Binding and unwinding—How T antigen engages the SV40 origin of DNA replication

Cell, Vol. 60, 161-164,

January

26, 1990, Copyright

0 1990 by Cell Press

Binding and Unwinding How T Antigen Engages the SV40 O rigin of DNA Replication James A. Borowiec,’ Frank B. Dean, Peter A. Bullock,t and Jerard Hurwitz Program in Molecular Biology and Virology Memorial Sloan-Kettering Cancer Center New York, New York 10021

Initiation of DNA synthesis from a specific site in a duplex DNA molecule presents a complex challenge for the host replication machinery. The origin of replication (ON) must first be recognized by a site-specific DNA binding protein that then causes, either by itself or in conjunction with other factors, sufficient gross changes in the duplex structure of the DNA to allow the replication machinery access to the base-pairing positions. Various eukaryotic and prokaryotic model systems permit an analysis of this process with purified proteins. Studies of simian virus 40 (SV40), a tumor virus containing 5.2 kb of circular doublestranded DNA, have been particularly useful because the presynthesis reactions are catalyzed predominantly by a single protein, SV40 large tumor antigen (T antigen). Tantigen, a multifunctional 82 kd phosphoprotein, is the sole viral protein required for SV40 DNA replication; all other factors are provided by the host cell. In addition to its role in SV40 DNA replication and regulation of viral gene expression, T antigen can also lead to the transformation of susceptible cell lines. Studies of various mutant T antigen proteins have shown that the replication and transformation functions of T antigen can be separated. The development of a cell-free system that catalyzes SV40 DNA replication in vitro coupled with the use of expression systems that overproduce T antigen has allowed detailed analysis of the individual steps carried out by T antigen prior to initiation of DNA synthesis. Recent reviews summarizing the SV40 DNA replication system have appeared (Challberg and Kelly, 1989; Stillman, 1989). This review will focus on events preceding initiation of DNA synthesis. Binding of T Antigen to SV40 ori The initial step in the pathway of SV40 DNA replication is binding of T antigen to DNA sequence elements comprising the ori. T antigen can bind to two strong sites in the vicinity of SV40 ori(sites I and II), and under certain conditions to weaker sites (collectively termed site Ill) on the late-gene side of the ori region. Of the three sites, only site II is contained within the minimal 64 bp region comprising the core ori; this region is both necessary and sufficient to allow initiation of viral DNA replication (Figure 1). The presence of sites I and Ill can augment core ori activity to various degrees depending on replication conditions (Guo et al., 1989). A common feature of each site is the presence of 5’-G&GGC-3’ sequences, although the number

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and orientation of the sequences and their spacing differ at each site. The four conserved sequences in site II are found in a 27 bp perfect inverted repeat that contains two GAGGC sequences in each arm of the palindrome. The critical GAGGC sequences in site I are organized as two direct repeats separated by an AT-rich tract. Biochemical and genetic evidence shows that the GAGGC repeats serve as the recognition site for T antigen. Scanning transmission electron microscopy has further demonstrated that a minimum of a single T antigen monomer can bind to each GAGGC repeat (Mastrangelo et al., 1985). The SV40 core ori has been subject to detailed genetic investigation, and these studies indicate the presence of three domains critical for SV40 DNA replication (Deb et al., 1986a, and references therein; Figure 1): the central GAGGC element, a 10 bp region partially overlapping an imperfect inverted repeat (early palindrome), and a 17 bp region rich in adenines and thymines (AT-tract). The latter two flanking regions undergo structural transitions during initial stages of SV40 DNA replication (see below). Binding of T antigen to SV40 core ori is stimulated loto 15fold by ATP (Dean et al., 1987b; Deb and Tegtmeyer, 1987; Borowiec and Hurwitz, 1988a). DNAase I protection analysis shows that T antigen covers the complete core ori sequence. The cleavage pattern is striking, because loss of DNAase I cleavage sites on both strands over the entire region indicates close apposition of T antigen to all faces of the DNA helix. Moreover, electron micrographs of the ATP-dependent complex show that T antigen is organized into a two-lobed structure at the ori region. The complex of largest mass found in significant numbers by scanning transmission electron microscopy contains 12 monomers of T antigen, although complexes of lower mass were found. That this number is significant is supported by the observation that ATP can cause T antigen to form multimers even in the absence of DNA, with a mass equivalent to six T antigen monomers (Mastrangelo et al., 1989). This suggests that the ATP-dependent complex is composed of two lobes, each containing a hexamer of T antigen. These results suggest that T antigen, in the presence of ATP, forms a complex around the core ori sequence with each hexameric lobe of the complex surrounding the DNA (Figure 2A). Specialized nucleoprotein structures also form at E. coli oriC and bacteriophage h or&, with the DNA

/+

Site I w

COREORKIiIN -I (Site II)

Figure 1. Features of the SV40 ori Region The T antigen binding site I (T-AS I), early palindrome * Present address: Department of Biochemistry, New York University Medical Center, New York, New York 10016. 7 Present address: Department of Biochemistry, Tufts University, Boston, Massachusetts 02111.

element (EP),

central element containing the GAGGC repeats (GAGGC),and ATtract (A/T) are shown. The orientation and approximate positions of the GAGGC repeats in binding sites I and II are indicated by short arrows. SV40 sequence positions are given above.

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Figure 2. Model of the ATP-Dependent Core ori

T Antigen Complex with SV40

(A) The double hexameric structure of T antigen bound to ori. (8) Cutaway view of the complex. Shown are the melted region in the early palindrome element (dark rhomboid), numerous protein-DNA contacts in the central region containing the GAGGC repeats, and distorted double-stranded DNA in the AT-tract. Aspects of this model have been previously described (Mastrangelo et al., 1989).

wrapping around numerous molecules of dnaA and h 0 proteins, respectively (see references in Bramhill and Kornberg, 1988; Dodson et al., 1989). This type of nucleosome-like wrapping of SV40 ori around T antigen does not occur, because electron microscopic analysis showed no significant shortening of the length of linear DNA molecules upon formation of the ATP-dependent complex. Structural Changes in SV40 ori The ATP-dependent binding of T antigen to SV40 ori induces structural distortions of the ori DNA (Borowiec and Hurwitz, 1988b; Figure 28). Two regions flanking the central GAGGC element become hypersensitive to either methylation by dimethyl sulfate or oxidation by potassium permanganate. These regions are virtually superimposable on the early palindrome element and the AT-tract critical for ori function. The early palindrome element rather than the AT-tract appears to be the site of DNA melting. Dimethyl sulfate and potassium permanganate probing show that T antigen denatures approximately 8 bp in the distal arm of the early palindrome within core ori. Efficient melting is supported by a number of adenine nucleotides, including ADP and adenosine 5’-[8,r-imidoltriphosphate (AMPPNP), which indicates that ATP hydrolysis is not required to disrupt the early palindrome region. Recently, Parsons et al. (1990) demonstrated that high levels of T antigen can induce significant melting of the early palindrome element even in the absence of the other two critical elements of the SV40 ori, to approximately 25% of the melting observed with the complete core ori. T antigen may therefore weakly recognize the structure of the early palindrome element in the ATP-dependent complex and use these contacts to denature the DNA. T antigen-induced melting in the early palindrome element occurs in a region of ori that is strongly biased toward purines on one strand and pyrimidines on the other. Such sequences are inherentlv unstable (Wells, 1988, and refer-

ences therein), and this property may be utilized by T antigen to melt the early palindrome element. Polypurinepolypyrimidine asymmetry is an important feature of the early palindrome element, as suggested by the presence of this asymmetry in related polyoma viruses. Sequences with polypurine-polypyrimidine tracts also occur in ori sequences of other viruses (e.g., the longer arm of ori, sequence of herpes simplex virus type 2), which suggests that these tracts may also serve to initiate the melting reaction at other ori sequences. The second region that undergoes a structural traneition within ori is the 17 bp AT-tract. This tract has an intrinsic structural anomaly even in the absence of T antigen. DNA fragments containing this tract have abnormal electrophoretic migration (Deb et al., 1986b) typical of a stable bend in the DNA. The tract undergoes further changes within the T antigen ATP-dependent complex, which can be detected by probing with potassium permanganate, a reagent that reacts efficiently with nucleotides in regions with altered DNA structure. The structural change does not represent denaturation of the DNA duplex because the amount of dimethyl sulfate methylation of internal hydrogen bonding sites is approximately 5% of that occurring within the early palindrome. The exact nature of the conformational change within the AT-tract remains unclear, although two possibilities include a further increase in DNA bending or, alternatively, an overall untwisting of the DNA helix (defined as an increase in the number of base pairs per helical turn). The overall effect of these structural alterations can be detected by changes in the topological distribution of covalently closed DNA molecules containing ori (Roberts, 1989). Thus, duplex DNA within the ATP-dependent T antigen complex is subject to severe destabilizing forces. Roughly 50% of core ori DNA is either melted or double-stranded DNA with significant distortion. The strong correlation between these structurally altered regions and the critical domains defined genetically suggests that the primary role of the two flanking regions in SV40 ori is to undergo structural changes required for ori function. Bidirectional Unwinding of DNA from ori After T antigen forms the ATP-dependent complex, ori becomes completely denatured, and T antigen further unwinds the DNA duplex in a bidirectional manner. With purified proteins, this reaction generates highly underwound DNA on circular DNA molecules and requires neither DNA synthesis nor negative DNA supercoiling. Mutations in ori affect DNA replication and DNA unwinding similarly, suggesting that the unwinding reaction is an integral component of SV40 DNA replication (Dean et al., 1987a). The DNA unwinding reaction was predicted by two earlier observations: the detection (using T antigen-specific antibodies) of T antigen at the forks of SV40 DNA replication intermediates (Stahl and Knippers, 1983) and the demonstration that T antigen is an ATP-dependent DNA helicase capable of displacing oligonucleotides bound to singlestranded DNA (Stahl et al., 1986). In vitro, the ori-dependent DNA unwinding reaction requires a single-stranded DNA binding protein (SSB) to generate extensive regions of single-stranded DNA. This

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tral GAGGC element, as well as to the early palindrome. These binding events lead to rapid alterations in the ATtract and denaturation of the early palindrome. It is not known whether these events occur prior to or concomitant with the formation of the double hexamer of T antigen at core ori. However, all of these reactions, including hexamer formation, can occur with ADP or nonhydrolyzable ATP analogs (AMP-PNP, ATPyS) and are independent of SSB; none of these reactions occurs with isolated T antigen hexamers formed in the absence of DNA. The next stage in the presynthesis reaction is T antigen-induced DNA unwinding, which is dependent on ATP hydrolysis and SSB. With circular DNA, a topoisomerase that is capable of relaxing the positive superhelical turns formed by helicase action is also essential. The unwinding reaction proceeds bidirectionally from ori, converting the duplex DNA to single strands that are then sequestered by SSB. Structural changes induced in ori by T antigen have notable similarities with presynthesis reactions occurring at oriC and orih (e.g., Bramhill and Kornberg, 1988; Dodson et al., 1989, and references therein). Each ori sequence has a lengthy AT-rich tract and multiple sequence repeats that bind numerous copies of an ori binding protein, resulting in a large multimeric complex. The binding of these initiation factors causes structural distortions of the DNA structure. These multimeric complexes can, in conjunction with other proteins (notably E. coli dnaB protein), lead to the formation of highly underwound DNA molecules. However, comparison of the presynthesis reactions also reveals differences. The ori binding proteins at oriC and orih (dnaA and I. 0 proteins) are wrapped by ori DNA sequences, while in the case of SV40 ori, DNA within the complex is wrapped by T antigen. Differences in functions may contribute to the observed structural differences of the protein-DNA complexes. In addition to its role as an ori binding protein, T antigen also acts as a DNA helicase migrating through the DNA, while complexes of DNA with dnaA or ?, 0 protein act as structures that position the dnaB protein, the helicase for these systems. Second, while the AT-region in prokaryotes appears to serve as the initial melting sequence, SV40 appears to exploit a polypurine-polypyrimidine tract within the core ori to nucleate the initial melting of the DNA helix. Third, the DNA unwinding reaction in prokaryotic systems requires DNA supercoiling, while the analogous reaction for SV40 works efficiently on linear DNA templates. It appears that single-stranded regions of DNA needed for DNA synthesis are generated using similar strategies in prokaryotic and eukaryotic organisms. In each system,

requirement is likely fulfilled in the natural host by the SSB initially isolated from human cells as a protein essential for SV40 DNA replication (variously termed HeLa SSB, RF-A, RP-A, and referred to here as human SSB; Wobbe et al., 1987; Fairman and Stillman, 1988; Wold and Kelly, 1988). While SV40 DNA replication has an absolute requirement for human SSB, this protein can be replaced in the DNA unwinding reaction by a number of different SSB proteins (e.g., E. coli SSB, adenovirus DBP etc.), suggesting the absence of a specific interaction between human SSB and T antigen at this step. When covalently closed circular DNA molecules are used, detection of unwound DNA also requires a topoisomerase to relieve the positive superhelicity generated during unwinding. Recently, a requirement has been reported for a third factor that is identical to the catalytic subunit of cellular protein phosphatase IIA (Virshup et al., 1989). The requirement of protein phosphatase IIA for DNA unwinding, however, is only observed using T antigen isolated from cells infected with the recombinant adenovirus vector. T antigen must be phosphorylated at threonine-124 to be active in SV40 DNA replication (McVey et al., 1989). The mutation of this threonine, as well as other serine residues, to alanine blocks replication (Schneider and Fanning, 1988). Thus, the phosphorylation state of T antigen plays a critical role in SV40 DNA replication. When acting as a DNA helicase, T antigen moves in the 3’-+5’ direction on the strand to which it is bound (Goetz et al., 1988; Wiekowski et al., 1988). This indicates that during SV40 DNA replication, T antigen would translocate on the parental strand, which serves as the leading-strand template. Although T antigen has been located at the forks of SV40 replication intermediates, unequivocal evidence that T antigen is the only DNA helicase essential for replication is lacking. During ori-dependent DNA replication using crude cytoplasmic extracts of human cells, significant amounts of underwound DNA are formed. Furthermore, the earliest labeled DNA structure detectable during replication in vitro is associated with the underwound species, which can be chased into mature DNA products (Bullock et al., 1989). Therefore, production of underwound DNA appears to be a prerequisite for initiation of DNA synthesis. Overview of Events Occurring at ori before Initiation of DNA Synthesis From the studies described (see also Tsurimoto et al., 1989) a model incorporating the many roles of T antigen can be proposed (Figure 3). A critical initial event during SV40 DNA replication is the ATP-dependent binding of T antigen to core ori. It is likely that this event includes binding of T antigen monomers to pentanucleotides in the cen-

Binding SV4ll

AT-tract chaws

DSA

Unwinding

-IO&S SSB ATP

ADP + PI

DNA

Synlhe\i=?

and EP

DNA untwisnng Double hexamer formation

rNTPs DNA pal a/DNA primase DNA pal 6 Accessory factors

Figure 3. Schematic Model for T Antigen-Dependent Reactions Occurring during Initial Steps of SV40 DNA Replication

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specialized protein-DNA complexes form that induce changes in the DNA helix. In addition, sequence domains within origins whose intrinsic instability facilitates structural changes (Kowalsky and Eddy, 1989) function to activate replication origins for the initiation of DNA synthesis. These complicated reactions serve to prepare the DNA for subsequent steps of DNA replication, namely, the binding of specific proteins that actually carry out the initiation of DNA synthesis. References Borowiec, J. A., and Hurwitz, J. (1988a). Proc. Nab. Acad. Sci. USA 85, 64-68.

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