ATP-dependent minor groove recognition of TA base pairs is required for template melting by the E1 initiator protein

Abstract

Template melting is an essential step in the initiation of DNA replication, but the mechanism of template melting is unknown for any replicon. Here we demonstrate that melting of the bovine papillomavirus type 1 ori is a sequence-dependent process which relies on specific recognition of TA base pairs in the minor groove by the E1 initiator. We show that correct template melting is a prerequisite for the formation of a stable double hexamer with helicase activity and that ori mutants that fail to melt correctly are defective for ori unwinding and DNA replication in vivo. Our results also indicate that melting of the DNA is achieved by destabilization of the double helix along its length through multiple interactions with E1, each of which is responsible for melting of a few base pairs, resulting in the extensive melting that is required for initiation of DNA replication.

FIG. 1.

(A) Permanganate reactivity of the wt ori probe in the absence of nucleotide or in the presence of ADP or ATP. Permanganate reactivity assays were performed by incubating the bottom-strand (lanes 1 to 8) and top-strand (lanes 9 to 16) labeled probes with E1 in the absence of nucleotide (lanes 2 and 10) or in the presence of ADP (lanes 3 to 5 and 11 to 13) or ATP (lanes 6 to 9 and 14 to 16). In the absence of nucleotide, 800 fmol of E1 was used, and in the presence of nucleotide 200, 400, and 800 fmol of E1 was used. After 30 min at room temperature, the samples were treated with 6 mM KMnO₄ for 2 min. For lanes 1 and 9, the top- and bottom-strand probes were treated with KMnO₄ in the absence of E1. (B) Summary of the permanganate reactivity of the wt ori probe. Black bars indicate the positions and relative levels of permanganate reactivity.

FIG. 2.

Permanganate reactivities of ori probes with altered sequences. (A) Permanganate reactivity assays in the presence of ATP were performed with the top and bottom strands of probes AT8 (lanes 1, 2, 9, and 10) and TA8 (lanes 3, 4, 7, and 8) and the bottom strand of T14 (lanes 5 and 6). The probes were incubated in the absence of E1 (lanes 1, 3, 5, 7, and 9) or in the presence of 800 fmol of E1 (lanes 2, 4, 6, 8, and 10) in the presence of ATP and treated with permanganate as described in the legend to Fig. 1. (B) Permanganate reactivity assays in the presence of ATP were performed with the top (lanes 1 to 8) and bottom (lanes 9 to 12) strands of the A16 (lanes 1 to 4, 11, and 12) and wt (lanes 5 to 10) templates. For lanes 2 to 4 and 6 to 8, 200, 400, and 800 fmol of E1 was used, respectively. For lanes 10 and 12, 800 fmol of E1 was used. (C) E1 complex formation on the wt and A16 probes. EMSA was performed on the wt (lanes 1 to 9) and A16 (lanes 10 to 18) probes. Three quantities of E1 (30, 60, and 120 fmol) were used in the presence of ADP (lanes 2 to 5 and 11 to 14) or ATP (lanes 6 to 9 and 15 to 18). For lanes 1 and 10, no E1 was added.

FIG. 3.

Permanganate reactivities of probes with TT insertions. (A) The A16 ori probe was modified by insertion of pairs of T's at different positions in the A16 sequence, as shown in panel C. These probes were tested for permanganate reactivity in the presence of 800 fmol of E1 as described in the legend to Fig. 1. For lanes 1, 3, 5, 7, 9, and 11, no ATP was added. For lanes 2, 4, 6, 8, 10, and 12, 5 mM ATP was added. (B) The bracketed part of the gel in panel A was scanned, and the scans for lanes 2, 4, 6, 8, 10, and 12 were aligned. (C) Summary of the permanganate reactivities generated with the TT-substituted A16 probes. The positions of the substitutions are boxed and shaded. Black bars indicate the positions and levels of permanganate reactivity observed on the A16 probe. The gray bars correspond to the pattern obtained after subtraction of the A16 pattern from each lane. (D) Bottom-strand labeled probes for the A16 template and the TT insertions T12/13, T14/15, T16/17, and T20/21 in the A16 context were tested for permanganate reactivity in the absence (lanes 1, 3, 5, 7, and 9) or presence (lanes 2, 4, 6, 8, and 10) of 800 fmol of E1 in the presence of ADP. (E) Summary of the permanganate reactivities generated by E1 on TT-substituted templates in the presence of ADP. For comparison, the permanganate reactivity on the wt template (from Fig. 1) is also shown.

FIG. 4.

(A) Comparison of permanganate reactivities of the A16 and T16/17 probes. Permanganate reactivity assays were performed on the A16 and T16/17 templates in parallel. E1 was incubated with the probe in the absence of nucleotide (lanes 1 and 8), in the presence of ADP (lanes 2 to 4 and 9 to 11) or in the presence of ATP (lanes 5 to 7 and 12 to 14). In the absence of nucleotide, 800 fmol of E1 was used, and in the presence of ADP or ATP, 200, 400, and 800 fmol of E1 was used. (B) Summary of the permanganate reactivities generated by E1 on the A16 and T16/17 templates in the presence of ADP and ATP. For comparison, the permanganate reactivity on the wt template (from Fig. 1) is shown. (C) The template T6(A16) was generated by insertion of six T residues into the A16 context at positions 12 to 17. Permanganate reactivity assays were performed in the presence of ATP on the bottom-strand labeled probe in the absence (lane 1) or presence (lane 2) of 800 fmol E1. (D) Permanganate reactivity of the T16/17 template. Permanganate reactivity assays were performed using the top-strand labeled T16/17 template. E1 (400 and 800 fmol, respectively) was incubated with the probe in the presence of ADP (lanes 2 and 3) or ATP (lanes 4 and 5) and treated with permanganate as described in the legend to Fig. 1. For lane 1, no E1 was added. Below the gel is a summary of the observed permanganate reactivity. (E) Schematic representation of distal and proximal T-dependent melting showing that TA bp (bold) direct melting 5 or 6 bp away from the inserted TA bp.

FIG. 5.

Melting requires minor groove recognition of TA bp. Permanganate reactivity assays were performed on E1 templates with substitutions at the 14-15 and 16-17 positions, as indicated in the figure. For lanes 1, 3, 5, 7, 9, 11, and 13, no E1 was added. For lanes 2, 4, 6, 8, 10, 12, and 14, 800 fmol of E1 was added in the presence of ATP. The bracket indicates the position of T-induced melting. (B) Schematic representation of the minor and major grooves of AT, IC, and GC bp.

FIG. 6.

In vivo DNA replication and unwinding are dependent on six TA bp. (A) Mutant ori's, as shown in panel B, were generated and tested for transient DNA replication in vivo by cotransfection of the respective mutant ori plasmids with expression vectors for E1 and E2. Two and three days after transfection, low-molecular-weight DNAs were prepared, and replicated DpnI-resistant plasmid DNA was detected by Southern blotting. (B) Sequences of the two sets of ori mutants that were tested in panel A. The top set is based on the wt ori sequence, and the bottom set is based on the A16 ori sequence. The substitutions compared to the parent template are indicated in shaded boxes. The level of in vivo DNA replication, relative to that with the wt ori, is indicated for each mutant.

FIG. 7.

The DH formed on the A16 probe is unstable. EMSA was performed using the wt (lanes 1 to 9) or A16 (lanes 10 to 18) 84-bp ori probe. Three quantities of E1 (30, 60, and 120 fmol) were used in the presence of ADP (lanes 2 to 4, 9 to 11, 16 to 18, and 23 to 25) or ATP (lanes 5 to 7, 12 to 14, 19 to 21, and 26 to 28). For the left half of the figure, lanes 1 to 14, the samples were loaded directly into the gel. For the right half of the figure, lanes 15 to 28, 0.2% deoxycholate was added to the samples prior to their being loaded into the gel. (B) ori fragment unwinding assays. ori fragment unwinding assays were performed by incubating the wt, A16*, and A16 probes with E. coli SSB in the absence of E1 (lanes 1, 7, and 13) or in the presence of 30, 60, 120, 240, or 480 fmol of E1 (lanes 2 to 6, 8 to 12, and 14 to 18). Unwinding (ssDNA) was detected as a specific ssDNA-SSB complex by EMSA.

FIG. 8.

Models for template melting. (A) Four different components of ATP-dependent template melting. Distal and proximal T-dependent melting accounts for the majority of permanganate reactivity. In addition, permanganate reactivity of the E1 BS and of proximal T-independent melting can be observed under specific conditions (see Fig. 4A). (B) Six TA bp in the wt ori direct melting. Within a 9-bp window, the presence of TA bp results in melting of the TA bp (distal T-dependent melting). The TA bp within this window also induce melting half a helical turn away (proximal T-dependent melting). (C) Model for how multiple E1 DNA interactions generate large-scale melting. The E1 molecules in the DT bind in a helical arrangement, wrapping around the DNA duplex. Each E1 molecule contacts DNA at two positions, separated by a one-half turn of the helix. These contacts correspond to distal and proximal T-dependent melting. Together, these six contacts account for the ∼15 to 18 bp of permanganate reactivity observed in the left half of the ori. For clarity, only two of the three staggered E1 molecules are shown.