIG. 3. E1 DT and DH formation. wt E1 and E1 point mutants were tested for DT and DH formation in EMSA using an 84-bp ori probe. Twoquantities of E1 (6 and 12 ng) were used in the absence of nucleotide, in the presence of 2 mM ADP or in the presence of 2 mM ATP as indicatedabove the lanes. The positions of E1 DT (E16) and E1 DH (E112) are indicated. (A) wt E1, D478A, D479A, and Y534A were tested; (B) wt E1and N523A were tested.


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other. Two of these mutants (D478A and N523A) also showeda completely unexpected phenotype since these proteins, evenin the absence of nucleotide, are capable of binding DNA. Aparticularly striking result is the trimer formation in Fig. 2B,which clearly demonstrates that D478A and N523A are fullycompetent for trimer formation in the absence of nucleotide. Itis also interesting that the phenotype that we observe in Fig. 3,in contrast to the results in Fig. 2, only partially mimics nucle-otide binding. Here, although D478A and N523A clearly showthat they can bind DNA in the absence of nucleotide, a ladderis formed instead of the DT, and DT formation requires ad-dition of ADP. This result indicates that the nucleotide bindinghas at least two functions in these assays, one is to activateDNA binding in a generic way and the other is specifically toallow or to stimulate DT formation and that these two func-tions can be separated. Only the first of these functions ismimicked by the D478A and N523A mutations.

Currently, we cannot propose a structural model for how theD478A and N523A mutations result in a conformation thatmimics the ATP-bound state for either of these mutants. It iswell established that the conserved residues in Walker B (D478and D479) are involved in hydrolysis of ATP and, consistentwith such a function neither of the alanine substitutions, canhydrolyze ATP, although both can bind nucleotide. The Sensor1 residue, N523, is generally involved in contacting Walker Band the �-phosphate of ATP. The normal function of theseresidues in AAA� proteins does not explain the ability ofthese mutations to mimic the nucleotide-bound state. Further-more, neither of these residues is in a radically different con-formation in the structures generated in the absence or pres-ence of nucleotide (3).

These data therefore fail to provide a mechanism for hownucleotide binding results in generic activation of DNA bind-ing. Our data also do not explain why the E1DBD in thecontext of the full-length E1 is inactive for DNA binding unlessnucleotide is present. A possibility that would explain thesephenomena is that the failure of E1 to bind DNA in theabsence of nucleotide might be caused by a physical obstruc-tion of the E1 DBD. Such an obstruction would require thatanother part of E1 could interact with the DBD and blockDNA binding. Because the blockage would occur only in theabsence of nucleotide, the region responsible for the blockagepresumably would be affected by nucleotide binding, such asthe E1 oligomerization and helicase domain, which is locatedimmediately C-terminal to the E1 DBD. In this scenario, thetwo residues D478 and N523 in the helicase domain would beinvolved in interaction with the E1 DBD in the absence ofnucleotide.

To determine whether the E1 DBD interacts physically withthe E1 oligomerization and helicase domain, we used twoversions of GST E1DBD (aa 142 to 308 and aa 159 to 303) toattempt to pull down the C-terminal half of E1 (aa 308 to 605),which contains the oligomerization and helicase domains andbinds nucleotide (Fig. 5). We labeled the C-terminal half of E1by in vitro phosphorylation using the protein kinase CK2 and[�-32P]ATP. This results in phosphorylation on residue S584 inE1. CK2� is autophosphorylated and gives rise to a band thatmigrates slower than 308-605 (lane 1, input). As a negativecontrol we performed pulldown assays in the absence andpresence of ATP (lanes 2 and 3) using GST 1-60, which con-

FIG. 4. E1 binds ATP with a very short half-life. (A) Schematicdescription of the experiment shown in Fig. 4B. E1 was incubated inthe absence or presence of nucleotide. The samples were then diluted20-fold. Probe was then added, and the sample then loaded on anEMSA gel. In sample 1 in each set, the binding reaction without ADPwas diluted without ADP resulting in a sample without ADP. In sam-ple 2 in each set, the binding reaction was diluted without ADP re-sulting in a sample containing 0.1 mM ADP. In sample 3 in each set,the binding reaction was diluted with 2 mM ADP resulting in a samplecontaining 2 mM ADP. (B) wt E1, N523A, and D478A were incubatedas described in the scheme in panel A and analyzed by EMSA using the39-bp trimer probe. Lane 10 contained probe alone.


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tains the N-terminal 60 residues of E1 fused to GST. As ex-pected, the GST 1-60 fusion protein could bring down neitherCK2� nor 308-605. We next used the two GST E1 DBD fu-sions, 142-308 and 159-303, and performed pulldown assays inthe absence of nucleotide (lanes 4 and 7) in the presence ofADP (lanes 5 and 8) or in the presence of ATP (lanes 6 and 9).Interestingly, the 308-605 fragment was efficiently pulled down

by both of the GST E1DBD fragments in the absence ofnucleotide (lanes 4 and 7). In the presence of ADP the inter-action was reduced twofold for both GST E1DBD fragments(lanes 5 and 8) and in the presence of ATP the interaction wasreduced fourfold for both GST E1DBD fragments (comparelane 4 to lane 6 and lane 7 to lane 9). Importantly, these resultsdemonstrate that the E1 DBD interacts efficiently with theoligomerization and helicase domains and that this interactionis affected by the presence of nucleotide consistent with themodel presented above. We currently do not understand whythe interaction between the E1 DBD and the 308-605 fragmentis not completely abolished by the presence of nucleotide.Furthermore, the D478A mutation did not appreciably affectthe interaction with the E1 DBD (data not shown). Conse-quently, although some of the pulldown results are consistentwith the model presented above, other results such as theresidual interaction in the presence of ATP and the failure ofthe D478A mutation to disrupt the interaction indicate thatour model lacks some important components. The resolutionof these questions will require further analysis of the interac-tion between the E1 DBD and the E1 helicase domain.

ACKNOWLEDGMENT

This research was supported by grant RO1 AI 072345 to A.S.

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13. Liu, X., S. Schuck, and A. Stenlund. 2007. Adjacent residues in the E1initiator beta-hairpin define different roles of the beta-hairpin in Ori melting,helicase loading, and helicase activity. Mol. Cell 25:825–837.

14. Sanders, C. M., and A. Stenlund. 1998. Recruitment and loading of the E1initiator protein: an ATP-dependent process catalyzed by a transcriptionfactor. EMBO J. 17:7044–7055.

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FIG. 5. E1DBD interacts physically with the E1 oligomerizationand helicase domain. (A) GST pulldown experiments were carried outwith the E1DBD GST fusions 142-308 (lanes 4 to 6) and 159-303 (lanes7 to 9) and a GST fusion of the 60 N-terminal residues of E1 (lanes 2to 3). The bait was the E1 helicase domain fragment 308-605 labeledby phosphorylation at residue S584 with [�-32P]ATP. The pulldownexperiments in lanes 2, 4, and 7 were carried out in the absence ofnucleotide. The pulldown experiments in lanes 5 and 8 were carriedout in the presence of ADP, and the pulldown experiments in lanes 6and 9 were carried out in the presence of ATP. Lane 1 contains 1% ofthe input material used in the pulldown experiments. The upper bandin the input lane corresponds to autophosphorylated CK2�, while thelower band corresponds to E1 308-605. (B) Diagram illustrating themodel for nucleotide dependent DNA binding by E1. In this modelthe E1 DBD interacts with the E1 helicase domain in the absence ofnucleotide, and this interaction prevents DNA binding by the E1 DBD.In the presence of ATP, the nucleotide causes a conformationalchange in the E1 helicase domain, which results in the release of theE1 DBD, allowing the DBD to bind DNA.


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21. Sun, S., L. Thorner, M. Lentz, P. MacPherson, and M. Botchan. 1990.Identification of a 68-kilodalton nuclear ATP-binding phosphoprotein en-coded by bovine papillomavirus type 1. J. Virol. 64:5093–5105.

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lomavirus e1 helicase: fluorescence anisotropy studies and characterizationof a dimerization-defective mutant protein. J. Virol. 77:5178–5191.

23. Titolo, S., A. Pelletier, A. M. Pulichino, K. Brault, E. Wardrop, P. W. White,M. G. Cordingley, and J. Archambault. 2000. Identification of domains ofthe human papillomavirus type 11 E1 helicase involved in oligomerizationand binding to the viral origin. J. Virol. 74:7349–7361.

24. Titolo, S., A. Pelletier, F. Sauve, K. Brault, E. Wardrop, P. W. White, A.Amin, M. G. Cordingley, and J. Archambault. 1999. Role of the ATP-binding domain of the human papillomavirus type 11 E1 helicase in E2-dependent binding to the origin. J. Virol. 73:5282–5293.

25. Yang, L., R. Li, I. J. Mohr, R. Clark, and M. R. Botchan. 1991. Activation ofBPV-1 replication in vitro by the transcription factor E2. Nature 353:628–632.

26. Yang, L., I. Mohr, E. Fouts, D. A. Lim, M. Nohaile, and M. Botchan. 1993.The E1 protein of bovine papillomavirus 1 is an ATP-dependent DNAhelicase. Proc. Natl. Acad. Sci. U. S. A. 90:5086–5090.

27. Yang, L., I. Mohr, R. Li, T. Nottoli, S. Sun, and M. Botchan. 1991. Tran-scription factor E2 regulates BPV-1 DNA replication in vitro by directprotein-protein interaction. Cold Spring Harbor Symp. Quant. Biol. 56:335–346.


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mimic the nucleotide-bound state bovine papillomavirus e1 initiator

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