Creating a novel origin of replication through modulating DNA-protein interfaces

Abstract

Background: While the molecular mechanisms of DNA-protein specificity at the origin of replication have been determined in many model organisms, these interactions remain unknown in the majority of higher eukaryotes and numerous vertebrate viruses. Similar to many viral origins of replication, adeno-associated virus (AAV) utilizes a cis-acting origin of replication and a virus specific Replication protein (Rep) to faithfully carry out self-priming replication. The mechanisms of AAV DNA replication are generally well understood. However, the molecular basis of specificity between the Rep protein and the viral origin of replication between different AAV serotypes remains uncharacterized.

Methodology/principal findings: By generating a panel of chimeric and mutant origins between two AAV serotypes, we have mapped two independent DNA-Protein interfaces involved in replicative specificity. In vivo replication assays and structural modeling demonstrated that three residues in the AAV2 Rep active site are necessary to cleave its cognate origin. An analogous origin (AAV5) possesses a unique interaction between an extended Rep binding element and a 49 aa region of Rep containing two DNA binding interfaces.

Conclusions/significance: The elucidation of these structure-function relationships at the AAV origin led to the creation of a unique recombinant origin and compatible Rep protein with properties independent of either parent serotype. This novel origin may impact the safety and efficacy of AAV as a gene delivery tool. This work may also explain the unique ability of certain AAV serotypes to achieve site-directed integration into the human chromosome. Finally, this result impacts the study of conserved DNA viruses which employ rolling circle mechanisms of replication.

Figure 1. Cloning and characterization of chimeric ITRs.

(A) Sequence and structure of ITR2 (black) and ITR5 (blue) shown with incorporation of SfiI sites for cloning (green). Length in nt of ITR elements indicated above brackets. RBE is boxed. RBE' is indicated by a hatched circle. Nicking stem is extruded with arrow indicating the nicking site and hatched box indicating the trs. The four initial chimeric ITRs generated are shown (right). (B) Replication assay and quantitation of chimeric Reps. Replication products from the indicated ITR and either Rep2 or Rep5 were analyzed by Southern blot. Monomeric (m) and dimeric (d) replicating species are indicated. The level of replication of each sample was measured by densitometric analysis and compared to wt replication.

Figure 2. Relation of nicking stem height and sequence to Rep-ITR specificity.

(A) Sequence of nicking stem in an otherwise ITR2 context. Arrow indicates trs site. Brackets indicate height of putative stems in nt from the base of the stem to the putative nicking site. Predicted ΔG values for the hairpins are below. Southern blot analysis of the ITRs replicated by Rep2 or Rep5 are shown below. (B) Quantitation of the Southern blots relative to wt replication from (A). (C) Same as (A), except nicking stems indicated were used in an ITR5 context. (D) Quantitation of the Southern blots relative to wt replication from (C).

Figure 3. Effect of RBE-nicking stem spacing on Rep-ITR specificity.

(A) ITR2 mutants were synthesized with the indicated spacing between the RBE and nicking stem. (B) Southern blot analysis of the ITRs depicted in (A) replicated by either Rep2 or Rep5 (Left). Quantitation of Southern blots relative to wt replication (Right). (C) ITR5 mutants synthesized as in (A). (D) Southern blot analysis and quantitation of (C).

Figure 4. The ITR5 spacer acts as a RBE for Rep5.

(A) ITR5 mutants were synthesized with the indicated RBE and spacer sequence. Brackets indicate individual tetranucleotide repeats bound by Rep monomers. Both strands of the wt ITR5 sequence are shown to illustrate conservation with the GAGY motif (indicated by *). Only one strand shown on others. (B) Southern blot analysis of the ITRs depicted in (A) replicated by either Rep2 or Rep5 (Left). Quantitation of Southern blots relative to wt replication (Right). (C) ITR2 mutants were generated with the RBE and spacer sequences indicated. (D) Southern blot analysis and quantitation for (C).

Figure 5. Cloning and characterization of chimeric Reps.

(A) An alignment of the N-termini of Rep2 and Rep5. (*) represents conserved amino acids. (: and.) indicates conservative substitutions. Blue indicates residues implicated in RBE binding interactions. Pink indicates residues which participate in the endonucleolytic active site. Green indicates residues implicated in RBE' binding. (B) Chimeric Reps created and their ability to replicate ITR2 or ITR5 flanked vectors. Numbers indicate the aa position of the switch from one Rep to the other. (+) indicates the presence of replication, (−) indicates the absence. (C) Western blot for expression of the chimeric Reps. (D) Southern blot demonstrating replication of an ITR2 or an ITR5 vector by the chimeric Reps. Note that the ITR5 vector is 500bp larger than the ITR2 vector. (E) Level of replication of the chimeric Reps relative to wt Rep2 or Rep5.

Figure 6. Characterization of Rep regions critical for ITR specificity.

(A) Chimeric Reps and their ability to replicate ITR2 or ITR5 flanked vectors. Numbers indicate the aa position of the switch from one Rep to the other. (+) indicates the presence of replication, (−) indicates the absence. Region 1 and 2 involved in Rep-ITR specificity are indicated. (B) Western blot for expression of chimeric Reps. (C) Southern blot demonstrating replication of an ITR2 or ITR5 vector by the chimeric Reps. Note that the ITR5 vector is 500 bp larger than the ITR2 vector. (D) Structural model illustrating the two Rep regions. Rep2 structure is blue, Rep5 is purple. The nucleophilic tyrosine is indicated. Black hatched circle indicates the predicted structural difference of region 1 in the major groove of the ITR. (E) Structural model as in (D). The nucleophilic tyrosine is indicated. (F) Detailed structural view of region 1. The side-chains of non-conserved residues from Rep5 (purple) and Rep2 (blue) are shown. Three Rep5 residues implicated in RBE' binding are indicated. (G) Detailed structural view of region 2. Side chains of active site residues are shown in black. Side chains of non-conserved residues in this region are shown for Rep2 (blue) and Rep5 (purple). The nucleophilic tyrosine is indicated, as is the adjacent Rep2 Asn-155.

Figure 7. Model of Rep-ITR specificity.

(A) Southern blot of Hirt DNA demonstrating replication of the indicated ITR vector by the indicated Rep. (B) Table indicating the presence (+) or absence (−) of replication of the gel from (A). (C) Model of a novel AAV origin of replication. The chimeric ITR can be replicated only by a chimeric Rep protein. Rep5 sequence in region 1 (blue) is required for the extended RBE of ITR5 (purple). Rep2 sequence in region 2 (yellow) is required to function on an ITR2 nicking stem (cyan).