Palindrome regeneration by template strand-switching mechanism at the origin of DNA replication of porcine circovirus via the rolling-circle melting-pot replication model

Abstract

Palindromic sequences (inverted repeats) flanking the origin of DNA replication with the potential of forming single-stranded stem-loop cruciform structures have been reported to be essential for replication of the circular genomes of many prokaryotic and eukaryotic systems. In this study, mutant genomes of porcine circovirus with deletions in the origin-flanking palindrome and incapable of forming any cruciform structures invariably yielded progeny viruses containing longer and more stable palindromes. These results suggest that origin-flanking palindromes are essential for termination but not for initiation of DNA replication. Detection of template strand switching in the middle of an inverted repeat strand among the progeny viruses demonstrated that both the minus genome and a corresponding palindromic strand served as templates simultaneously during DNA biosynthesis and supports the recently proposed rolling-circle "melting-pot" replication model. The genome configuration presented by this model, a four-stranded tertiary structure, provides insights into the mechanisms of DNA replication, inverted repeat correction (or conversion), and illegitimate recombination of any circular DNA molecule with an origin-flanking palindrome.

FIG. 1.

(A) Schematic representation of the PCV1 origin, indicating potential base pairing of the flanking inverted repeats. The locations of the PCR primers are indicated below the PCV1 genome. The genome sequence (1,759 nucleotides) and coordinates (1, 2, 3, etc.) are based on GenBank accession number AY184287 (2). The nucleotide coordinates (3′, 4′, 5′, etc.) are arbitrarily assigned to show the nucleotide complementarity of the palindromic sequences. The octanucleotide containing the presumed nick site (AGTATT↓AC) is boxed and indicated in bold. The palindrome is divided into six regions (right arm, RD3, RD7, and RD10; left arm, LD3, LD7, and LD11). The six-nucleotide tandem repeats located at nucleotides 13, 19, 30, and 36 (not perfect at nucleotide 38 and indicated by an asterisk) are in boxes. Relevant nucleotide sequences are assigned arbitrary positions (l-m-n-o-p-q-r-s and u-v-w-x-y-z) to assist in retracing the templates used during replication. (B) The rolling-circle melting-pot replication model (adopted from reference 4). (i) PCV1 origin after Rep binding to the octanucleotide (prior to nicking) with the plus- and minus-strand genomes in close proximity to each other. The destabilized environment (i.e., the melting pot) is enclosed by a dotted circle. (ii) Schematic representation of the DNA templates available during initiation of DNA replication after removal of the secondary structure in the model. The leading strand (L) displaces strand a and uses strand a′ or strand b as the template. (iii) Schematic representation of the DNA templates available during termination of DNA replication after removal of the secondary structure in the model. The leading strand (L) displaces strand b and uses the newly synthesized strand a_N or strand b′ as the template. The plus-strand genome is indicated in black, the minus-strand genome is indicated in blue, and the potential base-pairing opportunities available for the current round of DNA replication are indicated in red.

FIG. 2.

Immunochemical staining of Rep-associated antigens in PK15 cells transfected with deletion genomes. The input genome is indicated in each panel.

FIG. 3.

Input genomes and recovered viruses from left-arm mutations. For the input genome, the location of the engineered deletion and the potential configuration(s) of each genome are presented at the left of each panel. The octanucleotide sequences are enclosed in boxes, and the inverted repeat sequences regenerated via template strand switching are shaded but not labeled. “Illegitimate” nucleotides are circled. Deletions are indicated by dotted ovals. The dotted line in the palindrome indicates possible duplication of the preceding sequence. The C₃ and C₁₀ nucleotides of vRD3.5 are indicated by asterisks. The number of examples (specific subclones/total number recovered) of each recovered virus, determined by sequencing cloned PCR fragments, is indicated in parentheses.

FIG. 3.

Input genomes and recovered viruses from left-arm mutations. For the input genome, the location of the engineered deletion and the potential configuration(s) of each genome are presented at the left of each panel. The octanucleotide sequences are enclosed in boxes, and the inverted repeat sequences regenerated via template strand switching are shaded but not labeled. “Illegitimate” nucleotides are circled. Deletions are indicated by dotted ovals. The dotted line in the palindrome indicates possible duplication of the preceding sequence. The C₃ and C₁₀ nucleotides of vRD3.5 are indicated by asterisks. The number of examples (specific subclones/total number recovered) of each recovered virus, determined by sequencing cloned PCR fragments, is indicated in parentheses.

FIG. 3.

Input genomes and recovered viruses from left-arm mutations. For the input genome, the location of the engineered deletion and the potential configuration(s) of each genome are presented at the left of each panel. The octanucleotide sequences are enclosed in boxes, and the inverted repeat sequences regenerated via template strand switching are shaded but not labeled. “Illegitimate” nucleotides are circled. Deletions are indicated by dotted ovals. The dotted line in the palindrome indicates possible duplication of the preceding sequence. The C₃ and C₁₀ nucleotides of vRD3.5 are indicated by asterisks. The number of examples (specific subclones/total number recovered) of each recovered virus, determined by sequencing cloned PCR fragments, is indicated in parentheses.

FIG. 4.

Input genomes and recovered viruses from right-arm mutations. See the legend to Fig. 3 for details.

FIG. 4.

Input genomes and recovered viruses from right-arm mutations. See the legend to Fig. 3 for details.

FIG. 4.

Input genomes and recovered viruses from right-arm mutations. See the legend to Fig. 3 for details.

FIG. 5.

Input genomes and recovered viruses from double-arm mutations. See the legend to Fig. 3 for details.