Interactions Required for Binding of Simian Virus 40 T Antigen to the Viral Origin and Molecular Modeling of Initial Assembly Events

The purified T-antigen origin binding domain binds site specificallyto site II, the central region of the simian virus 40 core origin.However, in the context of full-length T antigen, the originbinding domain interacts poorly with DNA molecules containingjust site II. Here we investigate the contributions of additionalcore origin regions, termed the flanking sequences, to originrecognition and the assembly of T-antigen hexamers and doublehexamers. Results from these studies indicate that in additionto site-specific binding of the T-antigen origin binding domainto site II, T-antigen assembly requires non-sequence-specificinteractions between a basic finger in the helicase domain andparticular flanking sequences. Related studies demonstrate thatthe assembly of individual hexamers is coupled to the distortionsin the proximal flanking sequence. In addition, the point inthe double-hexamer assembly process that is regulated by phosphorylationof threonine 124, the sole posttranslational modification requiredfor initiation of DNA replication, was further analyzed. Finally,T-antigen structural information is used to model various stagesof T-antigen assembly on the core origin and the regulationof this process.

INTRODUCTION

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Introduction

Recent studies have established many of the factors requiredfor initiation of DNA replication in eukaryotic organisms (reviewedin references 3 and 75). However, at the molecular level, thisprocess is still poorly understood. In higher eukaryotic organismsthis situation reflects, in part, the failure to identify sequencesthat define origins of replication (26). As a result, littleis known about the protein-DNA interactions that are essentialfor initiation of replication. A further limitation is thatstructural information is available for only a few of the proteinsinvolved in initiation of eukaryotic DNA replication.

Therefore, basic issues related to initiation of eukaryoticDNA replication have been addressed by using viral model systems.One of the more useful viral model systems for studying DNAreplication is simian virus 40 (SV40) (reviewed in references8, 25, and 61). The SV40 origin of replication is well defined(reviewed in references 5 and 8), as is the viral initiatorprotein, termed T antigen (T-ag), a 708-amino-acid phosphoproteincontaining several domains (8, 25, 61). Moreover, the interactionof T-ag with the core origin has been extensively studied (reviewedin references 8, 25, and 61). A further advantage afforded bythis system is that considerable information about the structureof T-ag has been obtained. For instance, the structure of theT-ag domain that site-specifically binds to the SV40 origin,the origin binding domain (T-ag-obd) (residues 131 to 260),was determined by nuclear magnetic resonance methods (41). Inaddition, crystallographic techniques were used to determinethe structures of the J domain-containing (11, 67) N terminusof T-ag (residues 7 to 117) (35) and the C-terminal helicasedomain (residues 251 to 627) (39). That this structural informationis of general interest to the replication field was demonstratedby recent studies, for example, those that established thatthe structure of the T-ag-obd is nearly identical to those ofthe papillomavirus E1 DNA binding domain (21), the DNA bindingdomain from adeno-associated virus (28), and the catalytic domainof Rep, the initiator protein of tomato yellow leaf curl virus(12). These observations have raised the possibility that viralinitiators interact with their origins via conserved mechanisms.Moreover, structural homology between viral and cellular J domainshave been reported (see references 11 and 35 and referencestherein). In addition, the recent determination of the structureof the C-terminal domain of T-ag has provided a wealth of informationregarding the organization and functioning of a eukaryotic helicase(39).

When T-ag monomers assemble on the core origin, they oligomerizeinto hexamers and double hexamers (15, 31, 44, 49); preformedhexamers may also bind to origin-containing DNA (72). When assembledinto a double hexamer, T-ag is able to function as a helicase(1, 14, 64, 68). Electron microscopy studies have provided additionalinformation regarding the structures of T-ag hexamers (43, 44,56, 74) and double hexamers (73). For example, they revealedthat T-ag hexamers are planar rings containing a central holethrough which DNA was proposed to pass (56, 73, 74), a conclusionsupported by more recent studies (39). They also establishedthe relative positions of T-ag domains within the hexamers;for instance, the N-terminal face of the hexamer contains theJ/T-ag-obds, and the C-terminal side contains domains requiredfor ATP hydrolysis and helicase activity (39, 73, 74). Lessis known about the structure of T-ag double hexamers; nevertheless,double hexamers assembled on the core origin have been imagedby negative-staining techniques (73). Among the findings ofthese studies is that the J/T-ag-obds are situated at the hexamer-hexamerinterface (73), a finding consistent with previous biochemicalstudies (76).

Regarding the protein-DNA interactions that take place duringassembly of T-ag hexamers on the core origin, the A1 and B2loops (41, 62, 83) within the T-ag-obd are required for site-specificbinding to individual GAGGC pentanucleotides within site II(34, 41, 70; reviewed in reference 8). Consistent with thesereports, the purified T-ag-obd can site-specifically bind tooligonucleotides containing just site II (36). In contrast,site II-based oligonucleotides are poor substrates for T-agassembly (36, 70). When assayed via electrophoretic mobilityshift assay (EMSA), T-ag assembly into hexamers requires siteII and the presence of either of the flanking sequences (36,66). Based on these studies, it was concluded that efficienthexamer formation on the core origin requires at least two T-ag-origincontacts: site-specific binding of the T-ag-obd to site II andan additional contact(s) between the flanking sequences andresidues in the helicase domain of T-ag (36, 51).

The protein-DNA interactions required for double-hexamer formationare complex, since certain of them appear to be regulated bycell cycle-dependent phosphorylation events (2, 47, 76). Forexample, T-ag's interactions with subfragments of the core originare regulated by phosphorylation of Thr124 (2), an essentialposttranslational modification required for initiation of DNAreplication (45, 47, 57). Moreover, peptides containing theCDK/NLS region of T-ag interact with DNA in a manner that isregulated by phosphorylation of Thr124 (37), which is additionalevidence that phosphorylation regulates certain T-ag interactionswith the origin.

To further characterize the protein-DNA interactions necessaryfor T-ag and T124A assembly on the core origin, we have analyzedthe amount of flanking sequence required for hexamer and double-hexamerformation. Molecular modeling techniques have also been usedto predict the region of the T-ag helicase domain that interactswith the flanking sequences during assembly on the viral origin.Moreover, to further explore the mechanism of DNA unwinding,we have examined whether the structural distortions in the coreorigin (7, 51) are coupled to double-hexamer formation or toearlier events during T-ag assembly. Results from these studiesare presented here.

MATERIALS AND METHODS

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Materials and Methods

Commercial supplies of enzymes, DNA, reagents, and oligonucleotides. T4 polynucleotide kinase was purchased from Gibco-BRL. PlasmidpBR322 DNA, used as competitor DNA, was purified by standardprocedures (54) and digested with HaeIII purchased from NewEngland Biolabs.

Oligonucleotides were synthesized on an Applied Biosystems 394DNA synthesizer, purified by electrophoresis through urea-10%polyacrylamide gels, and isolated as described previously (54,65). Double-stranded oligonucleotides, ³²P labeled at their5' termini, were prepared by standard procedures (54, 65).

Purification of wild-type T-ag and T-ag containing the T124A mutation. SV40 T-ag and the T124A mutant were expressed in insect (Sf9)cells by using baculovirus expression vectors. The wild-typevector was previously described (50), while the T124A expressionvector was developed by L. Chen, R. Upson, and D. Simmons (unpublisheddata). Proteins were purified by using immunoaffinity techniqueswith the PAb 419 monoclonal antibody (19, 60). Purified proteinswere stored in T-ag storage buffer (20 mM Tris-HCl [pH 8.0],50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol [DTT] [29], 0.1 mMphenylmethylsulfonyl fluoride, 0.2 µg of leupeptin perml, 0.2 µg of antipain per ml, 10% glycerol) (2, 79) andfrozen at -80°C until use.

EMSAs. T-ag-and T124A-based EMSAs were conducted under SV40 in vitroreplication conditions (79). The reaction mixtures (20 µl)contained 7 mM MgCl₂, 0.5 mM DTT, 4 mM adenylyl imidodiphosphate(AMP-PNP), 40 mM creatine phosphate (di-Tris salt [pH 7.6]),0.48 µg of creatine phosphate kinase, 5 µg of bovineserum albumin, 0.8 µg of HaeIII-digested pBR322 (6 pmol)(used as a nonspecific competitor), 25 fmol of double-strandedoligonucleotide ( $~$ 10⁶ cpm/pmol), and 0.5 µg of T-ag orT124A (6 pmol) as indicated. After a 20-min incubation at 37°C,glutaraldehyde was added (0.1% final concentration), and thereaction products were further incubated for 5 min. The reactionswere stopped by the addition of 5 µl of 6x loading dyeII (15% Ficoll, 0.25% bromophenol blue, and 0.25% xylene cyanol)(54) to the samples. The reaction products were applied to 4to 12% gradient polyacrylamide gels and electrophoresed in 0.5%Tris-borate-EDTA (pH 8.4) for $~$ 1.5 h (10 W). The gels were driedon Whatman 3MM paper and subjected to autoradiography.

KMnO₄ footprinting. The KMnO₄ footprinting technique (7), performed in 30-µlreaction mixtures, was conducted under replication conditions(79) as previously described (33). DNA substrates included thecore origin containing plasmid pSV01 ${Delta}$ EP and four derivativesof this plasmid in which the core origin was replaced with mutantorigins, each containing a single pentanucleotide. For any givenmutant origin, the other three pentanucleotides were replacedwith transition mutations. As in previous studies (33, 36),oligonucleotide 1 (5' TGAGCGGATACATATTTG 3'), 5' end labeledwith [ ${gamma}$ -³²P]ATP and T4 polynucleotide kinase (54), was used inthe primer extension reactions. Upon completion of the reactions,the samples were ethanol precipitated, washed with 80% ethanol,and electrophoresed for $~$ 2.5 h at 1,500 V and 40 mA on a 7% polyacrylamidegel containing 8 M urea. The locations of the modified residueswere determined by using a dideoxy sequencing ladder (55), witholigonucleotide 1 as the primer.

Molecular modeling of T-ag assembly on core origin subfragments. The molecular model of the T-ag monomer bound to DNA was generatedby using the computer program InsightII by MSI. Standard B-formDNA was assembled by using the sequence of the 64-bp SV40 coreorigin (see reference 16 and references therein). The T-ag-obd(41) (Protein Data Base [PDB] accession code 1TBD) was boundto pentanucleotide 1 by using the A1 and B2 loops (41, 83).Docking was based on recent studies indicating that B2 residueHis203 contacts the middle GC base pair in the GAGGC sequence(E. M. Bradshaw et al., submitted for publication) and an alignmentsuggested by the E1-obd-DNA costructure (22). The J domain (35)(PDB accession code 1GH6) was docked to the T-ag-obd by usingconstraints previously described by VanLoock et al. (74). TheT-ag helicase domain (39) (PDB accession code 1N25) was juxtaposednext to the C terminus of the T-ag-obd. The orientation of thehelicase domain was established (i) by aligning residues knownto be in the inner channel with flanking sequence DNA (39) and(ii) by using spatial constraints generated by previous electronmicroscopy studies of T-ag hexamers (74).

RESULTS

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Results

Analyses of the helicase domain-flanking sequence interactions required for hexamer formation. Efficient assembly of monomers of T-ag, or T124A, into hexamersrequires a pentanucleotide (p) and either of the adjacent flankingsequences in the origin (36, 66, 70): the early palindrome (EP)or the AT-rich region (AT). The experiments with the resultsshown in Fig. 1 and 2 were conducted to establish how much ofthe proximal flanking sequence (fs) is required for hexamerformation on any given pentanucleotide. (In the following sectionsthe abbreviation fs is used to refer to sequences derived fromthe EP, AT regions, and, in certain instances, site II.)

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FIG. 1. Experiments used to establish the sequences within the EP region required for hexamer formation on pentanucleotides 1 and 2. (A) Studies of the EP requirements for hexamer assembly on pentanucleotide 1. Panel 1, oligonucleotides used in these studies. In the DNA sequence, lowercase boldface letters are used to symbolize the transition mutations used to replace pentanucleotides 2, 3, and 4. The 48-bp p1 + EP oligonucleotide is the only core origin subfragment presented; however, the numbers with asterisks indicate the ends of additional oligonucleotides generated as a result of progressive truncations of the EP. The wedge symbolizes the progressive truncation of these sequences in this and subsequent figures. Panel 2, band shift reactions conducted with the indicated oligonucleotides. The reactions in lanes 2, 5, 8, 11, and 14 were conducted with T-ag (6 pmol); those in lanes 3, 6, 9, 12, and 15 were conducted with T124A (6 pmol); and those in lanes 1, 4, 7, 10, and 13 served as protein-free controls. The position of T-ag hexamers is indicated by H in this and subsequent figures. (B) Studies of the EP requirements for assembly on pentanucleotide 2. Panel 1, oligonucleotides used in this set of studies. Lowercase boldface letters indicate the transition mutations used to replace pentanucleotides 1, 3, and 4. The 48-bp p2 + EP oligonucleotide is the only core origin subfragment presented; however, the numbers with asterisks indicate the ends of additional oligonucleotides generated as a result of progressive truncations of the EP. Panel 2, band shift reactions conducted with the indicated oligonucleotides. The reactions in lanes 2, 5, 8, 11, and 14 were conducted with T-ag (6 pmol); those in lanes 3, 6, 9, 12, and 15 were conducted with T124A (6 pmol); and those in lanes 1, 4, 7, 10, and 13 served as protein-free controls.

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FIG. 2. Experiments used to establish the sequences within the AT-rich region required for hexamer formation on pentanucleotides 3 and 4. (A) Studies of the AT requirements for hexamer assembly on pentanucleotide 3. Panel 1, oligonucleotides used in these experiments. Lowercase boldface letters indicate the transition mutations used to replace pentanucleotides 1, 2, and 4. The 47-bp p 3 + AT oligonucleotide is the only core origin subfragment presented; however, the numbers with asterisks indicate the ends of additional oligonucleotides that were generated as a result of progressive truncations of the AT. Panel 2, band shift reactions conducted with the indicated oligonucleotides. The reactions in lanes 2, 5, 8, 11, and 14 were conducted with T-ag (6 pmol); those in lanes 3, 6, 9, 12, and 15 were conducted with T124A (6 pmol); and those in lanes 1, 4, 7, 10, and 13 served as protein-free controls. (B) Studies of the AT requirements for assembly on pentanucleotide 4. Panel 1, oligonucleotides used in these studies. Lowercase boldface letters indicate the transition mutations used to replace pentanucleotides 1, 2, and 3. While the 47-bp p4 + AT oligonucleotide is the only core origin subfragment presented, the numbers with asterisks indicate the ends of additional oligonucleotides generated as a result of progressive truncations of the AT. Panel 2, band shift reactions conducted with the indicated oligonucleotides. The reactions in lanes 2, 5, 8, 11, and 14 were conducted with T-ag (6 pmol); those in lanes 3, 6, 9, 12, and 15 were conducted with T124A (6 pmol); and those in lanes 1, 4, 7, 10, and 13 served as protein-free controls.

(i) Hexamer assembly on substrates containing truncations of the flanking sequences located 5' of the pentanucleotides. In an initial set of studies, the flanking sequence requirementsfor hexamer formation on pentanucleotide 1 were determined byusing a series of truncated forms of the 48-bp p1 + EP oligonucleotide(Fig. 1A, panel 1) and AMP-PNP (a nonhydrolyzable analog ofATP used in all of the experiments presented in Fig. 1 to 3).Inspection of Fig. 1A, panel 2 (lanes 1 to 3), confirms thatthe 48 bp p1 + EP oligonucleotide readily assembles T-ag andT124A hexamers (66). The experiments for lanes 4 to 15 wereconducted with oligonucleotides containing progressively largertruncations in the EP region. It is apparent from lanes 4 to6 that for either T-ag or T124A, 4 bp of the EP can be removedwithout a significant reduction in hexamer formation. However,inspection of lanes 7 to 15 reveals that DNA substrates smallerthan the 44-bp p1 + 17fs oligonucleotide supported T-ag or T124Aassembly at greatly reduced levels. Based on these studies,it is concluded that T-ag assembly on pentanucleotide 1 requiresthe GAGGC and sequences extending into the EP for an additional14 to 17 bp. In a second series of experiments, the flankingsequence requirements for hexamer formation on pentanucleotide2 were studied by using truncated derivatives of the 48-bp p2+ EP oligonucleotide (Fig. 1B, panel 1). Examination of Fig.1B, panel 2 (lanes 1 to 3), demonstrates that the 48-bp p2 +EP oligonucleotide is a substrate for hexamer formation. Theexperiments for lanes 4 to 15 were conducted with oligonucleotidescontaining progressively larger truncations in the EP region.Inspection of lanes 4 to 12 reveals that a considerable amountof the EP can be deleted without disrupting hexamer formationon pentanucleotide 2. In contrast, very little hexamer was formedon the 32-bp p2 + 11fs oligonucleotide. It is concluded thatT-ag assembly on pentanucleotide 2 requires the pentanucleotideand at least 12 to 15 bp of DNA proximal to this site.

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FIG. 3. Establishment of the extent to which sequences 3' to a given GAGGC pentanucleotide are important for hexamer formation. (A) Core origin-based oligonucleotides, containing pentanucleotide 1, the EP, and various amounts of site II, were used to establish the minimum flanking sequence, 3' of pentanucleotide 1, needed for hexamer formation. The 48-bp p1 + EP is the only oligonucleotide presented; however, the numbers with asterisks indicate the ends of additional oligonucleotides used in these studies. Lowercase boldface letters indicate the transition mutations used to replace pentanucleotides 2, 3, and 4. (B) A representative band shift experiment, conducted with the indicated oligonucleotides in the presence of AMP-PNP. The reactions in lanes 2, 5, 8, 11, and 14 were conducted with wild-type T-ag (6 pmol), while those in lanes 3, 6, 9, 12, and 15 were conducted with the T124A mutant protein (6 pmol). As a control, the reactions in lanes 1, 4, 7, 10, and 13 were performed without protein.

The contribution of the AT-rich region to hexamer formationon pentanucleotide 3 was examined in the next series of reactions.It is apparent from Fig. 2A, panel 2 (lanes 1 to 3), that hexamersreadily form on the single-pentanucleotide-containing 47-bpAT + p3 oligonucleotide (Fig. 2A, panel 1). To determine howmuch of the AT-rich region is required for assembly on pentanucleotide3, additional experiments were conducted with the truncatedderivatives of the 47-bp AT + p3 oligonucleotide (Fig. 2A, panel1). Inspection of Fig. 2A, panel 2, reveals that hexamers efficientlyform on truncated forms of the AT + p3 oligonucleotide (lanes4 to 12) (e.g., the 43-bp 22fs + p3, 39-bp 18fs + p3, and 35-bp14fs + p3 oligonucleotides). In contrast, assembly is greatlyreduced on the 31-bp 10fs + p3 oligonucleotide (lanes 13 to15). It is concluded that T-ag assembly on pentanucleotide 3requires the pentanucleotide and at least 11 to 14 bp of theAT region proximal to this site. Finally, the flanking sequencerequirements for hexamer formation on pentanucleotide 4 werestudied by using truncated derivatives of the 47-bp AT + p4oligonucleotides (Fig. 2B, panel 1). The data in Fig. 2B, panel2 (lanes 1 to 3), confirm previous experiments (2, 66) thatdemonstrated that T-ag and T124A hexamers readily form on the47-bp AT + p4 oligonucleotide. Further inspection of lanes 4to 6 reveals that the 43-bp 16fs + p4 oligonucleotide supportsT-ag and T124A hexamers. In contrast, hexamers assemble at reducedlevels on smaller subfragments of the origin (e.g., the 39-bp12 fs + p4 [lanes 7 to 9], 35-bp 8 fs + p4 [lanes 10 to 12],and 31-bp 4fs + p4 [lanes 13 to 15] oligonucleotides). Theseresults demonstrate that efficient T-ag assembly on pentanucleotide4 requires the GAGGC and at least 13 to 16 bp of the AT-richregion proximal to the pentanucleotide, a length requirementsimilar to that for hexamer formation at the other three pentanucleotides.

Finally, T-ag assembles in an ATP-dependent manner (15, 17).Therefore, to establish whether the interactions with the flankingsequences are altered by nucleotide hydrolysis, the experimentsin Fig. 1 and 2, which were conducted in the presence of AMP-PNP,were repeated in the presence of ADP, and very similar resultswere obtained. They were also repeated in the presence of ATPand in the absence of nucleotides (data not shown). However,as previously reported (2, 66), greatly reduced levels of hexamersare formed in the in the presence of ATP (a reflection of T-ag'shelicase activity [14, 27, 68]) or in the absence of nucleotide(a cofactor for hexamer formation [39]).

(ii) Sequences 3' to a given GAGGC pentanucleotide are not required for hexamer formation. When it is bound to the core origin, it is unlikely that T-agmakes extensive contact with origin sequences 3' to a givenpentanucleotide. This hypothesis is based, in part, on the observationthat double hexamers preferentially form on pentanucleotides1 and 3 (33, 73), two sites that are separated by only 7 bp.Therefore, the arrangement of pentanucleotide pairs (e.g., 1and 3) provides limited room for protein-DNA contacts 3' ofany given pentanucleotide.

To test this prediction, a series of oligonucleotides that hadprogressively larger truncations of site II DNA located 3' ofpentanucleotide 1 were synthesized (Fig. 3A). After radiolabeling(see Materials and Methods), the oligonucleotides were usedin an additional series of band shift experiments (Fig. 3B).Inspection of this Fig. 3B (lanes 1 to 9) reveals that hexamerformation on pentanucleotide 1 requires very little DNA 3' ofthe GAGGC sequence. Furthermore, the phosphorylation statusof Thr124 did not change the sequence requirements for T-agassembly. However, hexamers formed at reduced levels on a substrate(i.e., the 28-bp 3fs + p1 + EP oligonucleotide) containing threebase pairs 3' of pentanucleotide 1 (lanes 10 to 12). In addition,an oligonucleotide containing two residues 3' of pentanucleotide1 (i.e., the 27-bp 2fs + p1 + EP oligonucleotide) was a verypoor substrate for hexamer formation. Whether the 2- to 3-bp3' flanking region is required for protein-DNA interactionsor to prevent fraying the ends of the oligonucleotide has notbeen determined. Nevertheless, these experiments confirm thathexamer formation on a given pentanucleotide does not requireextensive protein-DNA contacts with sequences 3' of the GAGGCsequence.

(iii) Summary of sequence requirements for hexamer formation. To illustrate the common DNA requirements for hexamer formationon the core origin, the sequences that support hexamer assembly(Fig. 1 to 3) were aligned relative to the individual pentanucleotides(Fig. 4A). The boundary regions are shown; sequences betweena given pentanucleotide and those within its associated boundaryregion are necessary for hexamer formation. Relative to thepentanucleotides, the AT-rich boundary regions have been mappedto residues located 11 to 17 bp from the 5' ends of the GAGGCsequences. Nevertheless, it is noted that a possible commonfeature of the pentanucleotide-boundary region arrangement isa separation of 14 bp (corresponding to $~$ 47.6 Å). The relativelyexact arrangement of the pentanucleotides and their associatedboundary regions suggests that an equally distinct spatial arrangementexists in T-ag between the A1 and B2 loops in the T-ag-obd andthe DNA binding residues in the helicase domain (see below).In addition, the experiments conducted with the T124A mutantdemonstrated that the phosphorylation status of Thr124 doesnot alter the sequence requirements for hexamer formation, anobservation consistent with previous studies (2, 47).

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FIG. 4. Conserved sequence requirements for hexamer formation on individual pentanucleotides. (A) The four parental oligonucleotides (i.e., the largest origin subfragments used in the truncation studies) were aligned via their individual pentanucleotides. The boxes indicate the locations of the boundary regions. As noted in the text, a common feature of the boundary regions may be a distance of 14 bp from the end of a given pentanucleotide. Finally, the positions of origin residues 5216 and 5213 are indicated. As mentioned in Discussion, SenGupta and Borowiec (58) reported that ethylation of these residues inhibited T-ag binding. (B) Positions of pentanucleotides 1, 2, 3, and 4 relative to their associated boundary regions (underlined), projected onto B-form DNA. In these models, the pentanucleotides are projecting out of the page; the locations of the major and minor grooves, relative to the boundary regions, are indicated.

To gain further insights into the spatial relationship betweenthe pentanucleotides and boundary regions, their relative positionswere mapped on standard B-form DNA (Fig. 4B). To construct thesemodels, the individual pentanucleotides were oriented such thatthey face out of the plane of the paper. Inspection of Fig.4B, panel 1, reveals that in this orientation, the boundaryregion associated with pentanucleotide 1 is within a major groovethat also faces out of the plane of the paper. Likewise, theboundary region associated with pentanucleotide 4 is withina major groove that is coplanar with the pentanucleotide (Fig.4B, panel 2). In contrast, inspection of Fig. 4B, panels 3 and4, reveals that the boundary regions associated with pentanucleotides2 and 3 have a different topology; they are situated withinboth major and minor grooves. This arrangement is of interestgiven that it was previously demonstrated that hexamers preferentiallyform on pentanucleotides 1 and 4, while levels of hexamer formationon pentanucleotides 2 and 3 were relatively low (33). Theseobservations suggest that hexamers preferentially form on pentanucleotidesassociated with boundary regions situated primarily in the majorgroove.

(iv) Modeling the binding of a T-ag monomer to the SV40 origin of replication. Given recent advances in determining the structure of T-ag (seee.g., references 35, 39, 41, 73, and 74), the formation of aT-ag monomer on pentanucleotide 1 was modeled (Fig. 5). Stepstaken to assemble a T-ag monomer on core origin DNA, using theJ, T-ag-obd, and helicase domains, are described in Materialsand Methods. The length of a single hexamer has been reportedto be $~$ 100 (74) to 110 (73) Å ( $~$ 29 to 32 bp). In reasonableagreement, the monomer depicted in Fig. 5 covers $~$ 115 Å( $~$ 34 bp). However, Li et al. solved the structure of a fragmentof the helicase domain extending between residues 251 and 627(39). Given that T-ag contains 708 residues, the model is lacking81 residues from the C terminus. Furthermore, based on a numberof observations, including those presented in Fig. 3, the N-terminalJ domain was positioned such that it does not make significantcontacts with core origin sequences 3' of a given pentanucleotide(a feature of the model that is not readily apparent from Fig.5). The model also indicates that the interaction of the T-ag-obdwith the 5' end of pentanucleotide 1 terminates at the junctionbetween site II and the EP, a characteristic of the model thatis supported by previous phenanthroline-copper footprintingof T-ag-obd-origin complexes (33).

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FIG. 5. A model of a T-ag monomer docked to the 48-bp p1 + EP oligonucleotide. The positions of two SV40 core origin domains, site II and the EP, are indicated. Pentanucleotides 1 and 3 are shown in green, while pentanucleotides 2 and 4 are shown in yellow. The boundary region associated with pentanucleotide 1 is shown in black. Also depicted is the region of the EP that is melted upon T-ag binding (Fig. 9) (reviewed in reference 5). The N terminus of T-ag (residues 7 to 117) is represented in pink. The T-ag-obd (TBD) (residues 139 to 247) is in purple. The A1 and B2 binding loops, shown in light blue (reviewed in reference 8), were docked to pentanucleotide 1. The C-terminal domain (residues 266 to 627) is in dark blue. The basic finger (residues 503 to 523), depicted interacting with the boundary region associated with pentanucleotide 1, is in red. Finally, since the structures of the J domain, T-ag-obd, and C-terminal domain were solved separately, there is some uncertainty regarding the relative orientations of the individual domains. Moreover, there is no evidence that the J-domain-containing N terminus is actually in contact with origin DNA (see, e.g., Fig. 3); therefore, this model should not be interpreted as suggesting DNA binding.

The location of the boundary region associated with pentanucleotide1 is based on the studies presented in Fig. 1A. Regarding thelocation of the residues within the helicase domain that contactthe boundary region, it is noted that a finger, extending betweenresidues 503 and 523, is situated opposite the boundary region.Residues at the apex of the loop are highly basic (i.e., ₅₁₁KKHLNKR₅₁₇),a useful feature for DNA binding; a similar cluster of basicresidues is not present in any other region of the helicasedomain. Furthermore, rotation of the inner channel of the helicasedomain around duplex DNA, using the program Insight II, failedto suggest additional DNA binding motifs (data not shown). Finally,the distance between Arg154 in the A1 loop and the apex of thebasic finger (Lys512) is $~$ 51 Å, a distance that is similarto the 47.6-Å separation between the 5' ends of the pentanucleotidesand the nucleotides located 14 bp away within the boundary regions.

Studies of the flanking sequence requirements for double-hexamer formation on the core origin. Having analyzed the flanking sequence requirements for hexamerformation on individual pentanucleotides (e.g., pentanucleotide1), we performed a similar analysis of the flanking sequencerequirements for double-hexamer formation. Reasons for conductingthese experiments included a desire to establish whether theinitially bound hexamer, presumably bound to the pentanucleotidesproximal to the flanking sequences (e.g., 1 and 4) (66), modulatedthe flanking sequence requirements for the assembly of the secondhexamer (e.g., 3 and 2). In addition, these studies were performedboth with wild-type T-ag, phosphorylated at Thr124 (30), andwith the T124A mutant (45, 47, 57). The use of both proteinswas designed to provide further insights into how phosphorylationof Thr124 regulates T-ag-DNA interactions and the assembly ofdouble hexamers.

(i) Establishment of the length of the AT-rich region required for assembly of the second hexamer. An initial series of experiments was conducted with the 64-bpcore origin oligonucleotide and derivatives containing truncationsin the AT-rich region (Fig. 6A). The positions of the boundaryregions required for hexamer formation on pentanucleotides 3and 4 (Fig. 2) are indicated. As a positive control, T-ag andT124A double hexamers were formed on oligonucleotides containingthe 64-bp core origin (Fig. 6B, lanes 1 to 3). Double hexamersalso formed on origin subfragments containing limited truncationsin the AT-rich region (lanes 4 to 9) (i.e., the 60-bp 16fs +site II + EP and 56-bp 12 fs + site II + EP oligonucleotides).Since the 12 fs + site II + EP oligonucleotide lacks the pentanucleotide4 boundary region, it is concluded that this boundary regionis not needed for assembly of the second hexamer (compare lanes4 to 6 and 7 to 9), a finding consistent with preferential assemblyof T-ag double hexamers on pentanucleotides 1 and 3 (33, 66).On origin subfragments containing progressively larger truncations(e.g., the 52-bp 8fs + site II + EP and 48-bp 4fs + site II+ EP oligonucleotides), T-ag continued to form double hexamers(lanes 11 and 14). In contrast, on the same substrates, T124Amolecules preferentially formed hexamers (lanes 12 and 15).Thus, the second T-ag hexamer to assemble is less sensitiveto truncations in the AT-rich region than the second T124A hexamer(compare lanes 11 and 14 with lanes 12 and 15). Moreover, theseexperiments indicate that the boundary region associated withpentanucleotide 3 is required for assembly of the second T124Ahexamer but not for assembly of the second T-ag hexamer. Finally,it was previously demonstrated that double hexamers readilyform on the 64-bp AT + p(1,3) + EP oligonucleotide (33). Theexperiments in Fig. 6B were repeated with similar truncatedforms of the 64-bp AT + p(1,3) + EP oligonucleotide, and nearlyidentical results were obtained (data not shown).

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FIG. 6. Determination of how much of the AT-rich region is required for efficient double-hexamer formation on derivatives of the 64-bp core origin. (A) DNA substrates used in this set of experiments. The 64-bp core origin oligonucleotide is the only molecule depicted; however, the numbers with asterisks indicate the ends of additional oligonucleotides generated as a result of progressive truncations of the AT. The boundary regions associated with pentanucleotide 3 (solid rectangle) and pentanucleotide 4 (dotted rectangle) are indicated. (B) Band shift reactions conducted with the indicated oligonucleotides in the presence of AMP-PNP. The reactions in lanes 2, 5, 8, 11, and 14 were conducted with T-ag (6 pmol); those in lanes 3, 6, 9, 12, and 15 were conducted with T124A (6 pmol); and those in lanes 1, 4, 7, 10, and 13 served as protein-free controls. The positions of T-ag hexamers (H) and double hexamers (DH) are indicated.

(ii) Establishment of the length of the EP region required for assembly of the second hexamer. Additional studies were conducted with oligonucleotides containingthe 64-bp core origin and smaller derivatives possessing truncationsin the EP region (Fig. 7A); the boundary regions required forhexamer formation on pentanucleotides 1 and 2 are depicted.As previously demonstrated (see, e.g., Fig. 6B), T-ag and T124Adouble hexamers readily form on oligonucleotides containingthe 64-bp core origin (Fig. 7B, lanes 1 to 3). Similar levelsof double hexamers form on the 60-bp AT + site II + 17 fs oligonucleotide,although a slight increase in T124A hexamer assembly was observed(compare lanes 5 and 6). T-ag continues to form relatively highlevels of double hexamers on oligonucleotides that do not containthe pentanucleotide 1 boundary region (e.g., the 56-bp AT +site II + 13 fs and 52-bp AT + site II + 9 fs oligonucleotides[lanes 7 to 12]). In contrast, these same oligonucleotides areprogressively poorer substrates for T124A double-hexamer assembly(compare lanes 8 and 11 with lanes 9 and 12). Indeed, the 47-bpAT + site II + 4fs (also termed the AT + site II) oligonucleotide,lacking both boundary regions in the EP, supported reduced levelsof T-ag double hexamers (lane 14) and essentially no T124A doublehexamers (lane 15). Based on the data in Fig. 6 and 7, it isconcluded that the second T124A hexamer will form at normallevels only in the presence of a complete second assembly unit,i.e., the pentanucleotide and its associated boundary region.In contrast, the assembly of the second T-ag hexamer, phosphorylatedon Thr124 (30), does not have an absolute requirement for theboundary region associated with the second pentanucleotide.

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FIG. 7. Determination of how much of the EP-rich region is required for efficient double-hexamer formation on derivatives of the 64-bp core origin. (A) DNA substrates used in this set of experiments. The 64-bp core origin oligonucleotide is the only molecule presented; however, the numbers with asterisks indicate the ends of additional oligonucleotides generated as a result of progressive truncations of the EP. The boundary regions associated with pentanucleotide 1 (solid rectangle) and pentanucleotide 2 (dotted rectangle) are indicated. (B) Band shift reactions conducted with the indicated oligonucleotides in the presence of AMP-PNP. The reactions in lanes 2, 5, 8, 11, and 14 were conducted in the presence of T-ag (6 pmol), and those in lanes 3, 6, 9, 12, and 15 were conducted with T124A (6 pmol). The reactions in lanes 1, 4, 7, 10, and 13 served as protein-free controls. The positions of T-ag hexamers (H) and double hexamers (DH) are indicated.

(iii) Modeling of the protein-DNA interactions required for formation of T-ag and T124A dimers. The results from the double-hexamer assembly experiments presentedin Fig. 6 and 7 are summarized in Fig. 8. Regarding the assemblyof the two T-ag monomers that initiate double-hexamer formation(Fig. 8A), the T-ag-obd is depicted as a dimer that protectspentanucleotides 1 to 3, a conclusion supported by phenanthroline-copperfootprinting studies (33, 34). Furthermore, based on the experimentspresented here (Fig. 6B), and earlier studies of wild-type T-ag(2, 66), the model indicates that formation of the initial hexamerrequires flanking sequence contacts with the EP, while the secondhexamer assembles in a manner that is less dependent on theseinteractions. A summary of the data presented in Fig. 7B woulddepict the C terminus of the initial hexamer contacting theAT-rich region, while the second hexamer assembles in a mannerthat is relatively independent of its contacts with the EP region.These contrasting observations suggest that the interactionsbetween the C termini and the flanking sequences are dynamic.

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FIG. 8. Contrasting of the protein-DNA interactions required for T-ag and T124A double-hexamer formation. (A) A model of the interactions that take place during the docking of two T-ag monomers, phosphorylated on Thr124, to pentanucleotides 1 and 3 (66, 73). The A1 and B2 loops within the two origin binding domains (TBD) are bound to the pentanucleotides (double triangles). The helicase domain associated with T-ag bound to pentanucleotide 1 is shown making contact with its boundary region via its basic finger (rectangle-triangle interaction). In contrast, the second helicase domain, associated with T-ag assembled on pentanucleotide 3, has a lower affinity for its associated boundary region. This may stem from the orientation of the pentanucleotide 3 boundary region in a manner that prevents interactions with the major groove (Fig. 4B). Protein-protein interactions between the origin binding domains are symbolized by the solid triangles. (B) A model of the protein-DNA interactions that take place when two monomers of the T124A mutant dock to pentanucleotides 1 and 3 (66, 73). In contrast to T-ag, two basic finger-boundary region interactions are necessary for assembly (the symbols for the protein-DNA interactions are the same as in panel A). Furthermore, relative to T-ag, it is proposed that fewer protein-protein interactions take place between the two T-ag-obds and J domains, a proposal in keeping with previous studies (47, 76). Finally, T-ag double hexamers were reported to span $~$ 230 Å (73), corresponding to $~$ 68 bp of B DNA. Consistent with this estimate, previous DNase I footprinting studies indicated that double hexamers protect a region slightly larger than the 64-bp core origin (6). Moreover, these estimates are in reasonable agreement with predictions of the size of the double hexamer ( $~$ 230 Å [ $~$ 68 bp]) based on doubling the dimensions of the hexamer (Fig. 5) ( $~$ 115 Å [ $~$ 34 bp]).

The T124A mutant is a model for T-ag prior to cell cycle dependentphosphorylation (2, 23, 45-47, 57, 76). The present studiesdemonstrate that the flanking sequence requirements for theT124A mutant are essentially those needed for formation of twoindependent hexamers (Fig. 8B). Therefore, assembly of bothT124A hexamers requires all of the previously described T-ag-origininteractions. This observation is consistent with previous studiesindicating that when two T124A hexamers assemble on the coreorigin, they act as independent species (2, 47) and make weakor inappropriate protein-protein interactions (47, 76).

Flanking sequence distortions can be detected on substrates containing single pentanucleotides. The interaction between the boundary regions and the basic fingeris of interest from the point of view of the mechanism of DNAunwinding. It has been suggested that the structural distortionsin the core origin (7, 51) are generated by the opposing movementof the two hexamers within a given double hexamer (24, 39).However, if at least two DNA binding sites exist within a givenhexamer (the A1 and B2 loops and the basic finger within thehelicase domain), then opposing movement of T-ag domains withina single hexamer could contribute torque necessary for the originstructural distortions. Therefore, to examine the stage duringT-ag assembly at which the structural distortions take place,we tested whether hexamers assembled on single pentanucleotides(33, 66) can distort the proximal flanking sequence.

Standard KMnO₄ assays were used for these experiments, usingplasmids containing mutant core origins that have a single pentanucleotide(see Materials and Methods). After the addition of T-ag, KMnO₄assays were conducted as previously described (7). Results froma representative study, performed in the presence of AMP-PNP,are presented in Fig. 9. As a positive control, a reaction wasconducted with a plasmid containing the intact core origin (lane2); the previously described alterations in the AT and EP regions(7, 51) are indicated. As additional controls, the reactionsin the odd-numbered lanes were conducted with the indicatedplasmids in the absence of T-ag. Reactions performed with T-agand plasmids containing either pentanucleotide 4, 3, 2 and 1are displayed in lanes 4, 6, 8, and 10, respectively. It isapparent that T-ag hexamers formed on individual pentanucleotidesare able to distort the proximal flanking sequence at or nearwild-type levels.

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FIG. 9. pSV01 ${Delta}$ EP mutants, containing single pentanucleotides, are able to catalyze T-ag-dependent structural changes in the core origin. T-ag (1 µg) was incubated under replication conditions with pSV01 ${Delta}$ EP(core) (lane 2), pSV01 ${Delta}$ EP(p 4) (lane 4), pSV01 ${Delta}$ EP(p 3) (lane 6), pSV01 ${Delta}$ EP(p 2) (lane 8), pSV01 ${Delta}$ EP(p 1) (lane 10), and vector alone (control). Reactions in the odd-numbered lanes were conducted with the indicated plasmids in the absence of T-ag. After treatment with KMnO₄, the sites of oxidation were probed by primer extension reactions with a ³²P-labeled oligonucleotide (see Materials and Methods). The primer extension products were analyzed by electrophoresis on a 7% polyacrylamide gel containing 8 M urea. The locations of the EP, site II, and AT-rich regions are shown on the right. It is noted that the control reactions conducted with the pSV01 ${Delta}$ EP control mutant, containing transition mutations in all four pentanucleotides (lanes 12 and 13), lack significant structural distortions in the AT-rich and EP regions. However, a T-ag-independent distortion was detected over the mutant pentanucleotides. The basis for the structural distortion over this region is not currently understood. Finally, we are also uncertain about the cause(s) of the relatively low levels of distortions detected in the distal flanking sequences (lanes 4, 6, and 8) or the more substantial distortions in the distal flanking sequences associated with the pentanucleotide 1-containing substrate (lane 10).

DISCUSSION

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Discussion

While scanning duplex DNA for the SV40 core origin, T-ag interactswith the surrounding sequences via nonspecific contacts, a processthat requires the A1 and B2 loops in the T-ag-obd (41, 83) anda region previously mapped between residues 269 and 522 (40).When the origin is encountered, T-ag undergoes a transitionbetween nonspecific and site-specific binding. The protein-DNAinteractions required for site-specific binding to the origininclude contacts between the A1 and B2 loops and the GAGGC pentanucleotides(reviewed in reference 8) and additional interactions betweenresidues in the helicase domain and the flanking sequences (36,52, 58, 66, 70). In addition to nonspecific DNA binding andorigin recognition, the C terminus is also involved in subsequenthelicase activity and single-stranded DNA binding (81, 82).In view of the importance of the C terminus-DNA interactionsfor viral replication, we elected to further characterize thesequence requirements for binding of the helicase domain tothe SV40 origin. An additional reason for undertaking theseexperiments is the possibility that they might further our understandingof related interactions between the helicase domain and DNA.

Regarding the flanking sequence requirements for assembly ofT-ag on the viral origin, EMSA experiments were used to identifythe boundary regions necessary for efficient hexamer assembly.Subsequent molecular modeling studies indicate that a loop inT-ag centered on the highly basic (₅₁₁KKHLNKR₅₁₇) motif is situatedopposite the boundary regions. That this motif might play animportant role in DNA replication is supported by previous mutagenesisstudies (53), for instance, those that characterized the Lys516 $->$ Argmutation (42). While this conservative T-ag mutant bound tothe core origin, it was defective in replication. Moreover,T-ag molecules containing a deletion of residues 507 to 510did not support DNA replication in vivo (71). In addition, Pro522 is situated at the distal end of the loop; the C11A T-agmutation, Pro522 $->$ Ser, was markedly reduced in its ability tobind single-stranded DNA and partial duplex helicase substrates(48). Also of interest is that this basic finger is in closeproximity with the P-loop, the ATP binding site (39). Thus,the conformation of the basic finger could be regulated by ATPhydrolysis. A similar juxtaposition of the ATP and DNA bindingmotifs has been found in prokaryotic helicases (see e.g., references18, 38, and 63). These and related observations (see, e.g.,the data summarized in Fig. 5) support the hypothesis that thebasic finger in T-ag is critical for binding to the boundaryregion and for DNA replication. The relatively small size ofthe basic finger, and thus its limited contact with the flankingsequences, may help to explain why footprints were not detectedin previous in situ footprinting assays using phenanthroline-copperion (33). Finally, sequence alignment using the program DIALIGN2.2 revealed that the residues centered on the ₅₁₁KKHLNKR₅₁₇motif are conserved in many other initiators from the Papovaviridaeviral family (e.g., polyomavirus large T-ag and human Bk andJC [data not shown]). Furthermore, a nonspecific DNA bindingactivity is present in the helicase domain of papillomavirusE1 (references 69 and 78 and references therein). Therefore,a number of viral initiators may rely on basic fingers, or similarmotifs, for origin recognition.

It is of interest to consider possible features of the boundaryregion-basic finger interactions. It was previously establishedthat nonspecific contacts with the sugar-phosphate backboneare important for the T-ag-flanking sequence association (2,58, 59, 66). Of considerable additional interest is that thefour boundary regions overlap four patches of phosphate residuespreviously shown to be required for T-ag binding (two in eachflanking sequence, with each pair arranged on opposite strands)(58). Related experiments demonstrated, however, that the levelsof hexamer and double hexamer are significantly decreased onderivatives of the core origin containing transition mutationsin the flanking sequences (references 2 and 66 and data notshown). Therefore, in addition to contacts with the sugar-phosphatebackbone, T-ag assembly depends upon other properties of theflanking sequences. One likely property is suggested by theobservation that diethyl sulfate, an agent that ethylates guaninesat the N-7 (major groove), interfered with T-ag's interactionswith the EP (58). The two main sites of ethylation were at guaninessituated at positions 5213 and 5216; similar conclusions werederived from methylation protection studies (7, 51). As shownin Fig. 4A (top model depicting the 48-bp p1 + EP oligonucleotide),guanine 5216 lies within the boundary region associated withpentanucleotide 1, while guanine 5213 lies just outside thisregion. These observations provide additional evidence thatmajor groove contacts with the boundary regions play an importantrole in T-ag assembly. In addition, the finding that the flankingsequences in the SV40 core origin undergo conformational changesupon T-ag binding (reviewed in references 5, 9, 25, and 61)suggests an additional property. Indeed, the site in the EPthat is melted by T-ag (7, 51) overlaps the boundary regionassociated with pentanucleotide 1 (Fig. 5), and it was previouslyproposed that the primary function of the AT-rich and EP regionsis not to promote binding but to undergo structural changesrequired for the initiation of DNA replication (4). As previouslynoted, the purified T-ag-obd site specifically binds to thecore origin (34, 36) but does not catalyze structural distortionsin the flanking sequences (36), further evidence that the structuraldistortions in the flanking sequence require interactions withthe helicase domain. In light of these observations, it is proposedthat basic finger-boundary region interactions play a criticalrole in the induction of the conformational changes in the flankingsequences. Consistent with this proposal, studies with manyother DNA binding proteins have demonstrated that variationsin their target flanking sequences influence protein-DNA interactionsby affecting DNA conformation rather than by altering particularprotein-base or protein-phosphate contacts (reviewed in reference32).

T-ag assembly and the induction of the structural distortionsin the flanking sequences are coupled processes. However, theexact stage in the assembly process at which the distortionstake place is not known. Our experiments demonstrate that theflanking sequence distortions can be detected on plasmid substratesthat support only hexamer formation. Therefore, the structuraldistortions in the flanking sequences are early events thatdo not require double-hexamer formation. Consistent with thisproposal, Parsons et al. reported that T-ag assembled on theearly (e.g., pentanucleotides 1, 2 + EP) and late (e.g., pentanucleotides3, 4 and AT) halves of the core origin was able to catalyzethe distortions of the proximal flanking sequences (52). Whetherthe conformational changes in the flanking sequences occur asa result of monomer binding, as a result of hexamer assembly,or at some intermediate stage is presently not known. Nevertheless,at some stage in the assembly process, the basic finger-boundaryregion recognition interface (32) is likely to play a role inthe distortions in the flanking sequences. Furthermore, recognitionof the structurally distortable sequences is probably coupledto the recognition of the GAGGC sequences by the A1 and B2 loopsin the T-ag-obd.

Relative to hexamer formation, the sequence requirements fordouble-hexamer assembly are complex. When phosphorylated onThr124 (30), the second hexamer oligomerizes in a manner thatis relatively independent of the presence of the second flankingsequence (Fig. 6 and 7) (2, 66); thus, it has reduced affinityfor one of its associated flanking sequences (modeled in Fig.8A). It is proposed that much of the binding energy necessaryfor formation of the second hexamer is derived from protein-proteininteractions with the initially formed hexamer, a hypothesissupported by previous studies (47, 76). In contrast, our studieswith the T124A mutant indicate that prior to phosphorylationof Thr124, the interactions between the helicase domains andthe EP and AT-rich regions are maintained in each of the hexamers,a conclusion supported by previous experiments (2, 47). It followsthat at non-S phases of the cell cycle, two hexamers may assembleon the core origin in a manner that preserves their independentstatus. However, upon phosphorylation of Thr124, possibly bya cyclin-dependent kinase (25), one of the hexamers releasesits nonspecific interactions with the flanking sequence andinitiates critical protein-protein interactions with the secondhexamer. Finally, since double hexamers prefer to assemble onpentanucleotides 1 and 3 (33, 66), it is of interest that thepentanucleotide 1 boundary region is associated with a majorgroove while the pentanucleotide 3 boundary region is associatedwith both major and minor grooves (Fig. 4B). Therefore, relativelylow affinity of the basic finger for the pentanucleotide 3 boundaryregion may help to explain the results summarized in Fig. 8A.

How the double hexamer assembled on the core origin is ableto catalyze the replication protein A- and topoisomerase I-dependentformation of the unwound species termed form U (10, 14, 20,80) has yet to be determined. It was previously reported thatwhile double-hexamer formation requires only two pentanucleotides(e.g., pentanucleotides 1 and 3), DNA unwinding requires allfour (13, 33, 66). Therefore, at some stage in the assemblyprocess, the double hexamer must engage the second pair of pentanucleotides(e.g., pentanucleotides 2 and 4) and their associated flankingsequences. One possibility is that initiation of DNA unwindingrequires oscillating contacts between pairs of pentanucleotidesand flanking sequences. It is also possible that retrogrademovement of domains within a single hexamer (this study) orbetween double hexamers (24, 39) generates torque necessaryfor propagation of the initial structural distortions. Uponcompletion of assembly and ATP hydrolysis, basic fingers notbound to DNA (e.g., those in the second hexamer) could engageDNA in a non-sequence-specific manner. Indeed, given the orientationof T-ag double hexamers on the core origin (Fig. 8) (73) andevidence that the DNA is actively spooled through the double-hexamerhelicase (see, e.g., references 1, 64, and 77), it is possiblethat residues in the basic finger play a role in moving DNAtowards the center of the complex. This component of the T-aghelicase cycle would depend upon non-sequence-specific interactionswith DNA, a known feature of residues located in the helicasedomain (40).

ACKNOWLEDGMENTS

We thank David Cullinan, Gavin Schnitzler, Julie Kerner, AnuradhaKumar, and Margaret VanLoock for useful discussions and A. J.Bullock for comments on the manuscript.

This study was funded by a grant from the National Institutesof Health (9R01GM55397).

FOOTNOTES

* Corresponding author: Mailing address: Department of Biochemistry A703, Tufts University School of Medicine, 136 Harrison Ave., Boston, MA 02111. Phone: (617) 636-0447. Fax: (617) 636-2409. E-mail: Peter.BULLOCK{at}TUFTS.EDU.

REFERENCES

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