Figure 1.
(A) Growth recovery assay [20]. Black circles and triangles represent centromeres and telomeres, respectively. White open arrow represents the RPL20B gene (YOR312C) whose duplication is selected for. Yellow and pink boxes denote intra- (left) and one type of inter-chromosomal (right) duplications, respectively. A non-reciprocal translocation event between the right arm of chromosome XV and another chromosome (denoted “n”) is represented: for other types of inter-chromosomal SD (i.e. chimerical supernumerary chromosome and unequal reciprocal translocation, see [20] and [21]). SD size ranges are indicated below the double-headed arrows. (B) Uracil prototrophy recovery assay. Top: schematic representation of the right arm of chromosome XV spanning the RPL20B locus and the two flanking Ty3 LTRs (YORWsigma3 and YORWsigma4) located 115 kb apart from each others. 5′- and 3′-truncated are either inserted next (YORWsigma3) or replaces (YORWsigma4) Ty3 sequences. The “R”-labeled red box indicates the 58 or 401 bp overlap between the two truncated URA3 cassettes. Bottom: a functional URA3 gene restoring uracil prototrophy is generated through 115 kb direct-tandem duplication events involving the overlapping sequences. (C) Size distribution of intra-chromosomal SDs. The x and y-axis of the diagram indicate the strain background and the percentage of events recovered, respectively. Yellow, violet and blue bars represent the proportion of duplications larger, equal to and smaller than 115 kb, respectively (with the actual number of events analyzed indicated in the table below). (D) Phenomenology of SD formation. Protein names involved in the different steps are indicated to the left of the diagram. Red, orange and blue names represent proteins whose deletions abolish, reduce and increase SD formation, respectively. Light and medium grey boxes indicate the two alternative mechanisms of SD formation, BIR (Break-induced Replication) and MMIR (Microhomology/Microsatellite-induced Replication), respectively. CPT = camptothecin.
Table 1.
SD characteristics in different genetic backgrounds.
Figure 2.
Representative breakpoint sequences of non LTR-mediated duplications.
Only events leading to chimerical ORF are presented. a WT junctions are from [20]. Top and bottom sequences correspond to centromere-distal and -proximal sequences, respectively, followed by the name of the genetic element involved at the junctions. Shaded areas indicate the regions of sequence identity shared by these two sequences and correspond to the breakpoint per se. The coordinates in brackets correspond to the first nucleotide position within the shaded areas. For each strain, the middle sequence corresponds to the actual breakpoint sequence followed by a description of the chimerical genetic element recovered at the junction. Neighboring elements correspond to sequences known to participate or interfere with replication, with slow-zone corresponding to inflection point in the replication pattern (i.e. regions where fork progression slows down, [67]), ter site to termination regions, and ARS to autonomous replicating sequences. On the right, the schematic representations with orientated grey and black boxes represent the structure of the chimerical elements generated with the sizes of the corresponding chimerical ORFs (in aa). The contribution (in aa) of each of the two elements involved in the fusion is indicated above and below the corresponding boxes. Amino acids encoded by a frame different from that of the original elements are referred as “new”.
Figure 3.
Potential interplay between replication factories and SD formation.
The top two drawings illustrate the conservation of the physical distance between two forks within the same replication bubble as elongation proceeds. Red/blue and grey bubbles symbolize replicons located on two different chromosomes but co-localizing within the same replication factory. A broken fork can be repaired either in a Rad52-dependent (i) or in a Rad52-independent (ii) manner. Rad52-dependent annealing could be achieved through interaction with a sequence from either the same or a different replication bubble (symbolized by the sharp sign between the two replication factories) leading to intra- or inter-chromosomal SD. In a rad52Δ strain, SDs are on average shorter and almost exclusively intra-chromosomal (bottom right schematic representation), suggesting a Rad52-independent preferential association between sequences originating from the same replication bubble during the annealing step. A schematic representation of the resulting duplications is presented at the bottom.