Sequences flanking the pentanucleotide T-antigen binding sites in the polyomavirus core origin help determine selectivity of DNA replication.

0022-538X/95/$04.0010

Copyrightq1995, American Society for Microbiology

Sequences Flanking the Pentanucleotide T-Antigen Binding Sites

in the Polyomavirus Core Origin Help Determine

Selectivity of DNA Replication

L. LI,

_{B. L. LI,}

_{M. HOCK,}

_{E. WANG,}

AND

W. R. FOLK

*

Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211,

_and

Department of Biological Sciences, Columbia University, New York, New York 10027

Received 12 June 1995/Accepted 16 August 1995

Replication of the genomes of the polyomaviruses requires two virus-specified elements, the

cis

-acting origin

of DNA replication, with its auxiliary DNA elements, and the

trans

-acting viral large tumor antigen (T antigen).

Appropriate interactions between them initiate the assembly of a replication complex which, together with

cellular proteins, is responsible for primer synthesis and DNA chain elongation. The organization of

cis

-acting

elements within the origins of the polyomaviruses which replicate in mammalian cells is conserved; however,

these origins are sufficiently distinct that the T antigen of one virus may function inefficiently or not at all to

initiate replication at the origin of another virus. We have studied the basis for such replication selectivity

between the murine polyomavirus T antigen and the primate lymphotropic polyomavirus origin. The murine

polyomavirus T antigen is capable of carrying out the early steps of the assembly of an initiation complex at

the lymphotropic papovavirus origin, including binding to and deformation of origin sequences in vitro.

However, the T antigen inefficiently unwinds the origin, and unwinding is influenced by sequences flanking the

T antigen pentanucleotide binding sites on the late side of the viral core origin. These same sequences

contribute to the replication selectivity observed in vivo and in vitro, suggesting that the inefficient unwinding

is the cause of the replication defect. These observations suggest a mechanism by which origins of DNA

replication can evolve replication selectivity and by which the function of diverse cellular origins might be

temporally activated during the S phase of the eukaryotic cell cycle.

The process by which DNA replication is initiated at the

simian virus 40 (SV40) origin has been elaborated in

consid-erable detail (28, 29, 41, 58, 101, 102). The viral T antigen binds

to the origin in a sequence-specific and ATP-dependent

man-ner to form a double hexamer (3, 5, 16, 17, 19, 22, 23, 68, 80).

It deforms the origin and then, by acting in concert with a

single-stranded-DNA-binding protein (SSB or RPA) and

topo-isomerase I, induces extensive unwinding of the DNA duplex

(4, 8, 18, 34, 59, 79, 88, 99, 121). Concomitantly, DNA

poly-merase

a

/primase interacts with this complex to initiate

RNA-primed DNA synthesis (13, 27, 39, 76). The nascent initiator

DNA is elongated by the combined action of DNA polymerase

a

/primase and a DNA polymerase

d

(or

ε

) complex (61, 77,

102, 111, 112, 115, 118). Leading-strand synthesis is

subse-quently catalyzed by the DNA polymerase

d

(or

ε

) complex,

while lagging-strand synthesis requires repetitive priming by

the DNA polymerase

a

/primase and elongation by the DNA

polymerase

d

(or

ε

) complex (115, 118).

The series of events required for the assembly of the

initia-tion complex is coordinated by highly specific protein-protein

and protein-DNA interactions. Productive interactions

be-tween the T antigen, single-stranded-DNA-binding protein,

and DNA polymerase

a

/primase are required for primosome

assembly and primer synthesis (13, 35, 36, 59, 72, 93). Specific

interactions between T antigen and DNA polymerase

a

/pri-mase (14, 35, 37, 49, 97) have been suggested to be a basis for

species-specific selectivity of viral DNA replication (9, 40, 74,

75). The highly specific protein-DNA interactions required for

the assembly of an initiation complex occur through the

rec-ognition of the origin by T antigen (3–5, 20, 23, 26, 28, 33, 62,

79, 96, 103). The four pentanucleotide motifs (PuPuGGC) in

the central palindrome of the core origin help organize the T

antigen double hexamers (3, 16, 17, 23, 68, 79, 80). The spatial

organization of these four pentanucleotide motifs is critical,

presumably because the T antigen hexamers must be exactly

positioned relative to each other to unwind the origin and to

accommodate SSB and DNA polymerase

a

/primase (2, 9, 12,

17, 23, 81, 114, 119).

Most polyomavirus origins consist of a core with four

pen-tanucleotide motifs, flanked by a DNA-unwinding element and

an A/T-rich sequence (21, 62, 63, 103). Despite these

con-served features, the T antigen of one virus may not efficiently

replicate another’s genome. Species-specific interactions

be-tween T antigens and cellular proteins such as DNA

poly-merase

a

/primase and differences in the spatial organization of

the four pentanucleotide motifs provide several possible

rea-sons why the origins of the murine polyomavirus (mPy) and

SV40 are not replicated by the other’s T antigens. However,

even among viruses which infect the same species and which

have nearly identical central palindromes, as occurs with the

SV40, BK, and JC viruses, there are substantial differences in

the capacity of one’s T antigen to replicate the other’s origin

(1, 65–67, 75, 98). This provides an opportunity to explore the

selectivity of replication complexes and to assess how diversity

among cellular origins might be used to modulate DNA

initi-ation frequency.

To this end, we have studied the interactions of the T

anti-gen of mPy with its own origin and with the origin of the

primate lymphotropic polyomavirus (LPV). Although the mPy

and LPV origins are very similar and have virtually identical

central palindromes, we find that the mPy T antigen does not

help assemble a functional replication complex at the LPV

origin, even with cellular proteins which productively interact

* Corresponding author. Phone: (314) 4845. Fax: (314) 882-5635. Electronic mail address: [email protected].

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with the mPy T antigen. Our data reveal that the capacity of

mPy T antigen to replicate DNA is strongly influenced by

sequences on the late side of the core origin, which differ

between the mPy and the LPV origins. These sequences help

activate the Tag DNA-unwinding activity. These findings

sug-gest a mechanism by which replication of diverse origins can be

modulated and thereby regulate the DNA replication of a

cellular genome with many origins.

MATERIALS AND METHODS

DNAs.The mPy and LPV DNA constructs used in these experiments were cloned in plasmid pUC19 or in phage M13mp19, and the DNAs were purified by two rounds of centrifugation in CsCl-ethidium bromide gradients.

In vivo DNA replication assays.Swiss mouse 3T6 cells and human P3HR1 lymphoblastoid cells were obtained from the American Type Culture Collection. DNAs were introduced into 3T6 cells following precipitation with calcium phos-phate or DEAE-dextran (70, 113) or into P3HR1 cells by electroporation (106

cells per ml, 1,200 V). The transfected cells were then cultured for 48 h, and the DNAs were extracted. The replication products were analyzed after DpnI diges-tion as previously described (110) with mPy and LPV [32_{P]DNAs as hybridization}

probes. In some experiments, test DNAs were introduced into cells together with origin-defective helper DNAs capable of expressing either mPy T antigen (pSO11 [107]) or LPV T antigen (pLPV-ApaR_{). These helper DNAs have base}

substitutions in the mPy core origin (pSO11) or a small deletion at the ApaI site in the LPV core origin (pLPV-ApaR_{) which prevents replication of the helper}

DNA but permits the expression of viral T antigens required for replication of test DNAs.

Preparation of cell extracts.Murine FM3A cells were purchased from Cellex Biosciences Inc. (Coon Rapids, Minn.). Ten liters of cells harvested at 53105

cells per ml were washed twice with ice-cold phosphate-buffered saline and shipped on ice. The subsequent preparation of FM3A cell extracts was done by the procedure described by Wobbe et al. (120). Cells were disrupted in hypotonic buffer (20 mM HEPES [N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid, pH 7.5], 5 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol [DTT]) by Dounce

homogenization; the lysate was adjusted to 0.2 M NaCl and centrifuged at 50,000 3g for 30 min, and the supernatant was dialyzed for 3 h against 3 liters of dialysis

buffer (20 mM HEPES [pH 7.5], 1 mM DTT, 0.1 mM EDTA, 50 mM NaCl, 10% glycerol). The resulting extract was clarified by centrifugation at 50,0003g for

30 min and stored in aliquots at2808C.

Preparation of T antigens.mPy T antigen was purified from extracts of High Five (Invitrogen) insect cells infected with recombinant baculovirus pVL941-LT (a gift of B. Schaffhausen) by immunoaffinity chromatography (75) with the F4 monoclonal antibody (78). At 48 h postinfection, 2.53108

cells in 500 ml of culture were collected, washed, and frozen at2808C. Thawed cells were lysed by sonication in 25 ml of buffer A (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 10% glycerol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride) and centrifuged at 30,0003g for 30 min, and the supernatant was

mixed at 48C with 4 ml of protein G-Sepharose coupled with F4 antibody for 3 to 15 h. Bound T antigen was washed extensively and eluted from the packed column with a buffer containing 20 mM Tris-HCl (pH 8.5), 1 mM DTT, 1 mM EDTA, 10% glycerol, 500 mM NaCl, and 50% ethylene glycol. The peak frac-tions of T antigen were dialyzed overnight against 2 liters of dialysis buffer (10 mM HEPES [pH 7.5], 5 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 50% glycerol) and stored in aliquots at2808C. Generally, up to 3 mg of T antigen with a purity of over 90% was obtained from 1 liter of infected cells. The purified T antigen retained maximal activity for at least 6 months. As reported by Lorimer et al. (64), different preparations of T antigen purified by this procedure varied in purity and activity.

In vitro DNA replication assay.mPy DNA replication in vitro was assayed by the method of Murakami et al. (75) and Prives et al. (87). Standard reaction mixtures (50ml) contained 80 mM Tris-HCl (pH 7.8); 40 mM creatine phos-phate; 7 mM MgCl2; 0.5 mM DTT; 4 mM ATP; 200mM each CTP, UTP, and

GTP; 100mM each dATP, dGTP, and dTTP; 20mM [a-32_{P]dCTP (1 to 10}

cpm/fmol); 1mg of creatine phosphate kinase; 0.5mg of plasmid DNA; 0.2 to 0.4 mg of FM3A cell extract; and 1 mg of T antigen. Reaction mixtures were incubated for 2 h at 338C, and the reaction was terminated by the addition of an equal volume of 40 mM EDTA–1.0% SDS and digested with 20mg of proteinase K. DNA was purified by phenol-chloroform extraction and ethanol precipitation. After restriction with DpnI, the replication products were fractionated by elec-trophoresis through a 1.0% agarose gel, dried, visualized by autoradiography, and quantitated with a PhosphorImager. The in vitro replication of mPy ori DNA was totally dependent on T antigen. Maximal in vitro DNA synthesis occurred when 0.5 to 1.0mg of DNA template was incubated with 1.0mg of T antigen per reaction.

KMnO4footprinting.The conditions for KMnO4modification were slightly

modified from those used for SV40 DNA by Borowiec and Hurwitz (4). Binding of T antigen to the mPy origin of replication does not require ATP hydrolysis (64); consequently, an ATP analog, 59-adenylyl imidodiphosphate (AMPPNP), was used, as it gave stronger KMnO4signals than did ATP (data not shown).

The standard reaction mixture (30ml) contained 80 mM Tris-HCl (pH 7.8), 40 mM creatine phosphate, 7 mM MgCl2, 0.5 mM DTT, 4 mM AMPPNP, 1.0mg of

T antigen, and 0.5mg of DNA. Reaction mixtures were incubated at 338C for 30 min. Freshly prepared KMnO4was added to a final concentration of 20 mM, and

the reaction was incubated for an additional 30 s at 338C. The reaction was quenched by addingb-mercaptoethanol to 1.0 M. The mixture was diluted to 80 ml with water and desalted on a 1.0-ml Sephadex G-50 spin column. The eluate was divided into two 35-ml portions, and the modified DNA was denatured by incubation for 5 min at room temperature with 0.1 volume of 2 M NaOH–2 mM EDTA. The solution was neutralized with 0.1 volume of 3 M sodium acetate (pH 5.2), and the DNA was precipitated with ethanol. The resultant single-stranded DNA was dissolved in 10ml of 40 mM Tris-HCl (pH 7.5)–20 mM MgCl2–50 mM

NaCl–1 ng of a 59-end-labeled primer complementary to either the top or the bottom strand sequence about 100 bp away from the central palindrome. The primers were annealed to the DNA by heating for 2 min at 658C, followed by cooling slowly over 15 to 30 min. Primer extension was carried out at 378C for 30 min following the addition of 0.5 mM deoxynucleoside triphosphate (dNTP) mix, 10 mM DTT, and 1 U of Klenow fragment of DNA polymerase I. The reaction was terminated with 6ml of stop solution (96% formamide, 20 mM EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol FF), heated at 908C for 2 to 5 min, and loaded onto a 6% polyacrylamide sequencing gel. Sequencing ladders derived with the same end-labeled primers were run beside the footprints.

Duplex DNA-unwinding assay.To prepare duplex DNA substrates, pPyori and pLPVori DNAs were digested with EcoRI and HindIII, and pPyxho and pL29 DNAs were digested with AccI. A fragment lacking an origin was obtained by digestion of pPyxho DNA with NocI and XbaI. These fragments were gel puri-fied, labeled with one [a-32

P]dNTP together with the other three unlabeled dNTPs by the Klenow fragment of DNA polymerase I, and then purified by NucTrap columns (Stratagene). The measurement of T antigen unwinding ac-tivity with duplex DNA fragments was performed essentially as described by Wang and Prives (117). Reaction mixtures (15ml) contained 80 mM Tris-HCl (pH 7.8), 40 mM creatine phosphate, 7 mM MgCl2, 0.5 mM DTT, 4 mM ATP,

1mg of creatine phosphate kinase, 1.0 ng of ori(1) or ori(2) [32_P]DNA

frag-ments, 0.5mg of Escherichia coli SSB, 0.2mg of T antigen, and the indicated amounts of linear pBR322 competitor DNA. Mixtures were incubated at 338C with T antigen for 30 min, and the reactions were terminated by the addition of an equal volume of a solution containing 2% sodium dodecyl sulfate (SDS), 50 mM EDTA, and 2 mg of proteinase K per ml. The mixtures were further incubated at 378C for 30 min, treated with 2ml of loading buffer (20% Ficoll, 0.5% xylene cyanol, 0.5% bromophenol blue), and fractionated by electrophore-sis through 5% polyacrylamide gels containing 0.53TBE. DNA bands were visualized by autoradiography and quantitated with a PhosphorImager.

RESULTS

DNA sequence homologies among the core origins of

mam-malian polyomaviruses.

The core origins of replication of the

known primate-specific polyomaviruses SV40, BK virus, JC

virus, SA12, and LPV and the rodent-specific polyomaviruses

mPy, hamster polyomavirus, and K virus are highly

homolo-gous (Fig. 1). Notably, each has a central palindrome with four

PuPuGGC pentanucleotide repeats (the sequence initially

bound by the viral T antigen), an A/T-rich sequence on the late

side, and a sequence on the early side with either an imperfect

inverted repeat or a polypyrimidine-polypurine (Py/Pu) tract

which contains DNA-unwinding elements.

Previous studies have documented that the SV40 T antigen

will not replicate the mPy DNA origin in vitro (75, 104), nor

are the SV40 and the mPy core origins replicated in vivo by the

other’s T antigens (1, 51, 107). The topology of T antigen

binding to DNA might be responsible for some of this

repli-cation selectivity, as mutational analysis of the SV40 and mPy

origins has shown that altering the symmetry of the

pen-tanucleotide repeats profoundly affects DNA replication (2,

12, 17, 23, 81, 110, 114). While the central palindromes of the

origins of SV40, BK, JC, and SA12 viruses are nearly perfectly

symmetric about a single base between the second and third

pentanucleotide motifs, the second and third pentanucleotide

motifs of the central palindromes of the mPy, LPV, hamster

polyomavirus, and K viruses overlap (Fig. 1). Nevertheless,

even among the viral origins of SV40, BK, and JC viruses with

similar arrangements of pentanucleotide motifs, there are

pro-found differences in the efficiency with which the origins are

replicated by each other’s T antigens (66, 67, 98), indicating

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that features other than the arrangement of pentanucleotide

motifs in the central palindrome affect the replication

effi-ciency of naturally occurring origins. To further explore such

replication selectivity, we focused on the mPy and the primate

LPV viruses, which replicate in different species but whose

origins and T antigens are highly homologous.

Viral replication selectivity.

mPy infects mice and will

rep-licate to a limited extent in the cells of other rodents. There is

sparse evidence that it productively infects primates: a few

early studies suggested that it can be propagated in monkey

kidney cells (100, 108), and we have observed that it replicates

to a limited extent in certain monkey cells which express SV40

large T antigen (107). LPV was isolated from an African green

monkey and replicates in primate B lymphoblastoid cell lines

but not fibroblastic cells (83, 105). Its genome is more similar

to that of mPy than to those of the other known primate

polyomaviruses (47, 82, 84). The similarity of the mPy and LPV

core origins (Fig. 1) suggested to us that they might be

repli-cated by each other’s T antigen. However, in preliminary

ex-periments, we observed that this was not the case;

conse-quently, we analyzed mPy and LPV DNA replication in greater

detail to define those features important for this replication

selectivity.

Replication of mPy in vivo required a functional enhancer in

cis (31, 54, 91, 92, 106) (Fig. 2A, compare lanes 1 and 2), while

the LPV genome introduced into mouse 3T6 fibroblasts did

not replicate (Fig. 2A, compare lanes 3 and 6). In human

lymphoid P3HR1 cells, a reciprocal pattern of replication

se-lectivity occurred, in that LPV replicated efficiently while mPy

did not (Fig. 2B, compare lane 3 to 4). Neither genome lacking

an enhancer replicated in human cells (Fig. 2B, lanes 1 and 2).

This cell type/species selectivity for replication may have

several causes. One might be that the viral enhancer functions

only in certain cell types and thereby restricts replication (30,

91, 92). Another cause might be incompatibility between the

viral T antigen and the replication proteins of the heterologous

FIG. 1. Alignment of the core origins of DNA replication of the known mammalian polyomaviruses. Numbers indicate positions in the physical map of each virus (15, 24, 25, 45, 46, 69, 82, 94, 110). The large boxes enclose the central palindromes containing the four pentanucleotide repeats (shown by arrows). The A/T-rich sequences and the Py/Pu sequences are indicated by rectangles, the imperfect inverted repeats are indicated by the dashed arrows, and the DNA-unwinding element (DUE) is indicated by the shaded box. Core origin sequence differences between mPy and LPV origins are indicated by dots.

FIG. 2. In vivo replication of wild-type and chimeric mPy and LPV DNAs. (A) Replication of viral DNAs in mouse 3T6 cells. Lane 1, pPyE2(mPy DNA lacking enhancer); lane 2, pPyBglII (mPy DNA with BglII linker at late side of core origin at nucleotide [nt] 5293, used as a parent for pPyE2); lane 3, pPy (mPy wild-type DNA); lane 4, pPyxho (mPy DNA with XhoI linker at nt 5293, used as a parent for pL29); lane 5, pL29 (mPy DNA with LPV sequences from nt 185 to 153 substituted for the equivalent mPy sequences between nt 5293 and nt 30); lane 6, pLPV (LPV wild-type DNA); lane 7, control (transfection without DNA). (B) Replication of viral DNAs in human P3HR1 cells. Lane 1, pPyE2; lane 2, pLPVE2(LPV DNA lacking enhancer); lane 3, pPy BglII; lane 4, pLPV. (C) Replication in human P3HR1 cells of viral origins cloned in pUC vectors with LPV T antigen expression. Lane 1, pPyori (mPy DNA from nt 5051 to 204); lane 2, pLPVori9(LPV DNA from nt 5050 to 760); lane 3, pLPVori (LPV DNA from nt 1 to 677); lane 4, pAH (LPV DNA from nt 677 to 394 joined to mPy DNA from nt 30 to 204); lane 5, pAN (mPy DNA from nt 5051 to 30 joined to LPV DNA from nt 153 to 1). (D) Replication of viral origins cloned infM13mp19 vectors in mouse 3T6 cells with mPy T antigen expression. Lane 1,fAH (AH chimeric LPV/mPy DNA); lane 2,fAN (AN chimeric LPV/mPy DNA); lane 3,fM13mp19 (vector control); lane 4,fPyori (mPy origin DNA); lane 5,fLPVori (LPV origin DNA).

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host (1, 9, 40, 74, 75, 93). A third cause might be the presence

of inhibitory proteins such as p53, which might bind one T

antigen but not the other (7, 116).

We attempted to distinguish between these possibilities by

assessing replication of an origin together with helper DNAs

capable of expressing a viral T antigen in cells which provide

the cognate accessory proteins capable of functioning with that

T antigen. When the mPy origin-enhancer DNA was

intro-duced into human P3HR1 cells together with a helper DNA

capable of expressing the LPV large T antigen, replication of

the mPy origin-enhancer was determined to be as efficient as

replication of the LPV origin-enhancer (Fig. 2C, lanes 1, 2, and

3), indicating that the LPV T antigen can form a functional

initiation complex at the mPy origin. However, the LPV

origin-enhancer was not replicated in mouse 3T6 cells when

cotrans-fected together with the mPy T antigen (Fig. 2D, compare

lanes 4 and 5), suggesting either that there is a cis-acting block

upon replication or that the mPy T antigen does not interact

productively with the LPV core origin.

To further analyze the basis for this replication selectivity,

we turned to in vitro assays which permit each step in the

assembly of the replication complex to be analyzed separately.

In vitro replication of mPy and LPV DNAs.

We asked

wheth-er the mPy T antigen togethwheth-er with a murine FM3A cell extract

capable of replicating mPy DNA can replicate an LPV origin

in vitro. This analysis indicated that the LPV origin was

repli-cated only minimally (Fig. 3, compare pPyori with pLPVori),

confirming the in vivo analysis reported above. To define the

DNA sequences that restrict replication of the LPV origin, we

constructed several chimeric DNAs and assessed their capacity

to be replicated by the mPy T antigen and the FM3A cell

extract. Two chimeric DNAs were prepared by joining

se-quences cleaved at the ApaI sites in the middle of the central

palindromes of the mPy and LPV origins (Fig. 4). pAN DNA

contains the late half of the mPy core origin-enhancer joined to

the early half of the LPV origin, and pAH DNA contains the

late half of the LPV core origin-enhancer joined to the early

half of the mPy origin (Fig. 4). Assays of their replication in

vitro revealed that the pAN chimera supported replication

mediated by Py T antigen (approximately 75% of that of

pPyori DNA), whereas pAH supported only 5% or less of the

replication of pPyori DNA mediated by mPy T antigen (Fig. 3).

To more precisely define the LPV sequences which restrict

replication by the mPy T antigen, an additional DNA construct

(pL29), containing 36 bp of LPV sequence substituted for the

homologous mPy sequences on the late side of the core origin

(between the ApaI and PvuII sites), was prepared. It was not

replicated by the mPy T antigen together with the murine cell

extract, in contrast to the replication of the parent DNA

con-taining the mPy origin (pPyxho) (Fig. 3). This indicates that

sequences on the late side of the core origin determine

repli-cation selectivity.

Comparable results were obtained in an analysis of the

rep-lication of these chimeric DNAs in vivo. In mouse 3T6 cells,

the pAN DNA chimera was replicated by mPy T antigen but

the pAH DNA chimera was not (Fig. 2D, lanes 1 and 2), and

the chimeric pL29 DNA did not replicate in mouse 3T6 cells in

contrast to the replication of its parent pPyxho DNA (Fig. 2A,

lanes 4 and 5). Both the pAH and pAN DNAs were replicated

by LPV T antigen in human P3HR1 cells, in agreement with

the observation that the intact mPy origin-enhancer is

repli-cated in human cells by LPV T antigen (Fig. 2C).

The sequence differences between LPV and mPy which must

determine this replication selectivity include one transition in

the central palindrome and nine bases on the late side of the

core origin (Fig. 1). The base substitution in the central

palin-drome has been shown previously not to affect replication of

mPy DNA (110). Therefore, it must be the sequences between

the central palindrome and the late border of the core origin

that determine this replication selectivity. To further assess

how these sequence changes affect replication, we studied the

individual steps leading to the formation of an initiation

com-plex.

mPy T antigen induces structural changes in the origins of

mPy and LPV DNAs.

Early steps in the initiation of SV40 DNA

replication involve the specific binding of large T antigen to the

central palindrome, the assembly of a double hexamer with

extended contacts on the flanking sequences, and the

subse-FIG. 4. Structures of wild-type and chimeric origins of replication. pPyori contains the origin-enhancer of mPy DNA from BclI (nt 5051) to DdeI (nt 204). pLPVori contains the origin-enhancer of LPV DNA from AhaIII (nt 677) to

NcoI (nt 1). pAN consists of the mPy sequences from nt 5051 to nt 30 joined at

the ApaI site to the LPV sequences from nt 153 to nt 1. pAH consists of the LPV sequences from nt 677 to nt 394 joined at the ApaI site to mPy sequences from nt 30 to nt 204. pPyxho contains the entire mPy DNA genome with an 8-bp XhoI linker inserted at the PvuII site (nt 5293). pL29 contains the entire mPy DNA genome with a substitution of LPV sequences from nt 154 to nt 185 between the

ApaI and XhoI sites of pPyxho.

FIG. 3. In vitro replication of wild-type and chimeric mPy and LPV origin DNAs mediated by mPy T antigen in murine FM3A extracts. Reaction mixtures containing 0.5 mg of plasmid DNA with cell extract in the absence (2) or presence (1) of 1.0mg of mPy T antigen were incubated at 338C for 2 h and analyzed as described in Materials and Methods.

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quent structural distortion of these flanking elements (3, 4, 16,

52, 68, 79, 119, 121). During this process, the A/T-rich domain

on the late side of the core origin is untwisted, and an

eight-base region in the early palindrome domain on the early side is

melted. The latter site coincides with the DNA-unwinding

el-ement predicted by thermodynamic calculations (63) and with

the origin of bidirectional replication mapped in vivo (28, 55,

56). The degree of structural change induced by T antigen in

the two flanking regions of the SV40 core ori region has been

shown to correlate with the efficiency of initiation of DNA

replication (2). This paradigm is likely to be applicable to all

polymavirus origins, including that of mPy, whose T antigen

binds to the same motifs as does that of SV40 (33, 85, 110).

To determine whether similar structural changes in mPy

DNA are induced by mPy T antigen and whether sequences on

the late side of the core origin affect such changes, we used

KMnO

modification of DNA. KMnO

reacts poorly with

nor-mal double-stranded DNA; however, when DNA is melted or

bent sharply (such as by a DNA-binding protein), specific sites

in the DNA become accessible to KMnO

oxidation (4, 6). The

oxidized nucleotides interfere with elongation by the Klenow

en-zyme, and their location can be determined precisely by

compar-ison with sequence ladders on a denaturing sequence gel.

In the absence of mPy T antigen, minimal levels of KMnO

modification were detected within the mPy core origin region

(Fig. 5A, lanes 1, 3, 5, 7, 9, and 11). Upon addition of mPy T

antigen, striking enhancements of KMnO

modification were

observed in the Py/Pu region of the mPy core origin, coinciding

with the Py/Pu region which corresponds with the early

palin-drome (DNA unwinding element) of SV40. Notably, mPy T

antigen mediated similar patterns of KMnO

modification in

the pPyxho and the pL29 DNAs, even though the latter was not

replicated by mPy T antigen. Similarly, mPy T antigen

dis-torted the pAH, pAN, and pLPVori DNAs even though these

DNAs differed greatly in their replication efficiency with mPy T

antigen. The KMnO

modification of these DNAs caused by

mPy T antigen generally was much weaker than the KMnO

modification induced by SV40 T antigen reacting with SV40

DNA (our unpublished observations) and was extremely

diffi-cult to quantify reliably. This may be due to the weaker binding

of mPy T antigen to its core origin sequences. Nevertheless, in

numerous experiments, it was always apparent that mPy T

antigen distorted the early region of the core origin.

The ability of mPy T antigen to induce structural changes in

the opposite strand of these DNA constructs was also

exam-ined. A high background level of KMnO

oxidation throughout

the core origin was observed in the absence of T antigen (Fig.

5B, lanes 1, 3, 5, 7, 9, and 11). Addition of mPy T antigen

induced distortion of the A/T-rich region in all the DNA

con-structs, and as with the modification of the other strand, the

level of KMnO

modification did not correlate with the

repli-cation capacity of these DNAs (Fig. 5B, lanes 2, 4, 6, 8, 10, and

12).

In summary, these results show that mPy T antigen binds to

the mPy and LPV core origins and causes structural changes

on both sides of the central palindrome. The pattern and

extent of structural changes do not correlate with the capacity

of the origins to be replicated by mPy T antigen either in vivo

or in vitro. This suggests that steps subsequent to the distortion

of the core origin determine the capacity of these DNAs to be

replicated by the mPy T antigen.

Unwinding of heterologous origins by mPy T antigens.

Fol-lowing structural deformation of the viral core origin, the

he-licase activity of SV40 T antigen induces extensive unwinding

of the DNA in concert with a single-stranded-DNA-binding

protein and topoisomerase I (3, 8, 34, 52, 59, 79, 88, 99, 114,

FIG. 5. KMnO4footprints of wild-type and chimeric mPy and LPV origins.

The DNAs (0.5mg) were incubated at 338C for 30 min with or without mPy T antigen in the presence of 4 mM AMPPNP and then incubated with KMnO4for

30 s. Sites of KMnO4modification were determined by primer extension. (A)

Footprints of KMnO4-reactive sites with primers (PyL5190 and LPV520)

com-plementary to the bottom strands (the adenine-rich strand in the A/T region of the core origin). (B) Footprints of KMnO4-reactive sites with primers (PyE167

and LPV0) complementary to the top strands (the thymine-rich strand in the A/T region of the core origin). The locations of domains of the core origin of replication are shown to the left of each set of lanes.

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121). The bidirectional DNA helicase activity of SV40 T

anti-gen is closely correlated with its replication capacity (17, 34, 52,

71, 114).

Previous studies have revealed that the SV40 and mPy T

antigens unwind duplex ori(

1

) and ori(

2

) DNA fragments

with comparable efficiency but that addition of nonspecific

competitor DNA causes the T antigens to unwind duplex

DNAs in an origin-specific manner (52, 117). In agreement

with these observations, we found that the mPy T antigen

unwound a DNA lacking the mPy origin (ori) as well as DNAs

containing the mPy or LPV origin with comparable efficiencies

in the absence of nonspecific competitor DNA (Fig. 6A, lanes

2, 7, and 12); however, with 0.1

m

g or more of linear pBR322

competitor DNA, the mPy T antigen poorly unwound ori(

2

)

DNA and the LPV ori DNA, while unwinding of the mPy ori

DNA fragment was reduced only slightly (Fig. 6A and B).

Similar analyses of the unwinding of pL29 DNA and that of its

parent, pPyXHO, revealed that the pPyxho DNA was

un-wound efficiently, while the pL29 ori fragment was inefficiently

unwound (Fig. 6C and D). These data indicate that the origins

which support replication by mPy T antigen are more readily

unwound in the presence of nonspecific competitor DNA.

In conclusion, these experiments indicate that the failure of

the mPy T antigen to replicate the LPV origin correlates with

the efficiency with which the origin is unwound and that

se-quences on the late side of the core origin help determine the

capacity of the mPy T antigen to act as an efficient unwinding

protein.

DISCUSSION

The initiation of DNA replication at a specific site on a

circular duplex DNA requires the identification of the origin by

a sequence-specific protein(s) and, subsequently, opening of

the DNA and synthesis of primers from which the leading and

lagging strands can be extended. The polyomaviruses utilize

the T antigen to nucleate the assembly of a replication complex

responsible for this process. The pentanucleotide-binding sites

recognized by the T antigens of the various members of this

family of viruses are highly conserved, yet several viruses have

evolved significant replication selectivity. How can this be

ex-plained?

One reason occurs in the specific interactions between the T

antigens and the cellular proteins which interact to assemble a

functional initiation complex. There is abundant evidence,

al-beit somewhat conflicting, that the T antigens of these viruses

interact differentially with the DNA polymerase

a

/primases of

different species (9, 40, 74, 75, 93). Such protein-protein

inter-actions probably change in concert with evolution of the

rep-lication machinery of the host cell.

Another means by which replication selectivity might occur

is through changes in the cellular proteins capable of activating

or inhibiting origin function. These proteins are believed to

function by preventing nucleosome occlusion or by affecting

assembly or function of the initiation complex (29). The

as-semblage of cellular proteins present in a cell which are

capa-ble of acting upon a specific viral core origin, auxiliary and

enhancer sequences, or the T antigen undoubtedly will dictate

the replication efficiency of that origin.

Our experiments suggest a third basis for replication

selec-tivity, namely, that it can occur because of a deficiency in the

capacity of the viral T antigen to efficiently unwind an origin

and that the sequences in the origin help dictate this process.

Regulation of the T antigen unwinding activity has been

sug-gested to be a key control point for replication (32, 34, 76, 89,

90). Only 2 to 3% of the T antigen-origin complex undergoes

FIG. 6. Unwinding of duplex DNA by mPy T antigen. (A and C) Represen-tative autoradiographs of the unwinding of ori(2), mPy ori, and LPV ori DNAs (A) and pPyxho ori and pL29 ori DNAs (C) by mPy T antigen in the absence of or with increasing amounts of unlabeled linear pBR322 competitor DNA. ss and ds, single- and double-stranded, respectively. (B and D) Plots of duplex DNA unwound as a function of competitor DNA. Incubations with labeled ori(1) or ori(2) DNA fragment were performed as described in Materials and Methods in the absence and presence of the indicated amounts of unlabeled linear pBR322 competitor DNA. Unwinding was quantitated by PhosphorImager and normal-ized to 50% in the absence of competitor DNA. The indicated values are averages of multiple independent experiments. The bars represent the standard error of the mean.

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unwinding in vitro (76). Control of unwinding may occur by

phosphorylation or dephosphorylation of specific residues in T

antigen (11, 43, 71, 73, 86, 114). It is apparent from our results

that sequence differences between origins also can affect the

conversion of T antigen from an origin-binding protein to an

origin-unwinding protein.

The studies of Lynch and Frisque (65) also indicated that

sequences on the late side of a viral origin help determine

replication specificity (between JC and SV40 viruses), and this

was shown to occur at a point beyond T antigen binding. Our

results extend their analysis to additional viruses and focus

upon a specific step in the replication pathway. These

obser-vations expand the significance of the switch in T antigen

activity from an origin-binding protein to an unwinding

activ-ity, to include a means by which the efficiency of DNA

repli-cation from an origin might be constitutively regulated. One

obvious circumstance when this might be advantageous is in

the event that a cell is infected by two distinct viruses; each

virus would benefit were its own T antigen not capable of

activating the other’s origin.

Perhaps another, more profound consequence of such

rep-lication selectivity is in the opportunity it provides to

deter-mine the timing of initiation of cellular DNA replication after

an initiation complex has bound to an origin. For example,

eukaryotic chromosomes contain multiple origins which

repli-cate at distinct times in S phase, but the mechanism for such

temporal control is not understood. Quantitative assessments

show that specific yeast origins have efficiencies of activation

that range from less than 20% to greater than 90% per cell

cycle (38, 42, 44). One means for such control is likely to be

through specific sequences affecting the efficiency (and perhaps

the rate) at which the initiation complexes initiate unwinding

of the DNA.

Recent studies of the interaction of SV40 T antigen with the

SV40 origin have suggested that the generation of structural

changes in the origin is mediated primarily through the

sugar-phosphate backbone and that sequence changes in the flanking

A/T element or early palindrome which diminish replication

do so solely because they cause these sequences to be resistant

to the structural alterations induced by T antigen (95). Our

data are not in accord with this suggestion. We find that the

restriction of mPy T antigen by the LPV origin does not

cor-relate with the capacity of the mPy T antigen to induce

struc-tural deformation but does correlate with its capacity to induce

unwinding. Similar analyses of the interaction of SV40 T

an-tigen with mutant origins and of mutant T anan-tigens acting at

the SV40 origin have led to a similar correlation (71, 81). In a

complementary study Bhattacharyya et al. (1a) demonstrate

that mPy and SV40 T antigens both produce structural

alter-ations in each other’s origins, but nevertheless, these T

anti-gens fail to replicate the respective heterologous origin.

The correlation between replication selectivity and the

se-quences on the late side of the origin and the effect of these

same sequences upon unwinding suggest that the replication

defect is caused at least in part by inefficient unwinding.

Nev-ertheless, the apparent replication defect appears to be greater

than the unwinding defect, which suggests either that the

un-winding assay is less discriminating than the replication assays

or that other factors might also be involved in the replication

selectivity. Both reasons seem likely: the unwinding assay with

SV40 T antigen has previously been found to be less

discrim-inating with origin mutants than are replication assays (17),

and mPy T antigen is much more promiscuous about the DNA

sequences it chooses to unwind than is the SV40 T antigen

(117).

We also note that our in vitro unwinding assays with T

antigens and duplex DNA fragments have been carried out

under artificial conditions and that many proteins which help

participate in the replication complex are absent.

Incorpora-tion of these proteins into our assays might reveal more

spe-cifically how sequences of the core origin affect unwinding. For

example, it has been reported that the A/T-rich flanking motif

is required for the stimulation of T antigen-dependent origin

DNA unwinding by DNA polymerase

a

(76). It is this same

region which we have found to be important in the replication

selectivity by mPy T antigen and which is responsible for the

replication selectivity of JC virus T antigen with its origin (65).

Since the p48 primase subunit helps determine the

species-specific initiation of Py DNA replication (9, 40), we wonder

whether the primosome has a special affinity for sequences in

this region of the core origin, so as to more efficiently initiate

primer synthesis (a suggestion which parallels that proposed by

Schneider et al. [93]). These same sequences induce bending in

DNA and are also known to be capable of binding a variety of

cellular proteins which might affect T antigen and polymerase

a

/primase activity (10, 21, 48, 57, 60, 109). Additionally,

aux-iliary sequences beyond the core origin bind T antigen and

cellular activators and thereby promote DNA unwinding (52a,

53). Mutations of the A/T tract in the core origin can be

compensated for by changes in these auxiliary sequences (50);

hence, there are likely to be specific interactions between

them. Understanding these additional interactions should help

explain how activation of the DNA replication complex is

mod-ulated within the cell.

ACKNOWLEDGMENTS

We thank B. Schaffhausen and D. Pallas for gifts of baculovirus vectors and hybridomas; Carol Prives, J. Dudley, V. Bermudez, Wei-dong Guo, and X. Wang for advice; and Mark Hannink and David Pintel for comments on the manuscript. Sarah Scanlon, as always, helped ensure that everything worked.

Financial support was provided by USPHS grant CA38538.

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