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Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA

2006; Springer Nature; Volume: 25; Issue: 24 Linguagem: Inglês

10.1038/sj.emboj.7601452

1460-2075

Elena Bochkareva, Dariusz Martynowski, Almagoul Seitova, Alexey Bochkarev,

Plant Virus Research Studies

Article30 November 2006free access Structure of the origin-binding domain of simian virus 40 large T antigen bound to DNA Elena Bochkareva Elena Bochkareva Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Dariusz Martynowski Dariusz Martynowski Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USAPresent address: Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, ND10.136, Dallas, TX 75390-8816, USA Search for more papers by this author Almagoul Seitova Almagoul Seitova Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Alexey Bochkarev Corresponding Author Alexey Bochkarev Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Elena Bochkareva Elena Bochkareva Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Dariusz Martynowski Dariusz Martynowski Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USAPresent address: Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, ND10.136, Dallas, TX 75390-8816, USA Search for more papers by this author Almagoul Seitova Almagoul Seitova Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Alexey Bochkarev Corresponding Author Alexey Bochkarev Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Author Information Elena Bochkareva1,‡, Dariusz Martynowski2,‡, Almagoul Seitova3 and Alexey Bochkarev 1,2,3 1Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario, Canada 2Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA 3Structural Genomics Consortium, University of Toronto, Toronto, Ontario, Canada ‡These authors contributed equally to this work *Corresponding author. Banting and Best Department of Medical Research & Department of Medical Genetics and Microbiology, University of Toronto, 100 College Street, Rm 522, Toronto, Ontario, Canada M5G 1L5. Tel.: +1 416 946 0805; Fax: +1 416 946 0588; E-mail: [email protected] The EMBO Journal (2006)25:5961-5969https://doi.org/10.1038/sj.emboj.7601452 Present address: Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, ND10.136, Dallas, TX 75390-8816, USA PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The large T antigen (T-ag) protein binds to and activates DNA replication from the origin of DNA replication (ori) in simian virus 40 (SV40). Here, we determined the crystal structures of the T-ag origin-binding domain (OBD) in apo form, and bound to either a 17 bp palindrome (sites 1 and 3) or a 23 bp ori DNA palindrome comprising all four GAGGC binding sites for OBD. The T-ag OBDs were shown to interact with the DNA through a loop comprising Ser147–Thr155 (A1 loop), a combination of a DNA-binding helix and loop (His203–Asn210), and Asn227. The A1 loop traveled back-and-forth along the major groove and accounted for most of the sequence-determining contacts with the DNA. Unexpectedly, in both T-ag-DNA structures, the T-ag OBDs bound DNA independently and did not make direct protein–protein contacts. The T-ag OBD was also captured bound to a non-consensus site ATGGC even in the presence of its canonical site GAGGC. Our observations taken together with the known biochemical and structural features of the T-ag–origin interaction suggest a model for origin unwinding. Introduction The sequence of events that underlies the initiation of DNA replication has been derived from extensive biochemical and genetic experiments in both prokaryote and eukaryote systems (reviewed in DePamphilis, 1996; Bullock, 1997; Stenlund, 2003; Eichman and Fanning, 2004). In each of these systems, the initiation process begins with binding of specialized origin DNA-binding proteins (OBPs) to the origin (ori). The OBP serves two main roles: first, it binds to multiple recognition elements within the ori through higher order nucleoprotein interactions and induces local distortion or unwinding of the origin DNA; second, the OBP recruits to the ori the other components of the primosome, usually including an ATP-dependent DNA helicase. This helicase acts in concert with the OBP to unwind origin DNA. A mechanistic understanding of these events is limited owing to insufficient structural information about all the biochemically defined steps in early primosome assembly. Functional origins of most eukaryotic genomes have not been characterized, and for many years, simian virus 40 (SV40) has been a favored laboratory model for studies of the initiation of DNA replication (Fanning and Knippers, 1992; Bullock, 1997; Butel and Lednicky, 1999). The viral OBP, named large T antigen (T-ag), is a 708-amino-acid (80.2 kDa) protein that also acts as the viral replicative helicase (Figure 1A). Insofar as is known, the T-ag binds to the origin first as a monomer to its pentanucleotide recognition element (Gidoni et al, 1982; Scheidtmann et al, 1984; Runzler et al, 1987). The monomers are then thought to assemble into hexamers and double hexamers, which constitute the form that is active in initiation of DNA replication. When bound to the ori, T-ag double hexamers encircle DNA (Mastrangelo et al, 1989; Bullock, 1997). Hexamer assembly nucleates at T-ag recognition pentanucleotides (Parsons et al, 1991). Preformed T-ag hexamers are incapable of forming double hexamers on DNA (Huang et al, 1998). A model has been proposed in which double hexamer assembly occurs by successive binding of 12 monomers (Huang et al, 1998). Figure 1.Structural and functional elements of SV40 DNA replication system. (A) Sequences present in the 64-bp core origin of SV40 replication. Locations of the AT-rich region, the pentanucleotide box (PEN) and EP sites, numbered as in the genome of reference strain 776. Arrows depict the four GAGGC pentanucleotides—the binding sites for T-ag OBDs (blue ellipses). Eight nucleotides within the EP that melt upon the T-ag double hexamer assembly are highlighted in yellow. The 23 bp PEN element structure reported in this work is boxed. (B) Functional domains of SV40 large T antigen (T-ag). 'J Domain', region not required for in vitro DNA replication; 'OBD' origin DNA-binding domain, minimal region required for binding to SV40 Ori DNA; 'Helicase', minimal region required for the helicase activity; the numbers given are the amino-acid residues. (C) T-ag OBD structure (apo form) ribbon diagram showing the domain containing residues 134–259 (in figure it is 132–257). α-Helices are in cyan and β-strands in magenta. Position of Cys 216 and DNA-binding components 'A1 loop' and 'B2 element' are indicated. (D) Secondary structure elements of T-ag OBD. The β-strands are indicated by arrows and the α-helices by boxes. Amino acids contributing to DNA binding are labeled with '*'. Download figure Download PowerPoint The functions of T-ag are mediated by several structural and functional domains. The N-terminal region (J-domain) is not required for in vitro DNA replication (Chen et al, 1997; Kim et al, 2001). The origin-binding domain (OBD) maps to amino acids 131–259 (Arthur et al, 1988; Joo et al, 1997). Mutagenesis studies of this domain in the context of the full-length protein also suggest a role for this domain in oligomerization and DNA structural perturbations. Helicase activity is associated with the helicase domain (aa 271–627; Figure 1D) (Li et al, 2003). Coordinated action of the OBD and helicase domains is required for melting of the SV4O origin (Chen et al, 1997). The SV4O core origin of replication (ori) is 64 bp in length, and is required for both in vivo and in vitro replication (Bullock et al, 1989; Bullock, 1997). There are three functional regions of the ori: the early palindrome (EP), the 23 bp pentanucleotide palindrome (PEN), and a 17 bp A/T-rich domain (Figure 1B). The PEN contains a cluster of four GAGGC pentanucleotides (P1–4), depicted by arrows in Figure 1B, which are arranged in two pairs inverted towards each other. All four pentanucleotides are required for initiation of DNA synthesis, and the spacing between the GAGGC elements is critical; substitution of the single base pairs separating the pentanucleotides did not affect DNA replication, whereas duplicating the same base pairs drastically reduced replication (Bullock, 1997). T-ag binds to the SV40 ori through the PEN element; it binds to the GAGGC pentanucleotide with high affinity (57–150 nM) (Titolo et al, 2003b). The OBD is also capable of binding in a non-sequence-specific way to both dsDNA and ssDNA, albeit with lower affinity (about 10-fold lower affinity for random dsDNA) (Titolo et al, 2003b). Origin unwinding by T-ag is dependent on the three functional regions of the origin, including four T-ag binding sites arranged as in PEN. T-ag binds cooperatively to the PEN element, in the presence of ATP oligomerizes into a double hexamer (SenGupta and Borowiec, 1994; San Martin et al, 1997; Gomez-Lorenzo et al, 2003) and induces melting of 8 nt within EP (Borowiec and Hurwitz, 1988) (Figure 1B). Electron microscopy studies revealed that the double hexamer spans about 240, or 120 Å per hexamer, along the DNA axis (Valle et al, 2000), which is consistent with the biochemical observation that T-ag protects 74 bp DNA (Fanning and Knippers, 1992). Dissection of the ori functional regions revealed that a single pentanucleotide is sufficient to mediate an assembly of T-ag hexamer (Joo et al, 1998) and the presence of two sites, 1 and 3 (Figure 1B), is sufficient for the formation of two T-ag hexamers (T-ag double hexamer). However, neither one nor two pentanucleotides is sufficient for unwinding of the SV40 ori (Joo et al, 1998). Only in the presence of all four sites, appropriately spaced, can T-ag partially melt 8 bp of the SV40 ori through its helicase activity (Borowiec and Hurwitz, 1988). The current state of structural knowledge about how OBPs from different species bind to their cognate DNA origins is limited. NMR and X-ray structures of the T-ag OBD in apo form are available (Luo et al, 1996; Meinke et al, 2006). In the crystal structure (Meinke et al, 2006), T-ag OBD adopts a left-handed spiral with six OBDs per turn and pitch of 35.8 Å per turn. Apo structures for OBD are also known for Epstein–Barr virus (EBV) OBP, EBNA1 (Bochkarev et al, 1995), bovine papilloma virus (BPV) E1 (Enemark et al, 2000), and the Rep proteins from adeno-associated virus 5 (AAV-5) (Hickman et al, 2002) and tomato yellow leaf curl virus (TYLCV) (Campos-Olivas et al, 2002). The crystal structures of EBNA1, BPV E1, and E2 (E1 binding partner on the origin) were also determined in complex with DNA (Hegde et al, 1992; Bochkarev et al, 1996; Enemark et al, 2002), but structural basis for T-ag binding to DNA remains unknown. The mechanisms of origin unwinding are best described for the bovine papilloma viruses (BPV). Like for SV40, the BPV origin also contains four binding sites arranged in two pairs and is bound by an OBP (E1) that contains both a DNA-binding and a helicase domain. E1 is a monomer in solution and dimerizes upon binding the origin. Assembly of the double hexameric E1 helicase proceeds in an ordered manner via a double-monomer, double-dimer, double-trimer, and finally a double-hexamer. Assembly of the E1 protein along this pathway is associated with a helix–to–ring transition (Schuck and Stenlund, 2005). Most studies on the SV40 system suggest general functional similarities with the BPV system, with minor differences. SV40 has a different arrangement of the binding sites in the origin and a higher affinity of its T-ag OBD to a single binding site (57–150 nM) than does E1 for its sequences (∼500 nM) (Titolo et al, 2003a, 2003b). In addition, the initial loading of E1 onto the ori is mediated through cooperative binding of E1 (the affinity for two properly spaced sites is ∼32 nM) but also through interactions with the viral transcription factor E2 (Sanders and Stenlund, 2001; Titolo et al, 2003a). Here, we report the high-resolution structure of the T-ag OBD in its apo form (1.5 Å) and the structure of the functional PEN palindrome DNA bound by T-ag OBD (1.65 Å). We also present a medium-resolution (2.3 Å) structure in which T-ag OBD was captured bound to the DNA in a nonspecific binding mode. Taken together with the structures of the T-ag helicase domain, the T-ag OBD spiral hexamer, and with biochemical data available in literature, our data suggest a molecular model for the T-ag double hexamer and for SV40 origin unwinding. Results Structure solution T-ag OBD was crystallized in two crystal forms belonging to the p21 and p212121 space groups and diffracted to 1.5 and 2.5 Å, respectively. The p21 structure was phased by a single isomorphous replacement with anomalous scattering (SIRAS) using a HgCl derivative and refined against 1.5 Å data to R=17.3% and R_free=19.5% (Figures 1C and D). The p212121 structure was solved by molecular replacement using the p21 structure as a model and refined against 2.5 Å data to R=21.6% and R_free=29.3%. The T-Ag OBD was also crystallized in complex with two different origin fragments. The first complex was with a 23 bp PEN palindrome (PEN-4; with a single base overhang at the 5′-end) and belonged to space group c2 and diffracted to 1.65 Å. The structure was solved by molecular replacement using the apo form T-ag OBD structure as a search model. The structure was refined against 1.65 Å data to R=20.1% and R_free=25.1% (Figure 2). Figure 2.Structure of four T-ag OBDs bound to the 23 bp PEN box of ori DNA (PEN-4). (A) Four copies of OBD are represented as ribbon models and colored as in Figure 1C. DNA is shown as a stick model. (B) Same structure, PEN-4, viewed along the DNA axis. OBDs bound to sites 1 and 3 are colored in cyan and those bound to sites 2 and 4 are in red. (C) A schematic diagram showing the contacts of T-ag OBD with the GAGGC pentanucleotide. The bases are numbered 1–5 starting from the 5′ end of the recognition element. The amino acids are boxed and the trace of polypeptide backbone is indicated with a thick dark line. The broken lines indicate hydrogen bonds. 'W' for water molecules. (D) Conformational changes in T-ag OBD induced by DNA binding. A superposition of free and bound OBD was generated as discussed in the text. Shown is the Cα trace of the free (blue and yellow for the P21 and P212121 crystal forms, respectively) and bound (red) domain. Important amino acids are labeled. Maximal shift of the loops are indicated with dashed lines, and the size of the shift is indicated in Å (large numbers). Download figure Download PowerPoint The second OBD/DNA complex comprised a T-ag OBD mutant with a Cys216 to Ser substitution (C216S) and a palindromic DNA that contained sites 1 and 3 (PEN-2). Site 2 in this DNA, which would be oriented on the opposite face of the DNA to the Site 1/3 palindrome, was mutated to ATgAT in order to create a perfect palindrome (Figure 3A). T-ag OBD (C216S) bound to this mutated DNA, was crystallized in space group p43212 and diffracted to 2.3 Å. The structure was solved by molecular replacement using the apo form T-ag OBD structure as a search model and refined against 2.3 Å data to R=22.4% and R_free=29.8% (Figure 3B). Additional crystallographic statistics are given in Supplementary Table 1. Figure 3.Non-sequence-specific binding of T-ag OBD to PEN-2. (A) DNA used for cocrystallization of the complex. Arrows depict two GAGGC pentanucleotides, sites 1 and 3. Mutated site 2 is crossed out. Non-canonical binding sites bound by T-ag OBD in the structure are shown in red. (B) T-ag OBD–PEN-2 complex structure. Two copies of OBD are represented as ribbon models. One monomer is colored according to the secondary structure; α-helices in cyan and β-strands in purple. The other monomer is rainbow colored from blue (N-terminus) to red (C-terminus). Download figure Download PowerPoint Structure overview T-ag OBD in the p21 apo crystal structure is similar to that in the NMR (PDB Id: 1TBD) (Luo et al, 1996) and crystal (PDB Id: 2FUF) (Meinke et al, 2006) structures reported previously. The RMS deviation between the apo structure and NMR structures is 1.2 Å with 121 Cα atoms and between the two X-ray structures of T-ag OBD is 0.9 Å with 122 Cα atoms. The structures of T-ag and BPV E1 OBDs are also similar (PDB Id: 1F08) (Enemark et al, 2000); the RMS deviation among 70 Cα was 1.8 Å. The two crystallographically independent molecules of T-ag OBD in the unit cell were connected by a disulfide bond between a cysteine on each molecule (Cys 216–Cys 216). Cys 216 is completely conserved across the polyoma virus family and the mutation C216G is defective in DNA unwinding (Wun-Kim et al, 1993). The p212121 and p21 structures were very similar (RMS deviation of 0.5 Å with 126 Cα atoms). The structures differed in that a Cys 216–Cys 216 disulfide bond was observed only in the p21 but not in the p212121 structure, and the loop containing Cys 216 had a slightly different conformation (discussed below). In the T-ag OBD/PEN-4 complex, four OBDs bind individually to the four GAGGC elements (Figure 2A). Surprisingly, contrary to what was anticipated from biochemical studies and the structure of the BPV E1-DNA complex, the T-ag OBDs bound to PEN-4 elements were not dimers and indeed were not even in direct contact. Two pairs (sites 1, 3 and sites 2, 4) of T-ag OBDs were oriented face-to-face on approximately opposite (upper and lower) sides of the DNA (Figure 2B). As predicted from mutagenesis studies, the protein–DNA contacts are mediated by two DNA-binding elements: a DNA-binding loop (S147–T155), known in the literature as 'A1 loop' (Simmons et al, 1990), and a DNA-binding helix with a portion of the loop ('B2 element') N-terminal to it (H203–N210), with additional contribution from Asn 227. The A1 loop travels back-and-forth in the major groove and accounts for most of the sequence-determining contacts with the DNA (Figure 2C). Protein–DNA interaction There are two crystallographically independent T-ag monomers in the T-ag/PEN-4 co-crystal structure. Direct protein–base contacts are virtually identical in both. Five residues of T-ag OBD make a total of eight direct, and one water-mediated, hydrogen bonds with the five bases in the major groove of the pentameric DNA recognition element. Specific protein–base recognition is provided by two arginines, Arg 154 and Arg 204, and two flanking guanines, G1 (the first G in the GAGGC pentamer) and the G complementary to C5. Arg 154 forms two sequence-specific hydrogen bonds with G1 (Figure 4A). Arg 204 forms two similar contacts with the G complementary to C5 (Figure 4G). Ser 152 binds specifically to A2 (Figure 4C). Asn 153 contributes three hydrogen bonds to specific interaction with G3 and G4 (Figure 4E and F). Figure 4.Structural details of T-ag interaction with DNA bases in the PEN-2 and PEN-4 structures. (A) Sequence-specific interaction of Arg 154 with G1 (the first G in the GAGGC pentamer) in the PEN-4 structure. A representative electron density map (shown in blue) is superimposed on the model. (B) Nonspecific interaction of Arg 154 with A1 (A in position of G1) in the PEN-2 structure. A representative electron density is superimposed on Arg 154. (C) Sequence-specific interaction of Ser 152 with A2. (D) Nonspecific interaction of Ser 152 with the T2 in the PEN-2 structure. Sequence-specific interactions, which involve (E) the G3 and Asn 153, (F) the G4 and Asn 153, and (G) the G (complementary to C5) with Arg 204. The protein and DNA are shown as stick models and colored by atom type; yellow for carbon, blue for nitrogen, red for oxygen, and purple for phosphorus. A representative electron density as captured from 2Fo−Fc map is shown with contours drawn at the 1.25σ level. Hydrogen bonds are indicated with red dashed lines, and the length of the bonds is indicated in Å. Download figure Download PowerPoint T-ag OBD also makes extensive nonspecific contacts to DNA. There are 10 direct and water-mediated hydrogen bonds to phosphates (Figure 2C). Nonspecific contacts to phosphate groups are mediated by Ser 147, Val 151, Phe 150, Thr 155, His 203 (ND1), Asn 210, and Asn 227. Four direct H-bonds and one water-mediated contact are originated from the A1 loop (Ser 147, Val150, Phe151, and Thr 155). Three direct and one water-mediated contacts are originated from the B2 element, and one comes from Asn 227. Our structural studies are consistent with the DNA-binding properties of T-ag OBD in solution as determined at near-physiological conditions (Titolo et al, 2003b). These studies showed that binding of multiple T-Ag OBDs to the origin was not cooperative. Similarly, in the structures, we did not observe any interaction between different OBDs in the protein–DNA complex. In contrast, the full-length T-ag does bind cooperatively to the origin as a double hexamer (Valle et al, 2006 and references cited there). Comparing the structure of T-ag OBP in the presence and absence of origin DNA Structural differences between the p21 apo and DNA-bound form of the T-ag OBD (aa 134–253) were identified by superimposing the two structures (Figure 2D, red and blue). The RMS deviation between the two forms was 0.59 Å with all (119) Cα atoms included. The biggest conformational change was detected in the DNA-binding loop (centered on alanine 151) and the loop containing Cys216 (C216 loop), which forms a disulfide bond in the apo structure. The conformational changes seen in the DNA-binding loop A1 were centered on phenylalanine 151 and involved residues 150 through 152. The maximal shift between the bound and free conformations for Phe 151 was 4.4 Å (Val150—3.7 Å; Ser152—1.7 Å). C216 forms a disulfide bond in one crystal form of the T-ag OBD. This residue has been implicated in T-Ag function; a mutant T-ag with C216G substitution was not functional in origin DNA unwinding, although the mutated protein bound the ori with wild-type affinity, oligomerized into hexamer, and even unwound an ori-containing linear DNA (Wun-Kim et al, 1993). C216 is on the surface of the OBD important for cooperative interactions between hexamers in assembly on the origin (Weisshart et al, 1999). Our observation that the C216 was capable of forming a disulfide bond perhaps suggests a mechanism for its importance. However, we do not believe that its ability to form a disulfide bond is relevant for DNA binding for the following reasons. First, the C216 region is on the opposite side of the DNA binding face of the OBD more than 15 Å away. Second, although the orientation of the loop that contains the C216 residue is different in our various T-Ag structures, the orientation does not correlate with DNA-binding (Figure 2D). In one of the crystal forms of the protein not bound to DNA, the C216 loop is in the same position as in the DNA-bound form. Thus, we can conclude first that the C216 loop is quite flexible and second that its position in the crystal structure is more a function of the crystallization conditions than whether the protein is bound to DNA or not. The DNA structure The DNA bends slightly around each OBD. The extent of the bending around binding sites 3 and 4 and sites 1 and 2 was equal and opposite; the sum of the four bends was equal to zero. The binding of the T-ag OBD alone to its origin therefore appears insufficient to alter the DNA structure and enable origin unwinding. It is also known that unwinding cannot be initiated by the helicase domain alone, confirming that origin melting likely results from an interplay among the ori, OBD, and helicase (Li et al, 2003). Nonspecific binding in the PEN-2 structure In the T-ag/PEN-2 complex structure, site 2 was mutated to ATgAT to create a palindromic DNA sequence more suitable for crystallization (Figure 3A). T-ag OBD was expected to bind to sites 1 and 3 because of the known affinity for these binding sites, and because of their co-localization on the same face of the DNA. Although a two-site origin cannot be melted by the T-ag double hexamer, it is sufficient for double hexamer assembly. We expected that the structure of the T-ag OBD bound to this element would provide insight into the protein–protein interactions that preceded double hexamer assembly. Surprisingly, T-ag OBD was bound not to the canonical GAGGC sequences in sites 1 and 3, but to an abutting non-canonical sequence—ATGGC (Figure 3B). Although we cannot exclude that this binding may be an artifact of crystallization, the PEN-2 crystal structure does reveal aspects of the plasticity of the DNA binding, a plasticity that might be exploited during origin unwinding. The general character of the non-canonical binding was similar to that observed in the binding to the consensus sequence. However, there was a significant difference in the interactions with the two 'non-canonical' bases (AT). In binding to this non-consensus site, Arg 154 moved away from A1 and formed a hydrogen bond with the phosphate group of this nucleotide (Figure 4B). Ser152, which makes a hydrogen bond with canonical A2 (Figure 4C), is oriented away and does not form an H-bond with the non-canonical T2 base (Figure 4D). In short, the crystallographic experiments showed that the T-ag OBD exhibits some structural plasticity in its interactions with DNA, enabling it to bind to non-consensus sequences, albeit with lower affinity. Model of SV40 ori bound by T-ag With the structures of the individual domains of the functional SV40 double hexamer in hand, as well as with the information about origin/T-ag interaction, we attempted to build an atomic model of the functional T-ag hexamer (putative intermediate form) on DNA (Figure 5). Our starting point was the PEN-4 structure (black), as this comprises the specific T-ag binding portion of a functional origin. In the crystals, symmetry-related DNA molecules (23 bp) were arranged in a pseudo-continuous DNA helix, enabling us to model an extended piece of DNA by adding two symmetry-related DNA molecules to the left-hand and right-hand side of the parent DNA. The length of the modeled DNA is 71 bp (23+1/1 overhang+23+1/1 overhang+23) with one base overhang on each side. The OBDs were positioned as found in the PEN-4 complex, with their C-terminal domains oriented towards the ends of the DNA. Two T-ag helicase hexamers (blue) were threaded on these DNA extensions and moved as close to OBDs as possible, but not so close as to make sterical clashes (PDB Id: 1N25) (Li et al, 2003). The N-terminal part of the helicase domain (residue 266) is oriented towards the C-terminus (residue 253) of the nearest T-ag OBD. In this orientation, the DNA is predicted to run through the central tunnels of the hexameric helicase. In the model, the T-ag double hexamer is predicted to contact 70–73 bp of DNA, which is in a good agreement with biochemical observation that T-ag protects 74 bp. The predicted location of the EP fragment melted by T-ag hexamer is inside the helicase domain, in proximity of the β-Hairpin which moves along the central channel and pulls DNA into the helicase for unwinding (Gai et al, 2004). Figure 5.Molecular model of an initial step in the SV40 DNA replication. The PEN-4 structure is shown in black, two hexameric helicase domains are in blue (PDB Id: 1N25), modeled DNA is shown as a stick model and colored per atom type (carbon in yellow, oxygen in red, nitrogen in blue, and phosphorus in purple). Position of the initially melted 8 nt fragment of EP with respect to the PEN box is highlighted with a yellow rectangle. Relative positions of the C-terminus in the OBD (aa 253) and N-terminus in the helicase domain (aa 266) are indicated. See text for more detail. Download figure Download PowerPoint Discussion A combination of the available structural and biochemical data suggests a model for origin unwinding. In the first step, four OBD domains bind to the four consensus sites in the origin (Joo et al, 1997) (Figures 2A and 6A). These binding events are thought to bring the associated helicase domains to the DNA and induce the assembly of the helicase domains into two hexamers around the DNA (Figures 5 and 6B). However, there are only four DNA-binding sites; yet, a total of 12 T-ag molecules must be recruited to form the two predicted hexameric helicases. Accordingly, the DNA-bound