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Sequential and ordered assembly of E1 initiator complexes on the papillomavirus origin of DNA replication generates progressive structural changes related to melting - PubMed

Sequential and ordered assembly of E1 initiator complexes on the papillomavirus origin of DNA replication generates progressive structural changes related to melting

Grace Chen et al. Mol Cell Biol. 2002 Nov.

Abstract

Multiple binding sites for an initiator protein are a common feature of replicator sequences from various organisms. By binding to the replicator, initiators mark the site and contribute to melting or distortion of the DNA by largely unknown mechanisms. Here we analyze origin of DNA replication (ori) binding by the E1 initiator and show sequential binding to a set of overlapping binding sites. The assembly of these initiator complexes is controlled by a gradual reduction in the dependence of interactions between the initiator and DNA and a gradual increase in the reliance on interactions between initiator molecules, providing a mechanism for sequential and orderly assembly. Importantly, the binding of the initiator causes progressive structural alterations both in the sites and in the sequences flanking the sites, eventually generating severe structural alterations. These results indicate that the process of template melting may be incremental, where binding of each initiator molecule serves as a wedge that upon binding gradually alters the template structure. This mechanism may explain the requirement for multiple initiator binding sites that is observed in many ori's.

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Figures

FIG. 1.
FIG. 1.

Schematic drawing of the 60-bp minimal origin of replication from BPV. The boxes highlight the sequences and positions of the six overlapping E1 BS (BS 1 to 6) as well as the position of the E2 BS.

FIG. 2.
FIG. 2.

Hydroxyl radical footprints of the E1 and E2 DBDs. (A) End-labeled BPV minimal ori fragments were incubated together with (i) the E2 DBD alone (70 ng) (bottom strand, lane 12, and top strand, lane 1), (ii) the E1 DBD and the E2 DBD (0.5 μg and 0.7 ng, respectively; bottom strand, lane 11, and top strand, lane 2), and (iii) a titration of E1 DBD alone (16, 8, 4, 2, 1, and 0.5 μg of E1 DBD; bottom strand, lanes 3 to 10, and top strand, lanes 3 to 8). Lanes 3 and 4 on the bottom strand contain the same amount of E1 DBD (16 μg) but different amounts of hydroxyl radical cleavage reagent. Similarly, lanes 5 and 6 both contain 8 μg of E1 DBD but different amounts of cleavage reagent. Lanes A+G, marker generated by cleavage at A and G in the probe. Free indicates lanes where no protein was added. Blue and green boxes, protections generated by the binding of two monomers of the E1 DBD; orange and red boxes, protections generated by the binding of additional molecules of E1 DBD. (B) Summary of hydroxyl radical footprints. Black circles over the boxed E2 binding site, protections produced by the E2 DBD; blue and green circles, binding by two monomers at either low concentrations of the E1 DBD alone or together with E2 DBD. At higher concentrations of the E1 DBD, the protections extend as shown (red and orange circles). (C) Protected sequences at both low and high concentrations of the E1 DBD projected onto a double-helix model. The protections shown in blue and green correspond to the protections from one dimer of E1. The binding of an additional E1 dimer protects sequences on the side face of the helix (red and orange).

FIG. 3.
FIG. 3.

Hydroxyl radical footprinting on wt and +3 templates. Footprinting was performed on the top strand of the wt (lanes 1 to 8) and the +3 (lanes 9 to 14) ori probes in the presence of E1 and E2 DBDs as indicated at the top. E1 DBD (0.8, 0.4, 0.2, and 0.1 μg) and E2 DBD (0.7 ng) were added in lanes 3 to 6 and 9 to 12. Lanes 7 and 13 contained 70 ng of E2 DBD alone. Below, the protections are shown projected onto the DNA sequence. Hatched boxes, protections generated by E2; open boxes, protections generated by the binding of two molecules of E1 DBD to the wt probe; black boxes, extensions of these protections observed on the +3 probe; gray boxes, protections shared between E1 and E2 DBDs. Arrow, hypersensitive site that appears upon the binding of four molecules of E1 DBD. Lanes A+G and Free are as defined for Fig. 2A.

FIG. 4.
FIG. 4.

(A) Gel shift analysis using four different-size ori probes. Probe A contains the complete minimal ori sequence (∼60 bp) from nt 7914 to 7927 and in addition contains 48 bp of flanking sequence upstream (left) and 25 bp downstream (right) of the ori fragment derived from the pUC19 polylinker. Probe B lacks the flanking polylinker sequences on the downstream side of the ori fragment. Probe C lacks the flanking sequences on the upstream side of the ori fragment as well as the A/T-rich region from the ori. Probe D contains only the sequences of the E1 BS and the E2 BS and lacks any flanking sequences as well as the A/T-rich region. Lane 2, 40 pg of E2 DBD added; lane 3, 5 ng of E1 DBD added. For each probe three fivefold titrations of E1 DBD were added (0.2, 1, and 5 ng) in the presence of 40 pg of E2 DBD. The compositions of the different complexes are indicated on the right. (B) Gel shift analysis using probes lacking flanking polylinker sequences on the downstream side of the ori fragment but with sequences of different lengths on the upstream side. Probe A is identical to probe B in panel A, and probe D corresponds to probe D in panel A. Probe B lacks flanking sequences on both sides but maintains the minimal ori sequence. Probe C, in addition, lacks part of the A/T-rich sequence. The quantities of E1 and E2 DBD were identical to those used in panel A.

FIG. 5.
FIG. 5.

DEPC interference analysis of the putative E14-E22 complex. Probe B from Fig. 4B was modified with DEPC, annealed, and subsequently used for EMSA with E1 and E2 DBDs. The binding reactions were scaled up 20-fold under the conditions used in lane 10 in Fig. 4B. The putative E14-E22 complex and the free probe were excised, and the DNA was treated with piperidine and compared to the free probe on a sequencing gel. The patterns of interference for the top and bottom strands for shown. The gel was analyzed by a Fuji imager and quantitated, and a scan of the corresponding lanes is shown in to the left. Below is a summary of the positions where interference can be detected. As a comparison, the interference patterns for E12-E22 bound to sites 2 and 4 and 1 and 3 are shown (6).

FIG. 6.
FIG. 6.

Potassium permanganate reactivity in the ori caused by binding of E1. A top-strand ori probe was incubated in the absence (−) or presence of increasing quantities of E1 protein (15, 30, and 60 ng). As a control, 60 ng of E1 was incubated in the presence of 5 mM ATP (+ATP). After 20 min at room temperature, potassium permanganate was added to 6 mM, and the reaction was terminated after 2 min by addition of 2-mercaptoethanol. After cleavage with piperidine, the resulting material was analyzed by 10% polyacrylamide gel electrophoresis. The positions of permanganate reactivity are indicated by arrows. Below, these positions are indicated in relation to the boxed E1 and E2 BS.

FIG. 7.
FIG. 7.

Model for the sequential assembly of E1 initiator complexes on the origin of DNA replication. See text for explanations.

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