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Journal of Virology, September 2008, p. 8849-8862, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00553-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evidence for a Structural Relationship between BRCT Domains and the Helicase Domains of the Replication Initiators Encoded by the Polyomaviridae and Papillomaviridae Families of DNA Tumor Viruses{triangledown} ,{dagger}

Anuradha Kumar,{ddagger} Woo S. Joo,{ddagger},§ Gretchen Meinke,{ddagger} Stephanie Moine, Elena N. Naumova, and Peter A. Bullock*

Department of Biochemistry, Tufts University School of Medicine, Boston, Massachusetts 02111

Received 12 March 2008/ Accepted 19 June 2008


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ABSTRACT
 
Studies of DNA tumor viruses have provided important insights into fundamental cellular processes and oncogenic transformation. They have revealed, for example, that upon expression of virally encoded proteins, cellular pathways involved in DNA repair and cell cycle control are disrupted. Herein, evidence is presented that BRCT-related regions are present in the helicase domains of the viral initiators encoded by the Polyomaviridae and Papillomaviridae viral families. Of interest, BRCT domains in cellular proteins recruit factors involved in diverse pathways, including DNA repair and the regulation of cell cycle progression. Therefore, the viral BRCT-related regions may compete with host BRCT domains for particular cellular ligands, a process that would help to explain the pleiotropic effects associated with infections with many DNA tumor viruses.


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INTRODUCTION
 
BRCT domains were discovered following characterization of the BRCA1 (breast cancer tumor suppressor protein) C terminus (58). Subsequent sequence-based genome searches revealed that BRCT domains are present in hundreds of additional proteins (57). The BRCT domains are frequently found as tandem repeats at the C termini of proteins; however, BRCT motifs also occur as monomers that are located at many different positions relative to the N and C termini (6, 46). Observations, such as those that demonstrated that the BRCT domains in BRCA1 are hot spots for oncogenic mutations (reviewed in reference 34), established the clinical importance of BRCT domains.

Members of the BRCT superfamily have been identified based on certain conserved sequence features; these include clusters of hydrophobic residues and two signature motifs (6) (discussed below). Structural criteria have also been used to define BRCT domains. For example, based on the structures of the BRCT repeats in the XRCC1 protein (120) and human BRCA1 (108), it was concluded that BRCT domains consist of a four-stranded parallel β-sheet flanked by two {alpha}-helices on one side ({alpha}1 and {alpha}3) and by a single {alpha}-helix ({alpha}2) on the other. However, there is some variability in terms of the structural features that define BRCT domains; for instance, it is not clear whether the poorly conserved {alpha}2 region is present in certain BRCT members (e.g., DNA ligase III [59] and the second BRCT repeat of rat BRCA1 [52, 59]). In addition, the BRCT repeats in 53BP1, a p53 interacting protein (47), have {alpha}-helical inserts between their β2 and β3 strands (i.e., {alpha}1') that are not found in other BRCT domains (52). Moreover, while the canonical BRCT domain contains a four-stranded parallel β-sheet, the REV1 protein is predicted to have only three parallel β-strands at the core of its BRCT domain (57).

The primary function of BRCT domains is to serve as scaffolds that recruit cellular proteins, many of which are involved in cell cycle checkpoints and DNA recombination, repair, and replication (reviewed in references 5, 6, and 46). Owing to the determination of several costructures, the residues in BRCT domains that serve as binding sites for individual protein ligands have been identified. For example, the surfaces on BRCT domains involved in binding to p53 (18, 52), {gamma}-H2AX (62, 97), and XRCC4 (21, 89) have been determined. In addition some BRCT domains are known to directly bind DNA, particularly DNA ends (57, 114, 115). Moreover, BRCT domains have also been implicated in transcriptional activation and chromatin unfolding (40, 111, 118). BRCT repeats also function as phosphopeptide binding modules during DNA damage-dependent signaling events (71, 82, 119), and costructural studies have helped to establish the nature of the interactions that take place between BRCT domains and particular phosphorylated ligands (12, 104, 109). Collectively, the analyses of BRCT domains bound to cellular ligands, such as that of the 53BP1 protein bound to the tumor suppressor protein p53 (18, 52, 64), have significantly extended our understanding of cellular transformation.

Fundamental insights into cellular transformation have also been obtained from the characterization of virally encoded oncoproteins (reviewed in references 26, 76, and 90). For example, studies of simian virus 40 (SV40) large T-antigen (T-ag) led to the identification of p53 (61, 67). In addition, the T-ags encoded by many polyomaviruses form complexes with the retinoblastoma (Rb) protein (16, 24). It is now possible to understand the molecular basis for T-ag's interactions with cellular proteins (e.g., p53 [64] and Rb [55]) because much of the structure of T-ag, including the J domain (55), origin binding domain (T-ag-obd [4, 70, 73, 74], and helicase domain [30, 63]) has been determined. Additional structures of viral oncoproteins, both alone and complexed to tumor suppressor proteins, have significantly enhanced our understanding of tumor formation (69).

According to the CATH system of classifying protein folds (78), the helicase domains within the initiators encoded by the Polyomaviridae and Papillomaviridae families contain a three-layered alpha-beta-alpha sandwich (37). More specifically, they contain a Rossmann fold (β-{alpha}-β-{alpha}-β) (83). Of interest, alpha-beta-alpha sandwiches are features of BRCT domains, as are Rossmann folds. Furthermore, DNA viruses are known to encode proteins that override DNA damage and checkpoint controls, pathways in which BRCT-containing proteins are known to be involved (98). Therefore, we examined whether characteristic features of BRCT domains are present within the initiators encoded by representative members of the Polyomaviridae and Papillomaviridae DNA tumor virus families; results from these studies are presented herein.


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MATERIALS AND METHODS
 
Three-dimensional (3D) alignments. (i) 3D structural coordinates of proteins. The following structures were downloaded from the Protein Data Bank (www.rcsb.org/pdb/) and used to create the alignments and figures in this paper: 1SVM, the helicase domain of T-ag in the ATP-bound conformation (30); 1KZY, the costructure of 53BP1 bound to p53 (52); 2H1L, the costructure of the T-ag helicase domain bound to the p53 DNA binding domain (64); 1CDZ, the DNA repair protein XRCC1 (120); 1WF6, DNA topoisomerase 2-binding protein 1 (TOPB1; T. Nagashima et al., unpublished data); 1TUE, the human papillomavirus type 18 [HPV18] helicase E1 bound to E2 (1); 2GXA, the E1 helicase of bovine papillomavirus [BPV] (25); 1U0J, adeno-associated virus type 2 (AAV2) Rep40 helicase (48); 1HEI, the hepatitis C virus RNA helicase (116); 1FNN, the eukaryotic initiation factor Cdc6 (68); 1PV4, the transcription termination factor Rho helicase (93); 1UBC, the bacterial recombination protein RecA (15); 1E0J, the phage T7gp4 helicase (92); 1IXS, the RuvB helicase (113); 1GL7, the bacterial conjugation protein TrwB (36); and 1NLF, Escherichia coli replication protein A (112). In addition to the structures of the BRCTs from 53BP1, XRCC1, and TopBP1 listed above, the remaining structurally solved BRCT domains listed in the Pfam database (http://pfam.sanger.ac.uk/family?acc=PF00533) were also downloaded and used in the structural and sequence alignments described in Fig. 4A.


Figure 4
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  		FIG. 4. Alignment of the amino acid sequences of 19 BRCT family members with those of the BRCT-related regions present in representative viral initiators. (A) The amino acid sequences of 19 BRCT family members are presented; the names of the individual proteins are listed to the left along with the corresponding residue numbers (h, human; r, rat). The secondary structural elements (based on the structure of the C-terminal BRCT of 53BP1; row 10) are presented at the top of the alignments. The Gly-Gly/Ala residues in motif 1 are highlighted in green. Motif 2 is situated in BRCT {alpha}3; residues at positions 1 and 5 in the W/F/Y-x-x-x-C/S consensus sequence are highlighted in orange. Residues located within the conserved hydrophobic clusters (i.e., M, V, I, L, W, F, Y, T, N, R, C, G, A, H, and P, as defined by Huyton et al. [46]) are highlighted in yellow. The conserved residues that comprise the phosphoserine binding pocket are highlighted in blue. Insertions of various sizes are present between β2 and β3, and the sizes of the insertions are given in parentheses; the {alpha}1' element of both 53BP1 BRCT repeats is located within this insertion. (B) Amino acid sequences of seven selected viral BRCT-related regions. The top line lists the sequences comprising the BRCT-related region in SV40 T-ag; the corresponding secondary elements are present above the sequence. The coloring of motif 1, surrogate motif 2, and the conserved hydrophobic clusters is identical to that in panel A. The extensive homology between SV40 T-ag and the T-ags encoded by BK virus (BKVT), JC virus (JCVT), and murine polyomavirus (MuPolT) enabled a similar alignment of their BRCT-related regions, performed using the program Clustal W (102). Also included are the sequences of the BRCT-related regions in HPV18 E1, BPV1 E1, and AAV2 Rep40. The structure-based homology studies indicate that the BRCT-related regions are disrupted between {alpha}9 and β4 by the indicated (*) sequences. Finally, the locations of sequences corresponding to the Walker A and B boxes and to the beta-hairpin are indicated.

(ii) Structural alignment of T-ag with the C-terminal BRCT domain in 53BP1. The Dali-Lite server (44, 45) was used to generate a preliminary structural alignment of the entire T-ag helicase domain (1SVM) with the C-terminal BRCT repeat (BRCT-b) of 53BP1 (1KZY chain C, residues 1868 to 1972). This superposition was visually inspected using the molecular graphics program PyMol (17). The structural alignment program LSQMAN (56) was then used to improve the superimposition between the two proteins. LSQMAN performs a best fit between the C{alpha}s of two structures via a least-squares superpositioning (56). Consecutive iterations of LSQMAN superpositions were performed until the root mean square deviation (RMSD) had been minimized. Data derived from this alignment, and from all of the additional alignments described in the following sections, are presented in Table S1 in the supplemental material.

(iii) Structural alignment of the BRCT domains in 53BP1 with other proteins. The process of structural and visual alignment was repeated for the following pairs of structures: T-ag helicase domain and the N-terminal BRCT repeat (BRCT-a) of 53BP1, HPV18 E1 helicase domain with the BRCT-b of 53BP1, BPV E1 helicase domain with the BRCT-b of 53BP1, and AAV2 Rep40 with the BRCT-b of 53BP1. The primary sequence alignments in Fig. 4B are based on these structural alignments (see Table S1, section A, in the supplemental material). Additionally, structures of the BRCT domains that have been determined (http://pfam.sanger.ac.uk/family?acc=PF00533) were aligned with the BRCT-b of 53BP1 using the Dali-Lite program. The primary sequence alignments of the structurally determined BRCTs in Fig. 4A are based on these structural alignments (see Table S1, section B, in the supplemental material).

Sequence alignments. Primary amino acid sequence alignments were created with Clustal X (101) and refined manually based on their structural alignments (performed as described above). Sequences analyzed included the 19 BRCT domains that have been structurally determined. The sequences of the structurally determined viral initiators of interest (i.e., SV40 T-ag, HPV18 E1, BPV E1, and AAV2 Rep40) were then aligned in a manner reflecting their structural homologies. Regarding the BK virus, JC virus, and murine polyomavirus sequences, whose structures have not been determined, their sequence alignments with SV40 large T-ag were performed with Clustal X. The final figure of the sequence alignments, Fig. 4, was generated using STRAP (33).

Site-directed mutagenesis. The vector pCMV-T-ag, containing sequences encoding wild-type SV40 T-ag, was previously described (7). The QuikChange site-directed mutagenesis kit (Stratagene) was used to mutate the Phe408 residue in SV40 T-ag, creating modified mutant vectors encoding valine, glycine, arginine, leucine, or aspartic acid at this position (F408V, F408G, F408R, F408L, and F408D, respectively). E. coli JM109 cells (Promega) were transformed with wild-type pCMV-T-ag, the mutated pCMV-T-ag vectors, or the empty parental pCMV-neo vector. Plasmid DNA was isolated from the JM109 cells using the Qiagen Plasmid Maxi kit and verified by DNA sequencing (84) prior to use in DNA transfections.

Protein level assays. U2OS cells (a human bone osteosarcoma cell line obtained from ATCC) were grown as recommended. A total of 10 µg of DNA (the pCMV-Tag or pCMV T-ag derivatives containing the F408 mutations described above) was transfected into a 70% confluent 100-mm plate of U2OS cells using the FuGENE-6 reagent (Roche Diagnostics). Transfection of the empty parental pCMV-neo vector served as a negative control. Forty-eight hours posttransfection cells were lysed in 50 mM Tris-HCl (pH 7.6), 300 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol, 1 mM Na orthovanadate, 50 mM NaF, a final 1x concentration of phosphatase inhibitor cocktail I (Sigma), and proteinase inhibitors (0.2 µg/ml of leupeptin and antipain). The protein concentration in each lysate was determined using a Bradford protein assay (Bio-Rad), and then equivalent aliquots were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. To determine the amount of T-ag in each lysate, Western blotting was conducted using the PAb 419 mouse monoclonal antibody followed by horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G and visualized with a chemiluminescent horseradish peroxidase substrate (Millipore). To confirm that equal amounts of protein were loaded in each well, the gel was also probed with anti-extracellular signal-regulated kinase (Sigma). Finally, the intensities of bands of interest were quantified using a FluorChem 8800 imager (Alpha Innotech).


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RESULTS
 
The T-ags encoded by the Polyomaviridae family contain helicase domains with BRCT-related folds. (i) Evidence for a BRCT-related fold in SV40 T-ag based on structural homology with a known BRCT domain. Since the helicase domains of the T-ags encoded by the polyomaviruses share certain architectural features with BRCT domains, we used the Dali-Lite server to compare the structure of a representative T-ag, encoded by SV40, with that of a canonical BRCT domain (the C-terminal repeat [BRCT-b] in 53BP1 [52]). The results of this preliminary structural alignment revealed that the SV40 T-ag helicase domain contains a subregion that is highly homologous to the BRCT-b of 53BP1. This initial structural alignment was further refined using the program LSQMAN (see Materials and Methods) (56). As can be seen from Fig. 1A, the fold of a region of the T-ag helicase domain (dark grey) (63), centered on the D2 subdomain (residues 397 to 550), is very similar to a representative BRCT fold (i.e., the BRCT-b of the 53BP1, residues 1868 to 1972 [18, 52]; magenta). Figure 1B presents a 90° rotation of Fig. 1A. For greater clarity, Fig. 1C presents just the relevant D2 subdomain of T-ag superimposed with the C-terminal BRCT repeat of 53BP1. Two regions of the T-ag D2 subdomain (63) that are structurally homologous to the BRCT fold (residues 401 to 445 and 522 to 548) are shown in blue. In the final superimposition of these two regions of T-ag with the BRCT-b domain in 53BP1, 52 residues were aligned with an RMSD of 2.4 Å and a Z score of 2.5 (see Table S1 in the supplemental material). Importantly, three β-strands within these regions of T-ag (β1, β4, and β5) align with three of the β-strands at the core of the 53BP1 BRCT domain (β1, β2, and β3). Furthermore, T-ag's {alpha}9 helix was closely aligned with BRCT {alpha}1 and T-ag's {alpha}12 aligned with a 310 {alpha}-helical loop in this BRCT, termed {alpha}1' (see the legend of Fig. 1). In addition, T-ag {alpha}8 is in close proximity to {alpha}3 in the BRCT-b of 53BP1. Finally, T-ag contains a 78-amino-acid "insert," extending from residues 446 to 521, that is not structurally homologous to the 53BP1 and interrupts the two regions of T-ag that do align with this representative BRCT.


Figure 1
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  		FIG. 1. Structural alignment of the BRCT-related region in the T-ag helicase domain with the C-terminal BRCT repeat in 53BP1. (A) A monomer of the T-ag helicase (63) (gray residues 251 to 627) was aligned (see Materials and Methods) with a canonical BRCT (magenta residues) present in the C terminus of the 53BP1 protein (52). The locations of T-ag subdomains D1 to D3 are indicated, as are reference secondary elements in T-ag (labeled in gray) and 53BP1 (labeled in magenta). (B) The identical alignment as in panel A but rotated 90°; the same reference structural elements are labeled. (C) A stereo figure of T-ag's D2 subdomain aligned with the C-terminal BRCT domain (BRCT-b) in 53BP1. The two regions of T-ag that align with the BRCT are blue, while the rest of the T-ag D2 subdomain, including the "insert," is gray. The 53BP1 BRCT-b is magenta. Individual structural elements from the T-ag helicase domain (63) and from 53BP1 (52) are indicated in gray and magenta, respectively. In the BRCT-b of 53BP1 (magenta) the c2 loop between β2 and β3 (120) has a 310 {alpha}-helical structure and is analogous to the helical loop in the first BRCT repeat (BRCT-a) of 53BP1, termed {alpha}1' (52). Hence, in the second BRCT repeat (BRCT-b) this c2 loop is also referred to as {alpha}1'.

(ii) Conserved features of the BRCT and T-ag helicase domains. It is apparent from Fig. 1A to C that a region within the T-ag helicase domain has an architecture, and thus a spatial arrangement of its secondary structural elements (79), similar to that of a canonical BRCT domain. Topology diagrams of BRCT-b in 53BP1 and the BRCT-related region in T-ag are presented in Fig. 2A and B. In BRCT-b of 53BP1, the secondary structural elements are connected in the order N'-β1-{alpha}1-β2-{alpha}1'-β3-{alpha}2-β4-{alpha}3-C' (Fig. 2A) (46). Of interest, T-ag elements β1, {alpha}9, β4, {alpha}12, and β5 align with elements β1, {alpha}1, β2, a1', and β3 of BRCT-b in 53BP1 (Fig. 2B) and are therefore referred to as BRCT related. However, the BRCT-related region in T-ag is disrupted between {alpha}9 and β4 by the previously described insertion and T-ag does not have structural elements corresponding to {alpha}2, β4, and {alpha}3 in the BRCT-b of 53BP1. Finally, the {alpha}-helix found at the {alpha}1' position in BRCT-b of 53BP1 is not a structural element that is typically found in BRCT domains. Nevertheless, the BRCT-related region of T-ag has an {alpha}-helix ({alpha}12) that aligns with this structural element.


Figure 2
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  		FIG. 2. Diagrams of the C-terminal BRCT (BRCT-b) of 53BP1 and the BRCT-related region in T-ag. (A) Colored in magenta is a topological map of the BRCT-b in the 53BP1 protein, depicting the standard N-β1, {alpha}1, β2, ({alpha}1'), β3, {alpha}2, β4, and {alpha}3-C arrangement in BRCT motifs (46) (secondary structures in parentheses are not features of canonical BRCT domains). Triangles indicate β-strands, and circles indicate {alpha}-helices. The approximate length of a given β-strand or {alpha}-helix is indicated by the correspondingly sized triangle or circle. The connecting lines that are drawn to these symbols indicate the approximate directions of the secondary structural elements; those drawn to the edge of the circle connect to the bottom of an {alpha}-helix, while those drawn to the center symbolize a connection to the top. Similarly, with the β-strands, connections drawn to the side of a triangle indicate a connection to the bottom of the β-strand, while connections drawn to the center of a triangle symbolize a connection to the top of the β-strand (106). In blue is a topology diagram depicting the BRCT-related region in the helicase domain of T-ag. The symbols are identical to those used to depict the BRCT-b in the 53BP1 protein. The gray symbols represent T-ag elements (i.e., β2, {alpha}10, β3, {alpha}11, and the β3'-β'' hairpin) that disrupt the BRCT-related region. Finally, in both figures, the green and orange balls represent motifs 1 and 2, respectively. (B) Diagram depicting the relationship between the 53BP1 BRCT-b domain (top line) and the BRCT-related region in T-ag. The residues identified as homologous by the LSQMAN alignment are boxed. T-ag lacks some of the canonical BRCT elements (i.e., {alpha}2, β4, and {alpha}3) and contains an insertion (consisting of β2, {alpha}10, β3, {alpha}11, and the β3'-β3" beta-hairpin) between {alpha}9 and β4. As in panel A, the green and orange spheres symbolize BRCT motifs 1 and 2, respectively. Also indicated within T-ag are the relative locations of the Walker A (A) and Walker B (B) motifs.

In view of the structural evidence indicating that T-ag contains a domain that is related to the BRCT domain in 53BP1, it was of interest to determine if similar alignments could be made with other BRCT domains. It is apparent from Fig. 3 that the BRCT-related region in T-ag is structurally homologous to additional BRCT domains, the expected result given that there is only slight variability in structure between different BRCT domains (59). In summary, these analyses revealed that there is significant structural homology between BRCT family members and a region of the T-ag helicase domain.


Figure 3
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  		FIG. 3. Structural alignment of BRCT domains. BRCT domains were structurally aligned to the BRCT-b of 53BP1 (magenta) and displayed in a ribbon representation. The structurally aligned BRCT-related region of T-ag is shown in blue and the insert region in grey. This alignment was described earlier and shown in Fig. 1. The other aligned BRCT domains are shown in various shades of pink to purple (see the key). Finally, selected secondary structural elements are labeled.

(iii) Further evidence for a BRCT-related region based on primary amino acid sequence alignments. (a) Introduction. While the topologies of BRCT domains are conserved, their amino acid sequences are highly divergent (46). Indeed, there is no one single amino acid residue that is absolutely conserved within the superfamily. However, certain sequence motifs have been described. Motif 1 consists of a conserved Gly-Gly/Ala pair (situated at positions termed P1 and P2) that is thought to provide flexibility for a tight turn between BRCT {alpha}1 and β2 (reviewed in reference 46). Motif 2 is located within BRCT {alpha}3; its consensus sequence consists of a Trp or closely related Phe or Tyr residue, which is usually followed by three small amino acids and then often a Cys or Ser residue (i.e., W/F/Y-x-x-x-C/S; where the W/F/Y residue is defined as position 1 [P1] and the C/S residue as P5 [see references 5, 6, 35, and 46 and references therein]). Structural studies of BRCT domains established that the bulky hydrophobic side chain (W/F/Y) projects into the core of the BRCT domain and interacts with hydrophobic residues in the β-sheet, thereby making the bulky hydrophobic residue in motif 2 integral to the stability of the BRCT fold (107). Additionally, a number of conserved hydrophobic residues are features of BRCT family members (6, 46). Therefore, it was of interest to determine whether these conserved features are present within the amino acid sequence of the BRCT-related region of the T-ag helicase domain.

(b) Structure-based alignments of amino acid residues. Having established the relationships between the secondary structural elements, it was possible to compare the amino acid sequences of representative BRCTs with the homologous region in T-ag and related viral initiators and to determine if the canonical BRCT sequence features were present. The primary sequence alignment of a representative group of 19 BRCT domains, whose structures have all been determined, is shown in Fig. 4A. The secondary structural elements of the BRCT domains are presented above the sequence alignments. The structurally based sequence alignments of the viral initiators are presented in Fig. 4B. The secondary structural elements in the BRCT-related region of T-ag are presented above the T-ag sequence. Furthermore, the T-ags encoded by the human pathogens BK and JC viruses, as well as polyomavirus, show a high sequence identity with SV40 T-ag in the BRCT region (70 to 80%) (28, 103). While the structures of the BK virus, JC virus, and polyomavirus T-ags have not been determined, their extensive sequence homologies indicate that the structures of their helicase domains are very similar to that of SV40 (10). Therefore, the corresponding amino acids of the BK virus, JC virus, and polyomavirus T-ags are positioned underneath the SV40 T-ag sequences to which they aligned. Also presented in Fig. 4B are structure-based alignments of the E1 proteins encoded by HPV18 and BPV1, as well as AAV2 Rep40. (Viral initiators that are not among the Polyomaviridae are considered in a subsequent section).

Figure 4A shows BRCT motif 1 (highlighted in green); as previously noted, this motif is generally a Gly-Gly/Ala pair located immediately following alpha helix 1. As can be seen from Fig. 4B, T-ag contains a Gly-Gly pair following the analogous {alpha}9 element. Motif 2 is situated within BRCT {alpha}3; the bulky hydrophobic (i.e., Trp, Phe, or Tyr) and Cys or Ser residues in the W/F/Y-x-x-x-C/S consensus sequence are highlighted in orange. As previously noted, T-ag is missing the three structural elements, including {alpha}3, that comprise the C' termini of BRCT domains. Thus, based on the amino acid sequence alignment, there is no direct evidence for motif 2. Nevertheless, since the BRCT {alpha}3 element bisects the {alpha}8 element in T-ag (Fig. 1 and 5), it is of interest that a homologue of the consensus sequence for motif 2 is present in T-ag {alpha}8 (T-ag contains a Phe residue in place of the more common Trp, followed by two small residues and then a Cys [F-x-x-C]; therefore, we have termed this "surrogate motif 2"). Moreover, when the sequences from the other polyomavirus family members are considered, it is clear that they contain, at the correct positions, many of the residues that match the consensus for motifs 1 and 2. However, in BK virus, only a single Gly was detected in motif 1. Furthermore, while murine polyomavirus contains a motif 1 homologue and a Phe two residues N-terminal to the "expected position" for surrogate motif 2, there is no evidence for the Cys/Ser residue.


Figure 5
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  		FIG. 5. Structure alignments used to establish the relative locations of known BRCT features within the T-ag BRCT-related region. (A) A slabbed, internal view of the LSQMAN alignment of SV40 T-ag with the C-terminal BRCT repeat (BRCT-b) of 53BP1 (as in Fig. 1C). For reference, 53BP1 {alpha}1 and T-ag {alpha}9 are indicated. The Gly-Gly pairs comprising motif 1 in 53BP1 and T-ag are shown as light and dark green spheres, respectively. (B) A view of the same alignment highlighting residues comprising motif 2 (W/F/YxxxC/S). The positions of {alpha}3 in 53BP1 and {alpha}8 in T-ag are indicated. The core hydrophobic residues at 53BP1 position 1 in motif 2, W1946 and T-ag F408, are shown as orange and red sticks, respectively. The conserved Cys1950 of 53BP1 and Cys411 of T-ag are shown as orange and red spheres, respectively. (C) Alignment of the N-terminal repeat (BRCT-a) of 53BP1 with large T-ag (see Materials and Methods), demonstrating that residues in the 53BP1 BRCT domain known to coordinate phosphoserine/threonine residues (i.e., T1737 and M1738, shown as brown sticks) are spatially related to those in the Walker A motif of T-ag (i.e., S430 and G431; shown as yellow sticks).

Following the initial identification of BRCT domains via one-dimensional alignments with the BRCA1 sequence, Callebaut and Mornon (6) extended the identification of BRCT domains by employing an algorithm designed to locate conserved hydrophobic patches (hydrophobic cluster analysis). Indeed, because of the low primary sequence identity between BRCT domains, the detection of these conserved hydrophobic patches is an important criterion for identification of BRCT domains (6). In the sequence alignment shown in Fig. 4A and B, these hydrophobic patches are highlighted in yellow. It is clear that the BRCT-related region within the T-ag helicase domain contains the conserved clusters of hydrophobic residues. Moreover, it is apparent from Fig. 4A and B that the hydrophobic patches are largely features of the β-sheets. Thus, most of these residues are located in the interior of the helicase domain. Finally, it is of interest that the hydrophobic patches are present in the other polyomavirus family members.

An additional distinguishing feature of BRCT repeats is that some of them are known to function as phosphoprotein recognition motifs, particularly when arranged as tandem repeats. Peptide selection studies established that BRCT domains bind specifically to peptides containing a pSer-X-X-Phe motif (71, 82, 119). Structural studies (12, 104, 109) demonstrated that a phosphoserine binding pocket is located in the BRCA1 BRCT N-terminal repeat that provides ligands necessary for coordination of the phosphate oxygen atoms. Related studies established that the phenylalanine residue is recognized by a hydrophobic groove that is situated at the interface between the N- and C-terminal BRCT repeats. The amino acid residues that comprise the phosphoserine binding surface in a number of BRCT domains (35) are Ser/Thr-Gly in the β1-{alpha}1 loop and Thr/Ser-X-Lys in {alpha}2 (Fig. 4A; shaded in cyan). Interestingly, the Walker A box in the viral initiator proteins, used to bind adenine nucleotides (85), is situated between T-antigen β1 and {alpha}9 (63), and it contains the Ser/Thr-Gly consensus motif (Fig. 4B). However, since the viral initiators lack an element equivalent to {alpha}2, there is no evidence for the Thr/Ser-X-Lys portion of the pSer recognition motif. Moreover, there is no evidence for a second BRCT repeat in the viral initiators; thus, no data were obtained regarding the hydrophobic groove situated between two tandem BRCT repeats or the side chains that are needed for coordination of the phenylalanine.

(iv) The relative spatial positions of motif 1, surrogate motif 2, and those utilized for phospholigand binding within the BRCT-related region in T-ag. The studies presented in Fig. 4A and B enabled the identification of amino acids in SV40 T-ag and related viral initiators that aligned with those in motifs 1 and 2 in known BRCT domains. They also suggest the possibility that the Ser/Thr-Gly motif, present in BRCT domains and involved in phosphoserine binding, is related to the adenine nucleotide binding Walker A motif in T-ag. To extend these analyses, we determined whether residues in individual motifs in the T-ag helicase structure are aligned in space with those in a representative BRCT domain (i.e., the C-terminal BRCT repeat [BRCT-b] in the 53BP1 protein). Based on the data presented in Fig. 4, T-ag residues Gly444 and Gly445 are analogous to those in motif 1 in BRCT domains. It is clear from Fig. 5A that glycines 444 and 445 lie in close proximity to the residues that comprise motif 1 in the representative BRCT domain from 53BP1 (light green spheres). As with other BRCT structures (120; reviewed in reference 46), the position of the Gly-Gly pair within the putative BRCT-related region of T-ag is located at the end of {alpha}9, enabling a sharp turn prior to the following β-strand.

Motif 2 is situated in BRCT element {alpha}3 (Fig. 5B); while T-ag does not have a direct homologue of this element (Fig. 4B), the T-ag {alpha}8 helix is in approximately the same spatial position as {alpha}3 in the BRCT-b of 53BP1 (Fig. 1C). However, as seen in Fig. 5B, these two helices are oriented at divergent angles. As a result, many of the residues in T-ag {alpha}8 do not spatially align with those in {alpha}3 of the BRCT-b of 53BP1. Nevertheless, the conserved bulky hydrophobic residue in the P1 position in 53BP1 motif 2, Trp1946, is in a similar spatial position as T-ag Phe408 in surrogate motif 2; indeed, the side chain of Phe408 is in close proximity to the side chain of Trp1946 (Fig. 5B, orange ring). This is of interest, given that the bulky hydrophobic amino acids in motif 2 are among the most highly conserved residues in BRCT domains (reviewed in reference 46), and they play a pivotal role in ensuring the stability of the BRCT fold (107). Moreover, given the W/F/Y-x-x-x-C/S consensus sequence for motif 2, it is of interest that T-ag residue Cys411 (side chain shown as red spheres) is in the same relative orientation to the P1 hydrophobic residue as Cys1950 of 53BP1 motif 2 (side chain shown as orange spheres) (Fig. 5B).

The S/T-G residues in the loop following β1 frequently form a portion of the phosphopeptide binding pocket in BRCT domains (Fig. 4A). In the 53BP1 BRCT (fifth row in Fig. 4A), a T-M pair substitutes for the S-G residues (52). Figure 5C shows the T-M residues of the 53BP1 BRCT phosphopeptide binding pocket (brown sticks); the SV40 T-ag residues that are comparable based on the sequence alignment, Ser430-Gly431, are shown as yellow sticks. In 53BP1, Thr1737 is directly coordinated to the phosphate group (52). In contrast, T-ag residues Ser430 and Gly431 are close to the bound ATP (the OH group of Ser 430 is ~3.5 Å from an oxygen in {gamma}-Pi [30]), but they do not directly contact the phosphates. Instead, T-ag residue Lys432 is coordinated to both {gamma}-Pi and β-Pi, while Thr433 and Thr434 make additional contacts to the triphosphate (30). Thus, residues in the T-ag Walker A box are in similar spatial positions to the BRCT residues needed for binding to the pSer portion of the pSer-X-X Phe motif; however, they are not identical, and thus their evolutionary relationship is equivocal. Nevertheless, the analyses presented in Fig. 4A and B and 5A and B indicate that many of the other sequence, topological, and structural features that define BRCT domains are also features of the helicase domains of T-ags encoded by the Polyomaviridae family.

Testing whether BRCT-based alignments position the DNA binding domain of p53 near its known binding site on T-ag. 53BP1 contains a tandem repeat of BRCT domains, and the costructure of the p53 DNA binding domain (DBD) bound to 53BP1 established that the N-terminal BRCT repeat and the linker region of 53BP1 constitute the binding surface for p53 (Fig. 6A1; PDB: 1KZY). The recent p53 DBD/T-ag helicase domain costructure (64) also identified the T-ag residues that interact with the p53 DBD (Fig. 6A2; PDB: 2H1L). These costructures enabled us to evaluate whether a BRCT-based structural alignment would correctly dock the p53 DBD to its binding site on T-ag.


Figure 6
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  		FIG. 6. Testing whether a BRCT-based alignment correctly places the p53 DNA binding domain on its known binding site on T-ag. (A) X-ray costructures containing the p53 DBD, depicted as ribbon diagrams. (A1) Costructure (PDB: 1KZY) of the p53 DBD (yellow), bound to 53BP1 (the N- and C-terminal BRCT domains are colored green and dark pink, respectively). (A2) Costructure (PDB: 2H1L) of the p53 DBD (pale blue), bound to the helicase domain of T-ag (gray; except for the BRCT-related regions, which are blue). (B) Results of the C-terminal and N-terminal BRCT-based docking experiments. In panels 1 and 2 the p53/T-ag costructure is shown in the same orientation as in panel A. The T-ag helicase domain is depicted as a ribbon diagram with a translucent surface (gray), and the structure of p53 derived from the X-ray costructure is shown in a ribbon representation (pale blue). The position of p53 from the docking experiments with the C-terminal (B1) or N-terminal (B2) BRCT of 53BP1 is shown as pale pink and pale green ribbons, respectively. For clarity, the structure of the 53BP1 BRCT tandem repeat is omitted from this view. The N and C termini of the T-ag helicase domain are labeled. (C) The p53 DNA binding domain interaction surface on T-ag: crystallographic versus predicted. Each of these three images shows a T-ag helicase domain monomer as a surface rendering (gray); the orientation of T-ag is rotated approximately 90° from the view shown in panel B. (C1) View showing the BRCT-related region of T-ag (dark blue) relative to the p53 interaction surface seen in the p53/T-ag costructure (pale blue). Also shown are four residues on T-ag identified by mutational studies that are important for binding full-length p53 (red). (C2) View showing the predicted p53 binding surface (pale pink), derived from the C-terminal docking experiments, overlaid on the p53 binding surface seen in the p53/T-ag costructure (pale blue). (C3) View showing the predicted p53 binding surface (pale green), based on the N-terminal docking experiments, overlaid on the p53 binding surface seen in the p53/T-ag costructure (pale blue).

Although the C-terminal BRCT repeat of 53BP1 does not contact the p53 DBD, it has greater structural homology with T-ag (based on the Dali-Lite and LSQMAN alignment scores; see Table S1 in the supplemental material); therefore, it was used in the initial "docking" studies. The BRCT-related region in T-ag (Fig. 6A2) was superimposed onto the C-terminal BRCT domain present in the 53BP1/p53 DBD costructure (Fig. 6A1) via the previously described LSQMAN alignment (see Materials and Methods). This superposition is presented in Fig. 6B1, with 53BP1 omitted for clarity. This figure shows that the BRCT-based structural alignment "docked" the p53 DBD (pink) onto the same binding face as the p53 DBD (blue) in the p53 DBD/T-ag costructure.

Given that the N-terminal BRCT makes more contacts with the p53 DBD, the analogous docking experiment was performed using the N-terminal BRCT of 53BP1. The results of this analysis are presented in Fig. 6B2 and reveal that the p53 DBD (pale green) is docked on a surface of T-ag that is very similar to that of the p53 DBD observed in the X-ray costructure (pale blue). While these BRCT-based docking methods both predict that the p53 DBD will bind the same face of T-ag, it is important to note that the orientations of the docked p53 DBD molecules are different (Fig. 6B1 and B2).

To more clearly demonstrate the results of this study, Fig. 6C presents three views of a surface rendering of a monomer of the T-ag helicase domain (grey) in which the residues that comprise the BRCT-related region, the binding surface for the p53 DBD as seen in the crystal structure, and those predicted by the "docking" studies, are colored differently. For ease of comparison, the T-ag residues that directly bind the p53 DBD as determined by the p53/T-ag costructure (64), are shown in each panel of Fig. 6C (pale blue). Also indicated in each of the three views are four residues (red) (Pro399, Asp402, Cys411, and Pro584) that, when mutated, abrogate binding with full-length p53 (54, 66). In Fig. 6C1, the residues that comprise the BRCT-related region of T-ag are shown (dark blue). The residues predicted to be part of the p53 binding surface, based on the C-terminal (pale pink) or N-terminal (pale green) BRCT docking studies, are presented in Fig. 6C2 and C3, respectively. Of considerable interest, both the C- and N-terminal BRCT-based docking procedures placed the p53 DBD close to the crystallography-determined binding site on T-ag; the degree of overlap between the predicted binding sites (pale pink and pale green) and the crystallographic binding surface (pale blue) are presented in Fig. 6C2 and C3, respectively.

The N-terminal BRCT-based alignment results in an interaction surface that includes a portion of the T-ag BRCT-related region. In addition, the "predicted" interaction surface overlaps partially with the location of mutants that are known to disrupt p53 binding (Fig. 6C; red). However, the p53 DBD does not make many contacts to the BRCT-related T-ag domain, either in the p53/T-ag costructure or the "docked" p53/T-ag models (Fig. 6C). The failure to achieve an exact alignment with the structurally determined binding site for the p53 DBD may reflect, in part, that 53BP1 and T-ag interact with the p53 DBD in slightly different manners. T-ag binds to the entire DNA binding surface of p53 (packing against three major DNA binding elements in p53, {alpha}-helix 2, loop 2, and loop 3 [9, 64]). In contrast, portions of the p53 DNA binding surface are exposed in the p53/53BP1 costructure (18, 52). Thus, the p53 DBD/53BP1 costructure is not a perfect model for predicting the p53 DBD/T-ag interface.

Evidence for the presence of BRCT-related regions in the initiators encoded by additional DNA viruses. Based on our studies of the T-ags encoded by the Polyomaviridae family, it was of interest to address whether the initiators encoded by other DNA viruses contain BRCT-related regions. The structures of the E1 helicase domains from HPV18 (1) and BPV1 (25) (Papillomaviridae family) have been determined. The structure of the AAV initiator Rep40 has also been reported (48, 49); therefore, while AAV is not a DNA tumor virus, Rep40 was also included in our analyses.

(i) Evidence for BRCT-related folds based on structural homologies. Additional LSQMAN-based structural homology studies of the C-terminal BRCT (BRCT-b) of 53BP1 and the E1 proteins encoded by HPV18 and BPV1, as well as AAV2 Rep40 (alignments not shown), were conducted. Inspection of Table S1 in the supplemental material (section A, row 2) reveals that a region of HPV18 E1 aligned with the 53BP1 BRCT-b for 42 residues with an RMSD of 2.6 Å and a Z score of 1.4. A similar analysis of BPV1 E1 established that 41 residues aligned with the 53BP1 BRCT-b with an RMSD of 2.3Å and a Z score of 1.5 (see Table S1, section A, row 3, in the supplemental material). For AAV, 46 residues aligned with an RMSD of 3.2 Å and a Z score of 0.8 (see Table S1, section A, row 4, in the supplemental material). Although these scores are poorer than those for the T-ag alignment, these analyses raise the additional possibility of a BRCT-related region in these proteins. In addition, as with T-ag, these BRCT-related regions are disrupted by analogous insertion regions (Fig. 4B).

(ii) Amino acid sequence alignments. The 3D-based alignments were used to generate the primary amino acid sequence alignments of HPV18 E1, BPV1 E1, and AAV2 Rep40 with a representative set of known BRCTs (Fig. 4B). As with SV40 T-ag, these amino acid alignments were then analyzed for the presence of the conserved BRCT sequence features. It is apparent from Fig. 4B that the HPV18 E1, BPV1 E1, and AAV2 Rep40 helicase domains contain the conserved hydrophobic patches (highlighted in yellow). Moreover, HPV18 E1 contains a Gly-Ala pair that aligns with those in motif 1, while BPV1 E1 contains a Gly-Gly pair. However, the sequence alignment of AAV2 Rep40 does not reveal a similar pair of residues that usually define motif 1 in BRCT domains. Furthermore, relative to that for the SV40 T-ag, the primary sequence alignments provided less evidence for the motif 2 consensus sequence (W/F/Y-x-x-x-C/S) in HPV18 E1, BPV1 E1, and AAV2 Rep40. For example, while all three of the viral helicases contain potential bulky hydrophobic groups at position 1, none of them contain the Cys/Ser portion of the consensus sequence. However, at the primary sequence level, certain previously identified BRCT domains also contain little evidence for a completely intact motif 2 (5, 57).

In view of the primary amino acid sequence alignments presented in Fig. 4B, additional structural analyses were conducted and viewed in terms of evidence for motif 1 and surrogate motif 2 in the HPV18 E1, BPV1 E1, and AAV2 Rep40 initiators. More specifically, we analyzed whether the conserved sequence motifs in the viral helicase domains localize in space with the corresponding motif in the BRCT-b of 53BP1. The HPV18 E1 Gly503-Ala504 pair had a spatial position similar to that of the residues comprising motif 1 within the BRCT-b in 53BP1 (Gly1895-Gly1896; superimpositions not shown). Furthermore, BPV E1 residues Gly451 and Gly452 were in positions similar to those in motif 1 in the BRCT-b of 53BP1. However, as expected from the sequence alignment of AAV2 Rep40 (Fig. 4B), there was no evidence for residues related to motif 1. Regarding motif 2, the E1 helicase domains from HPV18 and BPV1, as well as Rep40 from AAV2, have bulky hydrophobic residues, either Trp or Phe, at positions analogous to Trp1946 (i.e., the P1 residue in motif 2 of the BRCT-b domain in 53BP1 [data not shown]). However, both the sequence and structural alignments revealed that the Cys/Ser residue at the P5 position of surrogate motif 2 is not present in any of these viral initiators. It is noted, however, that the Cys/Ser residue is not as highly conserved as the bulky hydrophobic group (Fig. 4A; see Table S3, panels D and E, in the supplemental material) (6, 46).

Testing whether protein levels are altered by mutations introduced into T-ag surrogate motif 2 at the P1 position. In view of the analyses presented in Fig. 1 to 5, we initiated tests of the hypothesis that a BRCT-related region is present in the helicase domain of T-ag. Previous mutational studies established that the bulky hydrophobic residue in motif 2 is a critical structural component of the BRCT fold (23, 107). Based on these earlier studies, it was concluded that residues which preserve the hydrophobicity of the P1 residue maintain the BRCT fold (23, 95, 99), while the BRCT fold is disrupted by nonhydrophobic residues (107). Therefore, the P1 analog of surrogate motif 2 in T-ag, Phe408, was mutated to Val, Leu, Gly, Arg, or Asp (see Materials and Methods). The residues introduced at this internally located position (63) were designed to conserve the hydrophobic core (F408V and F408L), as well as disrupt it via the introduction of a charged residue (F408R and F408D) or a small residue (F408G) that creates an empty pocket.

In one series of experiments, we analyzed the protein levels of the Phe408 mutants upon expression in U2OS cells in the absence (Fig. 7) or presence of cycloheximide (data not shown; the results, however, were nearly identical to those in Fig. 7). These studies, which reflect protein stability, revealed that those mutations that preserved the hydrophobic core of the protein (i.e., F408V and F408L) had no significant effect on protein levels (lanes 4 and 5), a result consistent with previous studies of BRCT domains (23, 95). It is also apparent from Fig. 7 that the F408G and F408R mutations caused destabilization of T-ag (lanes 6 and 7, respectively). The analogous mutations in the N- and C-terminal BRCT repeats of BRCA1 (i.e., at W1718 and W1837, respectively) also led to protein instability (107). The F408D mutation had a dramatic effect on T-ag stability (lane 8). In contrast, although predicted to disrupt correct folding, the W611D mutation in XRCC1 was stable (99). Thus, when known BRCT domains were mutated at motif 2 position 1, they had stability profiles that were very similar, but not identical, to those of T-ags containing the same mutations at position F408. Moreover, in vivo replication assays showed that the stable T-ag mutants (e.g., F408V) supported DNA replication at wild-type levels (data not shown). Thus, a mutation strategy based on those used to demonstrate the essential role of the P1 residue in BRCT stability (95, 99) revealed that a hydrophobic residue at position F408 is essential for T-ag's stability and its ability to catalyze SV40 DNA replication.


Figure 7
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  		FIG. 7. Determination of the stability of T-ag as a function of mutation of residue F408. (A) U2OS cells were transfected with pCMV-T-ag or pCMV-T-ag derivatives containing one of the F408 mutations. Forty-eight hours posttransfection, lysates were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting to visualize wild-type and mutant T-ag levels. As a positive control, 200 ng of purified T-ag was loaded in lane 1. Lane 2 contained 300 µg of protein lysate from U2OS cells transfected with the pCMV vector. Lanes 3 to 8 contained 300 µg of total protein lysate from U2OS cells that expressed either wild-type (WT) T-ag or the F408V, F408L, F408G, F408R, and F408D mutants, respectively. As a protein loading control, the lower quarter of any given gel was removed and blotted for total extracellular signal-regulated kinase (Erk). (B) Quantification of three identical experiments for expression of wild-type T-ag or the F408 T-ag mutants.


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DISCUSSION
 
Since the initial characterization of BRCT domains in the BRCA1 protein, hundreds of additional BRCT domains have been identified (57). Structural studies have determined that the BRCT fold generally consists of a four-stranded parallel β-sheet flanked on one side by a pair of {alpha}-helices ({alpha}1 and {alpha}3) and on the other side by {alpha}2 and in isolated cases {alpha}1'. These secondary structural elements are arranged, topologically, into a Rossmann fold. In addition, certain sequence features define BRCT domains; these include motifs 1 and 2 and the conserved hydrophobic residues that form the core of the BRCT fold. Therefore, it is of interest that the helicase domains of the viral initiators examined herein contain regions that are structurally homologous to BRCT domains. Moreover, our studies demonstrate that the viral BRCT-related regions also contain many of the sequence motifs that define BRCT domains, for example, the conserved hydrophobic patches (6, 46) and those present in motif 1 (Gly-Gly/Ala). At the sequence level, the evidence for motif 2 (i.e., W/F/Y-x-x-x-C/S) is, however, more equivocal. Nevertheless, the viral initiators contain, in the same spatial positions, many of the residues that correspond to BRCT motifs 1 and 2. Furthermore, statistical analyses presented in the supplemental information indicate that the frequencies at which the consensus amino acids occur within motifs 1 and 2 in known BRCT domains are very similar to those at which they occur within motif 1 and surrogate motif 2 in the viral initiators. There is less evidence, however, that they are present in a control group of structurally solved AAA+ proteins, all of which contain Rossman folds (see Tables S2 and S3 in the supplemental material).

A view of the BRCT-related region, in the context of a T-ag hexamer, is presented in Fig. S1 in the supplemental material. Of interest, much of the BRCT-related region is exposed in a hexamer of the T-ag helicase domain. Thus, much of this surface would be available for interactions with cellular proteins, such as p53. In addition, BRCT domains are known to bind to DNA. Therefore, it is of further interest that the surface of the T-ag helicase domain makes extensive contacts with DNA (see reference 60 and references therein). Collectively, these observations indicate that BRCT-related regions are present in the initiators encoded by the Polyomaviridae and Papillomaviridae DNA tumor viruses. Of additional interest, AAV2 Rep40 contains a domain that is structurally homologous to BRCT domains. However, since it lacks the motif 1 consensus sequence, there is less evidence that Rep40 contains a BRCT-related region.

Based on our analyses, the BRCT-related regions in the viral initiators are disrupted by the insertion containing the β2, {alpha}10, β3, {alpha}11, and β3'-β3'' beta-hairpin elements (Fig. 4B). The insertions in the viral initiators are situated between {alpha}9 and β4, an interesting location given that BRCT domains tolerate insertions of considerable length between {alpha}1 and β3 (blocks B and C in reference 6), frequently between β2 and β3 (Fig. 4A). While the exact origin of the "insertion sequences" is not known, BLAST searches show they are present in many AAA+ proteins (data not shown). Of interest, the insertions contain residues that contribute critical functions to the viral initiators, for example, the Walker B box (91) and the beta-hairpin (63, 80). Based on these observations, it is possible that an important step in the formation of the viral initiator proteins was the insertion of residues from a AAA+ element into a BRCT repeat. According to this hypothesis, the Mg2+-binding Walker B box would enable the hydrolysis of ATP, while the beta-hairpin would enable a number of nucleotide-dependent DNA binding events (1, 25, 29, 60, 86, 87). It is apparent, however, that this hypothesis requires additional testing.

One of the advantages of studying viral initiators, particularly SV40 T-ag, is the wealth of mutational data that are available for consideration. Therefore, the T-ag mutational data were analyzed in terms of the hypothesis that a BRCT-related region is present within the helicase domain. Mutational studies of the T-ag residues that constitute motif 1, Gly444 and Gly445, have not been reported (81). Regarding motif 2, a destabilization of the BRCT fold and a concurrent loss of the BRCT's ligand-binding ability resulted when the hydrophobic Trp/Phe/Tyr residue was mutated to a less hydrophobic residue (23). In T-ag the conserved hydrophobic residue at position 1 of surrogate motif 2 is Phe408; a Phe408Tyr mutation was previously analyzed, but it did not have any effect on the ability of T-ag to form plaques or much effect on T-ag's ability to bind p53 (65). However, some BRCT domains are known to have a Tyr at this position (6) (see Tables S2 and S3 in the supplemental material), and therefore the putative BRCT may not have been significantly altered by this T-ag mutation. Indeed, our experiments demonstrated that T-ag is stable when hydrophobic substitutions are introduced at this position, a finding that is consistent with reports that a hydrophobic residue at this position is essential for the integrity of BRCT domains (23, 107). Furthermore, T-ag residue Cys411 corresponds to the P5 position within motif 2. Interestingly, the Cys411Arg mutant was characterized and was determined to be defective in p53 binding (66), a result in keeping with the loss of the p53 binding site (Fig. 6B). Lastly, mutations in T-ag in the residues situated in the hydrophobic patches (Fig. 4; yellow residues) have not been reported.

The other viral initiators have been subjected to fewer mutational studies. Nevertheless, a residue in BRCT-related motif 1 of HPV16 E1 was mutated (Gly496Arg) and the mutant was determined to be defective in terms of its ability to support DNA replication (117). Moreover, it was found to be defective for binding to Ubc9 and E1BP and slightly defective for oligomerization (117). In addition, the Gly496Arg mutation reduced E1's ability to interact with E2 (117). The costructure of the HPV18 E1 and E2 proteins revealed that the equivalent residue (Gly503) is not a component of the E2 binding site (1). Given that motif 1 enables a tight turn between {alpha}1 and β2, Gly496 in HPV16 E1 may play a structural role in the overall fold of this subdomain, an observation that would help to explain why the HPV16 E1 Gly496Arg mutation caused a defect in ligand binding. It will be of interest to determine the phenotypes of additional viral initiators that have mutations at residues analogous to those corresponding to motifs 1 and 2 in the BRCT-related regions.

In view of the observations presented herein, it is worth considering the selective advantage that a DNA tumor virus, such as SV40, would acquire by having a BRCT-related region within its initiator. It is now well established that SV40 transformation depends upon the interactions between T-ag and the p53 and Rb proteins (see, e.g., references 38 and 39; reviewed in references 2, 27, and 90). Therefore, it is of interest that BRCT-based alignments placed the p53 DNA binding domain very close to its known binding surface on T-ag (Fig. 6). This observation raises the possibility that a p53/BRCT interaction contributed to the evolutionary events that led to the current association between p53 and T-ag. In addition, it is not clear if inactivation of p53 and Rb proteins accounts for the full transformation potential of T-ag (2). Thus, transformation may depend upon additional, as yet undefined, interactions between T-ag and cellular proteins, some of which may be dependent on the BRCT-related region.

Furthermore, SV40 T-ag catalyzes events other than transformation; for example, to enable viral replication upon infection of permissive cells, T-ag promotes progression of the cell cycle into S phase (reviewed in references 2, 27, and 90). This process is associated with a number of events, such as (i) activation of host cell checkpoint responses (77), (ii) induction of ATM-mediated signaling events (88), (iii) T-ag-dependent inhibition of p53 transactivation (51, 75), and (iv) a reduction of the percentage of cells in the G1 phase of the cell cycle while increasing the percentage of cells in the S, G2, and M cell cycle phases (94). How SV40 replicates in the face of hostile checkpoint conditions is unclear (88). Nevertheless, by evolving a BRCT-related region, T-ag would enhance its ability to target proteins in pathways needed to support the viral life cycle. For example, the DNA polymerase {alpha}-primase complex, required to initiate DNA synthesis, binds to T-ag at an unknown site within the helicase domain (22, 31). The possibility that this binding event is dependent on the BRCT-related region is of interest given that BRCT domains are present in other DNA replication factors; for example, they are present in the essential replication factor RFC (57) and in DNA polymerases {lambda} and REV1 (32, 50). Finally, T-ag is also known to attenuate the G2 (8, 53) and spindle checkpoints (8, 14). Therefore, interactions with additional cellular factors and the BRCT-related region might account for some of T-ag's additional pleiotropic effects (e.g., its ability to disrupt DNA repair processes [19], promote genomic instability [96, 110, 121], alter somatic mutation rates [100], and stimulate cellular DNA synthesis [11, 20, 42, 43]). Similar arguments can be made for the T-ags encoded by JC virus, BK virus, and polyomavirus.

The E1 proteins are not critical factors for HPV18- and BPV1-dependent transformation (41). It is clear, however, that the helicase domains of the E1 proteins contain sites of interaction with both viral and cellular proteins. For example, the viral E2 protein competes with the DNA polymerase {alpha}-primase complex for E1, and residues located in the E1 helicase domain are critical for these interactions (3, 13, 72). Thus, while the BRCT-related regions in E1 proteins are not needed for transformation-dependent interactions, they may promote protein/protein interactions needed for processes such as viral replication.

The analyses presented herein establish that many of the structural and sequence motifs used to define BRCT repeats are present in the helicase domains of SV40 T-ag and the E1 proteins of HPV18 and BPV1. Proof that the viral BRCT-related regions function as BRCT motifs will require additional studies. However, if "BRCT-related" domains are proven to exist in these proteins, it would help to explain why viral initiators encoded by the Polyomaviridae and Papillomaviridae viral families target cellular proteins involved in such basic processes as DNA replication, DNA repair, and cell cycle control. Given that other DNA viruses are known to target the DNA repair machinery (reviewed in reference 105), it will be of interest to determine whether BRCT-related regions are features of the proteins encoded by these viruses. Additional structural and functional studies will enable these and related questions to be addressed.


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ACKNOWLEDGMENTS
 
This study was supported by a grant from the National Institutes of Health to P.A.B. (9R01GM55397).

We thank Andrew Bohm, Ole Gjoerup, Jacques Archambault, Brian Schaffhausen, Gary Sahagian, Matt Weitzman, and Paul Phelan for helpful conversations.


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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 Back

{triangledown} Published ahead of print on 25 June 2008. Back

{dagger} Supplemental material for this article may be found at http://jvi.asm.org/. Back

{ddagger} The first three authors contributed equally to this work. Back

§ Present address: Department of Public Health and Family Medicine, Tufts University School of Medicine, Boston, MA 02111. Back

Present address: Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10021. Back


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Journal of Virology, September 2008, p. 8849-8862, Vol. 82, No. 17
0022-538X/08/$08.00+0     doi:10.1128/JVI.00553-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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