In mammals, the putative involvement of circular intermediates has been only postulated in the generation of two translocations causing a specific phenotype by disruption of the acceptor site in the cattle genome. Whether this was a singular mutation event, a peculiar bovine feature, or a more common mechanism of genome evolution was not determined [15]. We provide evidence of a similar mechanism behind the generation of some large duplications fixed in the human genome.
Our data support the involvement of circular DNA intermediates and suggest a replicative interaction between the donor and acceptor sites in the generation of these duplications. The most parsimonious explanation for the A-B/C-D to B-A/D-C specific flip in sequence order observed between the ancestral and derivative cSDPs would be the circularization of the ancestral cSDP by the fusion of its end points A and D, and the opening of the circular intermediate for re-insertion at single and different breaking points (B/C) (Figure 1A) [11]. Alternative mechanisms previously suggested, such as transposition followed by inversion that separated the blocks, would place the blocks in inverted direction (B-A/C-D). Thus, a second inversion of exactly the remaining block would be required to generate the observed A-B/C-D to B-A/D-C flips.
Although not specific, additional features that could be related to the generation mechanism of these cSDPs include: (i) the absence of homology in the sequence regions overlapping the breaking junctions of the cSDPs ruling out a homologous recombination mechanism in the formation and in the integration of the circular intermediates; (ii) the presence of micro-rearrangements in the sequences overlapping the breaking junction: short deletions and/or insertions of 1 to-13 bp and/or micro-homologies of 1 or 2 bp; and (iii) a non-tandem location of the ancestral and derivative duplicates. Although the formation and/or insertion of the circular intermediate could only be predicted at the nucleotide level in eight cSDPs, the information provided by the scars left by the circular intermediate formation and integration suggests the implication of a non-replicative non-homologous end joining (NHEJ) mechanism in the formation of the intermediates and is compatible with either NHEJ or to replicative Microhomology-Mediated Break-Induced Replication (MMBIR) / Fork Stalling and Template Switching (FoSTeS) mechanism in its insertion. These informative scars, both in the fusion and insertion breakpoints, are similar to the ones determined in one of the two translocations generated by means of circular intermediates in cattle: a two bp micro-homology typical of NHEJ in the fusion breakpoint of the circular intermediate and micro-duplications and micro-deletions reminiscent of MMBIR in the opening of the intermediate [15]. Furthermore, like in the bovine translocation, the breakpoints of cSDPs mapped to interspersed non-homologous repeat elements suggesting a possible contribution of these elements in the duplication mechanism. On the other hand, the repetitive elements content within ancestral cSDPs matched that of the corresponding chromosomes which suggests repetitive elements within the cSDPs did not contribute to their formation [22].
Three main questions need to be answered: (i) how could a linear segment circularize by fusion of its proximal and distal ends, a requisite for the cSDPs specific flip in sequence, in absence homologous recombination or inverted repeats?; (ii) how could the circular intermediates integrate in the genome in absence of homologous recombination?; and finally (iii), how to account for the large genomic distance between the ancestral and derivative loci?
One possible explanation for the first two questions would be a mechanism like the one reported for chromoanasynthesis [23], localized chromosome rearrangements with variable gains in copy number particularly in cancer genomes. This model postulates that an unexcised interstrand crosslink could lead to breakage of the sister chromatid, with circularization of a retained fragment and integration of the fragment into the genome [23]. In this mechanism, the donor linear segment circularizes by the rejoining of the two ends of the broken chromatid, an event that in our proposed circular intermediate mechanism corresponds to the generation of the fusion point (A/D). Furthermore, this chromatid rejoining will produce the characteristic flip in sequence order observed in the cSDPs. The genome scar signals left by the rejoining of the broken ends A and D in the cSDPs as well as the ones reported in the bovine translocations, two bp micro-homologies, one bp insertions or between two directly adjacent nucleotides suggests a non-replicative mechanism by NHEJ, as previously proposed [15]. Nevertheless, sequence features at the breakpoints are insufficient to distinguish between the NHEJ and MMBIR/FoSTeS mechanisms [24]. In this sense, a replicative MMBIR-like mechanism and homology-directed repair in S-phase has been recently described to explain the formation of circular DNA from the CUP1 locus in yeast [ 25].
On the other hand, the absence of homology and the presence of only small deletions/insertions as genomic scars and micro-homologies at the integration points of the circular intermediates for cSDPs (breaking junctions B/C and α/β) as found in the bovine translocations suggests the involvement of a replicative MMBIR mechanism [15]. The replicative MMBIR/FoSTeS repair pathways have been implicated in various genomic rearrangements including chromoanasynthesis [23]. In this regard, chromoanasynthesis generated by mutagenesis in C. elegans produces two patterns of copy-number increase in the offspring: one pattern with copy number gain from 2 to 3, indicating a simple reintegration of a retained sister chromatid fragment; and a second pattern with up to fivefold copy-number increases of clustered chromosome regions that could be indicative of rolling circle replication mechanism [26, 27]. The copy number pattern of cSDPs of only two suggest the generation of the cSDPs occurred as discrete step by a simple and single reintegration of the recircularized fragment and not by a rolling circle mechanism [28].
The MMBIR/FoSTeS model proposes that after a replication fork stalls the polymerase can switch templates and, depending upon the relative location and orientation of the replication origins, results in directed or inverted tandem duplication, inversion, translocation, or more complex rearrangements [29-31].
Additionally, it has been proposed that, although the involved forks in MMBIR/FoSTeS could be separated by sizeable linear distances or in different chromosomes, they must be adjacent or in close proximity in three-dimensional space, perhaps within replication factories [32]. Further analyses of SDs in human and other species’ as well as in cancer cells and the study of non-recurrent de novo duplications in somatic cells with bioinformatic and experimental tools [4, 33] are needed to define the real role of these circular intermediates in genome plasticity during evolution, health and disease.