Origin-Dependent Inverted-Repeat Amplification: A Replication-Based Model for Generating Palindromic Amplicons
In each of the examples I–IV, initiation of replication from an origin near the sequence labeled E generates bidirectional forks that have progressed through the flanking sequences D and F. In the four scenarios depicted, either one or both of the replication forks becomes “closed” by ligation of the leading strand to the lagging strand. Open arrows indicate the direction that flanking replication forks move to expel the “closed” fork intermediate by branch migration. (I) In this example, both forks “close.” Replication forks approaching from either direction (open arrows) will release the hairpin-capped linear that contains genes D, E, and F. Subsequent replication of this “dog bone” molecule generates a dimeric, palindromic circular molecule that can reintegrate through homologous recombination into the original chromosome, or into a homolog if one is present, or by random integration elsewhere in the genome. These steps are described in detail in Figure 2. (II) In this example, the fork closest to the centromere “closes” while the telomere proximal fork remains active, fusing with distal replicons to allow replication of segments G, H, and the right telomere. The fork moving outward from the centromere (open arrow) will dislodge a fragment that has a hairpin at one end and a telomere at the other. Subsequent replication results in an isochromosome containing genes D–H. These acentric chromosomes are relatively stable in yeast , , and in human cells stability can be improved by the acquisition of a neocentromere  or by chromosome tethering , . (III) In this example, only the distal fork closes. Completion of replication by a fork from the telomere proximal side of the closed fork releases a similar intermediate as in (II). Replication of the fragment with a hairpin near gene F generates an isochromosome that contains the centromere and genes A–F. Cycles of BFB will occur until the break is healed by the addition of a telomere. (IV) This example begins as the one in (III); however, the fork near gene D suffers a ssDNA break. Repair of this break by ligation of the broken strand to the nascent strand generates a branched intermediate that can be resolved by a fork moving in from the region of genes G and H. Two aberrant chromosome fragments are generated by this resolution, both requiring telomere addition to become resistant to nucleases. The fragment containing the centromere is an “inv dup del” chromosome indistinguishable from a chromosome generated by BFB (III). The fate of the acentric fragment is expected to be similar to that of an acentric isochromosome.