The E1 initiator recognizes multiple overlapping sites in the papillomavirus origin of DNA replication

Abstract

A common feature of replicator sequences from a variety of organisms is multiple binding sites for an initiator protein. By binding to the replicator, initiators mark the site and contribute to melting or distortion of the DNA. We have defined the recognition sequence for the papillomavirus E1 initiator and determined the arrangement of binding sites in the viral origin of replication. We show that E1 recognizes a hexanucleotide sequence which is present in overlapping arrays in virtually all papillomavirus replicators. Binding of the initiator to these sites would result in the formation of a closely packed array of E1 molecules that wrap around the double helix.

FIG. 1

The E1 protein binds to the hexanucleotide sequence AACAAT. (A) A probe containing an 8-bp XhoI linker flanking half of the E1 palindrome with the E2 binding site in its natural position was used in EMSA with E1 and E2 DBDs. Under these conditions, a monomer of the E1 DBD binds cooperatively with E2 DBD to the probe. Transversion mutations were generated in the XhoI linker (B), the central 6 bp (C), and the right flank (D) and then tested by EMSA. The results are shown in graphical form in panel E. (B) Two transversion mutations in the XhoI linker (M1, lanes 9 to 16, and M2, lanes 17 to 24) were generated and compared to the wt probe for E1 binding. Three fourfold dilutions of E1 DBD in the absence (lanes 1 to 3, 9 to 11, and 17 to 19) or presence (lanes 4 to 6, 12 to 14, and 20 to 22) of the E2 DBD were used. Lanes 7, 15, and 23 contained E2 DBD alone; lanes 8, 16, and 24 contained probe alone. The position of the combined E1 and E2 DBD complexes and the E2 DBD complex are indicated by arrows. (C) Six transversion mutations were generated in the E1 half-palindrome and tested for E1 binding as described in panel B. Mutations C3 to G (lanes 6 to 10), A4 to T (lanes 11 to 15), and A5 to T (lanes 16 to 20) are shown in comparison to the wt (lanes 1 to 5). Only binding in the presence of E2 is shown. Lanes 1 to 3, 6 to 8, 11 to 13, and 16 to 18 contained both E1 and E2 DBDs. Lanes 4, 9, 14, and 19 contained E2 DBD alone; lanes 5, 10, 15, and 20 contained probe alone. (D) Three transversion mutations were generated in the right flank in positions 7 to 9 and tested for binding of E1 DBD as described above. Mutations A7 to T (lanes 6 to 10), A8 to T (lanes 11 to 15), and T9 to A (lanes 16 to 20) are shown in comparison with the wt (lanes 1 to 5). Lanes 1 to 3, 6 to 8, 11 to 13, and 16 to 18 contained both E1 and E2 DBDs; lanes 4, 9, 15, and 19 contained E2 DBD alone; lanes 5, 10, 15, and 20 contain probe alone. (E) Summary of the results of transversion mutations in E1 binding site. The effects of mutations is shown in a bar graph. The level of E1 binding to the wt sequence is set at 1.0.

FIG. 1

The E1 protein binds to the hexanucleotide sequence AACAAT. (A) A probe containing an 8-bp XhoI linker flanking half of the E1 palindrome with the E2 binding site in its natural position was used in EMSA with E1 and E2 DBDs. Under these conditions, a monomer of the E1 DBD binds cooperatively with E2 DBD to the probe. Transversion mutations were generated in the XhoI linker (B), the central 6 bp (C), and the right flank (D) and then tested by EMSA. The results are shown in graphical form in panel E. (B) Two transversion mutations in the XhoI linker (M1, lanes 9 to 16, and M2, lanes 17 to 24) were generated and compared to the wt probe for E1 binding. Three fourfold dilutions of E1 DBD in the absence (lanes 1 to 3, 9 to 11, and 17 to 19) or presence (lanes 4 to 6, 12 to 14, and 20 to 22) of the E2 DBD were used. Lanes 7, 15, and 23 contained E2 DBD alone; lanes 8, 16, and 24 contained probe alone. The position of the combined E1 and E2 DBD complexes and the E2 DBD complex are indicated by arrows. (C) Six transversion mutations were generated in the E1 half-palindrome and tested for E1 binding as described in panel B. Mutations C3 to G (lanes 6 to 10), A4 to T (lanes 11 to 15), and A5 to T (lanes 16 to 20) are shown in comparison to the wt (lanes 1 to 5). Only binding in the presence of E2 is shown. Lanes 1 to 3, 6 to 8, 11 to 13, and 16 to 18 contained both E1 and E2 DBDs. Lanes 4, 9, 14, and 19 contained E2 DBD alone; lanes 5, 10, 15, and 20 contained probe alone. (D) Three transversion mutations were generated in the right flank in positions 7 to 9 and tested for binding of E1 DBD as described above. Mutations A7 to T (lanes 6 to 10), A8 to T (lanes 11 to 15), and T9 to A (lanes 16 to 20) are shown in comparison with the wt (lanes 1 to 5). Lanes 1 to 3, 6 to 8, 11 to 13, and 16 to 18 contained both E1 and E2 DBDs; lanes 4, 9, 15, and 19 contained E2 DBD alone; lanes 5, 10, 15, and 20 contain probe alone. (E) Summary of the results of transversion mutations in E1 binding site. The effects of mutations is shown in a bar graph. The level of E1 binding to the wt sequence is set at 1.0.

FIG. 1

The E1 protein binds to the hexanucleotide sequence AACAAT. (A) A probe containing an 8-bp XhoI linker flanking half of the E1 palindrome with the E2 binding site in its natural position was used in EMSA with E1 and E2 DBDs. Under these conditions, a monomer of the E1 DBD binds cooperatively with E2 DBD to the probe. Transversion mutations were generated in the XhoI linker (B), the central 6 bp (C), and the right flank (D) and then tested by EMSA. The results are shown in graphical form in panel E. (B) Two transversion mutations in the XhoI linker (M1, lanes 9 to 16, and M2, lanes 17 to 24) were generated and compared to the wt probe for E1 binding. Three fourfold dilutions of E1 DBD in the absence (lanes 1 to 3, 9 to 11, and 17 to 19) or presence (lanes 4 to 6, 12 to 14, and 20 to 22) of the E2 DBD were used. Lanes 7, 15, and 23 contained E2 DBD alone; lanes 8, 16, and 24 contained probe alone. The position of the combined E1 and E2 DBD complexes and the E2 DBD complex are indicated by arrows. (C) Six transversion mutations were generated in the E1 half-palindrome and tested for E1 binding as described in panel B. Mutations C3 to G (lanes 6 to 10), A4 to T (lanes 11 to 15), and A5 to T (lanes 16 to 20) are shown in comparison to the wt (lanes 1 to 5). Only binding in the presence of E2 is shown. Lanes 1 to 3, 6 to 8, 11 to 13, and 16 to 18 contained both E1 and E2 DBDs. Lanes 4, 9, 14, and 19 contained E2 DBD alone; lanes 5, 10, 15, and 20 contained probe alone. (D) Three transversion mutations were generated in the right flank in positions 7 to 9 and tested for binding of E1 DBD as described above. Mutations A7 to T (lanes 6 to 10), A8 to T (lanes 11 to 15), and T9 to A (lanes 16 to 20) are shown in comparison with the wt (lanes 1 to 5). Lanes 1 to 3, 6 to 8, 11 to 13, and 16 to 18 contained both E1 and E2 DBDs; lanes 4, 9, 15, and 19 contained E2 DBD alone; lanes 5, 10, 15, and 20 contain probe alone. (E) Summary of the results of transversion mutations in E1 binding site. The effects of mutations is shown in a bar graph. The level of E1 binding to the wt sequence is set at 1.0.

FIG. 1

The E1 protein binds to the hexanucleotide sequence AACAAT. (A) A probe containing an 8-bp XhoI linker flanking half of the E1 palindrome with the E2 binding site in its natural position was used in EMSA with E1 and E2 DBDs. Under these conditions, a monomer of the E1 DBD binds cooperatively with E2 DBD to the probe. Transversion mutations were generated in the XhoI linker (B), the central 6 bp (C), and the right flank (D) and then tested by EMSA. The results are shown in graphical form in panel E. (B) Two transversion mutations in the XhoI linker (M1, lanes 9 to 16, and M2, lanes 17 to 24) were generated and compared to the wt probe for E1 binding. Three fourfold dilutions of E1 DBD in the absence (lanes 1 to 3, 9 to 11, and 17 to 19) or presence (lanes 4 to 6, 12 to 14, and 20 to 22) of the E2 DBD were used. Lanes 7, 15, and 23 contained E2 DBD alone; lanes 8, 16, and 24 contained probe alone. The position of the combined E1 and E2 DBD complexes and the E2 DBD complex are indicated by arrows. (C) Six transversion mutations were generated in the E1 half-palindrome and tested for E1 binding as described in panel B. Mutations C3 to G (lanes 6 to 10), A4 to T (lanes 11 to 15), and A5 to T (lanes 16 to 20) are shown in comparison to the wt (lanes 1 to 5). Only binding in the presence of E2 is shown. Lanes 1 to 3, 6 to 8, 11 to 13, and 16 to 18 contained both E1 and E2 DBDs. Lanes 4, 9, 14, and 19 contained E2 DBD alone; lanes 5, 10, 15, and 20 contained probe alone. (D) Three transversion mutations were generated in the right flank in positions 7 to 9 and tested for binding of E1 DBD as described above. Mutations A7 to T (lanes 6 to 10), A8 to T (lanes 11 to 15), and T9 to A (lanes 16 to 20) are shown in comparison with the wt (lanes 1 to 5). Lanes 1 to 3, 6 to 8, 11 to 13, and 16 to 18 contained both E1 and E2 DBDs; lanes 4, 9, 15, and 19 contained E2 DBD alone; lanes 5, 10, 15, and 20 contain probe alone. (E) Summary of the results of transversion mutations in E1 binding site. The effects of mutations is shown in a bar graph. The level of E1 binding to the wt sequence is set at 1.0.

FIG. 2

Summary of the effects of mutations in all positions on E1 binding. The six positions which showed significant effects on E1 binding in Fig. 1 were chosen for a complete mutagenesis, wherein the bases at all positions were changed into the three possible alternatives. The mutants were tested for binding of E1 DBD as shown in Fig. 1, and the results were quantitated and are shown as a bar graph.

FIG. 3

(A) The information from the mutagenesis experiments was used to identify other potential E1 recognition sequences in the ori based on sequence homology. These new putative sites, 1, 3, 5, and 6, are shown together with the known sites 2 and 4 as shaded boxes. (B) Sites 1, 3, 5, and 6 were inserted into the Xho probe, and binding was measured by EMSA in the presence of E2 DBD as described for Fig. 1. The results are summarized in the bar graph.

FIG. 4

E1 DBD binds to binding sites 1 and 3 when the E2 binding site is moved by 3 bp. (A) EMSA was performed to determine the ability of the E1 DBD to bind to the ori in combination with the E2 DBD by using a probe with the E2 binding site in the wt position (wt, lanes 1 to 8) or when the E2 binding sites was moved 3 bp through an insertion of 3 bp between the E1 and E2 binding sites (+3, lanes 9 to 16) or with a probe containing the +3 insertion and a point mutation in binding site 4 (B42+3, lanes 17 to 24). Lanes 1 to 3, 9 to 11, and 16 to 18 contained three twofold dilutions of E1 DBD alone. Lanes 4 to 6, 12 to 14, and 19 to 21 contained the same dilutions of E1 DBD in the presence of E2 DBD. Lanes 7, 14, and 23 contained E2 DBD alone; lanes 8, 15, and 24 contained probe alone. The arrows indicate the migration of the respective complexes. (B) DEPC interference analysis was performed on both strands of the B42+3 probe. The probes were modified with DEPC, and the complexes formed in the presence of E2 DBD alone (lanes 1 and 4) and both E1 and E2 DBDs (lanes 2 and 5) were isolated and subjected to cleavage with piperidine and analyzed on a sequencing gel. The positions of interference on both strands are indicated by filled circles. (C) Diagram of the positions of interference in the wt and +3 ori templates. The DEPC interference produced by binding of a dimer of E1 DBD in the presence of E2 DBD on the B42+3 probe is shown in comparison with the interference obtained for a complex formed on a wt probe (4). The B42 mutation is shown in boldface.

FIG. 5

Inosine substitutions indicate that E1 recognizes the major groove. Inosine substitutions were generated at the six positions corresponding to the E1 recognition sequence and tested for binding as described above. (A) I/C base pairs were generated at positions 1 (lanes 11 to 15), 2 (lanes 21 to 25), and 3 (lanes 26 to 30) on the top strand. Probes containing G/C base pairs at positions 1 and 2 (lanes 6 to 10 and 16 to 20, respectively) are shown for comparison. Lanes 1 to 3, 6 to 8, 11 to 13, 16 to 18, 21 to 23, and 26 to 28 contained both E1 and E2 DBDs. Lanes 4, 9, 14, 19, 24, and 29 contained E2 DBD alone; lanes 5, 10, 15, 20, 25, and 30 contained probe alone. (B) I/C base pairs were generated at positions 4 (lanes 11 to 15), 5 (lanes 21 to 25), and 6 (lanes 31 to 35) on the top strand. Probes with G/C base pairs at the corresponding positions are shown for comparison (lanes 6 to 10, 16 to 20, and 26 to 30, respectively). Lanes 1 to 3, 6 to 8, 11 to 13, 16 to 18, 21 to 23, 26 to 28, and 31 to 33 contained E1 and E2 DBDs. Lanes 4, 9, 14, 19, 24, 29, and 34 contained E2 DBD alone; lanes 5, 10, 15, 20, 25, 30, and 35 contained probe alone. (C) Summary of the results from inosine substitutions. The wt level of binding is set to 1.0.

FIG. 5

Inosine substitutions indicate that E1 recognizes the major groove. Inosine substitutions were generated at the six positions corresponding to the E1 recognition sequence and tested for binding as described above. (A) I/C base pairs were generated at positions 1 (lanes 11 to 15), 2 (lanes 21 to 25), and 3 (lanes 26 to 30) on the top strand. Probes containing G/C base pairs at positions 1 and 2 (lanes 6 to 10 and 16 to 20, respectively) are shown for comparison. Lanes 1 to 3, 6 to 8, 11 to 13, 16 to 18, 21 to 23, and 26 to 28 contained both E1 and E2 DBDs. Lanes 4, 9, 14, 19, 24, and 29 contained E2 DBD alone; lanes 5, 10, 15, 20, 25, and 30 contained probe alone. (B) I/C base pairs were generated at positions 4 (lanes 11 to 15), 5 (lanes 21 to 25), and 6 (lanes 31 to 35) on the top strand. Probes with G/C base pairs at the corresponding positions are shown for comparison (lanes 6 to 10, 16 to 20, and 26 to 30, respectively). Lanes 1 to 3, 6 to 8, 11 to 13, 16 to 18, 21 to 23, 26 to 28, and 31 to 33 contained E1 and E2 DBDs. Lanes 4, 9, 14, 19, 24, 29, and 34 contained E2 DBD alone; lanes 5, 10, 15, 20, 25, 30, and 35 contained probe alone. (C) Summary of the results from inosine substitutions. The wt level of binding is set to 1.0.

FIG. 6

Arrangement of the E1 binding sites in the BPV core ori. (A) Stereo diagram of the BPV ori DNA containing the E1 binding sites. E1 binding sites 1 to 4 are indicated by arrows. The first three bases on the top and bottom strands for each binding site are colored as shown in panel B. Binding sites 2 and 4 are shown in red, binding sites 1 and 3 are shown in blue, and the putative sites 5 and 6 are shown in green. (B) DNA sequence of the E1 binding region. The positions of binding sites 1 to 4 are indicated by solid arrows, and the positions of the putative sites 5 and 6 are indicated by stippled arrows. The three first bases on the top and bottom strands of each E1 binding site are color coded. E1 binding sites 2 and 4 are indicated in red, and E1 binding sites 1 and 3 are indicated in blue. The putative sites 5 and 6 are shown in green. Above the sequence is shown the positions of ethylation interference (30) with a dimer of E1 (E1E2-ori complex, filled circles) and two dimers of E1 (E1-ori complex, open circles). (C) Schematic diagram of the arrangement of the E1 molecules on the ori with the different E1 binding site arrangements. Binding of E1 to the binding sites 1-4 is indicated by filled symbols. Binding to the putative binding sites 5 and 6 is indicated by open symbols.

FIG. 7

Comparison of the E1 binding site arrangement in select papillomavirus origins of replication. The stippled arrows indicate putative E1 binding sites with significantly lower affinity based on the lack of conservation at positions that are important for binding of BPV E1.