Figure 1.
Homologous sequences are necessary and sufficient in cis to support RRIGA.
A) (i) Schematic of RRIGA arising from NAHR between YDRCTy2-1 at 515 kb and YDRCTy1-1 at 650 kb on Chromosome IV; (ii) Schematic of RRIGA arising from NAHR between 3′URA3 (RA3) and 5′URA3 (UR) fragments replacing YDRCTy2-1 and YDRCTy1-1, respectively (see Figure S1). Frequencies shown are for replacement of Ty and all adjacent LTR and tRNA sequences (version 2). B) (i) Sectoring frequencies (mean ± SEM, n = 3 to 5) before (0 hr) and after (3 hr) induction of re-replication in strains with endogenous Ty elements at 515 kb and 650 kb (YJL8100), with YDRCTy2-1 replaced by RA3 (YJL8355), with YDRCTy1-1 replaced by UR (YJL8359), or both Ty elements replaced with the respective URA3 fragments (YJL8363); (ii) Re-replication induced amplification frequency estimated by multiplying 3 hr sector frequency by fraction of sectors containing 515–650 kb amplification (see Figure S3).
Figure 2.
A selection-based assay for detecting RRIGA events.
A) Comparison of RRIGA frequencies (mean ± SEM) measured using sectoring assay (n = 4) or URA3 selection assay (n = 5) in strains (YJL8363/8364) with YDRCTy2-1 and YDRCTy1-1 replaced by URA3 fragments as described for Figure 1B. B) Isolates from URA3 selection assay have amplifications spanning the segment between URA3 fragments (Chromosome IV 515–650 kb). aCGH copy number analysis of Chromosome IV shown for 12 isolates selected before (0 hr) and 32 isolates selected after (3 hr) re-replication from YJL8112/8113 and YJL8363/8364. Chromosome IV schematic shows position and orientation of Ty elements (triangles), centromere (circle), and ARS317-ade3-2p re-initiation cassette (bar and vertical line). C) Isolates from URA3 selection assay have amplifications tandemly arrayed in loco in direct repeat. The unamplified parental amplicon and three possible orientations for tandem duplications in loco are shown schematically. Predicted PCR junction fragments are shown for five sets of primers that flank amplicon boundaries (+, PCR product expected; −, no PCR product expected). Representative PCR products are shown for parental strain YJL8363 and 10 re-replication-induced isolates from B. D) Amplicons appear to be chromosomally integrated but excisable. The 10 isolates tested by PCR in C were grown on non-selective media (YEPD), then replica plated to media lacking uracil (SDC-Ura) or media containing 5-FOA (SDC+5-FOA). Patch A, YJL8698 with extrachromosomal copy of URA3. Patch B, YJL6974 with integrated but excisable copy of URA3. Patch C, YJL8344 with integrated and un-excisable URA3.
Figure 3.
RRIGA is primarily mediated by single-stranded annealing (SSA).
A) Summary of genetic requirements for the major sub-types of homologous recombination. SSA = Single Stranded Annealing; GC = Gene Conversion; BIR = Break Induced Replication. “+” = required; “−” = not required; “+/−” = required in some but not all cases; “n.d.” = not determined. B) RRIGA amplification frequencies for WT (YJL8363/8364), dnl4Δ (YJL8407/8408), rad52Δ (YJL8409/8410); rad51Δ (YJL8412/8413), rad1Δ (YJL8415/8416), msh3Δ (YJL8418/8419), and pol32Δ (YJL8421/8422) strains using the URA3 selection assay. Difference in frequency after 3 hr and 0 hr induction of re-replication was normalized for differences in the amount of re-initiation (see Text S1 and Figure S4). Data are presented as the mean ± combined error (see Text S1). C) Model for RRIGA involving SSA-mediated NAHR. Arrow, amplified segment. Triangles, non-allelic homologous sequences.
Figure 4.
Re-replication induces double-stranded DNA breaks distal to flanking repetitive elements.
A) Strategy for mapping DSBs arising during re-replication using an I-SceI site near the re-initiating origin ARS317 as a physical reference point (see Text S1). The lengths of the small linear fragments generated by DSBs indicate the position of the DSBs relative to the I-SceI cut site. B) Representative Southern blot for DSBs induced by rightward moving re-replication forks. Re-replicating MC2A strains (YJL8425/YJL8426) were induced to re-replicate and at the indicated times chromosomal DNA was prepared, digested with I-SceI, size-separated by PFGE, Southern blotted, and probed for fragments extending rightward from the I-SceI site. Genomic DNA from non-re-replicating MpGAL control strains (YJL8427/8428) were processed in parallel. Unbroken: full-length Chromosome IV fragment from I-SceI site to right telomere. Bracket: fragments due to DSBs that map origin-distal to YDRCTy1-1. * unidentified DNA fragment present independent of re-replication. C) Distribution of DSBs induced by re-replication from ARS317 (see Text S1 and Figure S5). For each 2425 bp size range, the amount of re-replication induced fragmentation within that size range is displayed as a percent of the total amount of G2/M chromosomes before re-replication was induced. Positions are relative to ARS317 (at 0 kb) with positions of CEN4 (a), YDRCTy2-1 (b), and YDRCTy1-1 (c) indicated.
Figure 5.
Flanking repetitive elements must be re-replicated in order for RRIGA to occur.
A) Schematic showing relocation of the RA3 and UR elements (3′ and 5′ portions of URA3, respectively) used in the URA3 RRIGA selection assay to change their position relative to the re-initiating origin (ARS317, light blue line) and the distribution of re-replication forks (as inferred from the re-replication profile). B) Induced amplification frequencies (mean ± SEM, n = 3) for strains with the indicated amplicon boundaries as defined by the position of the URA3 fragments. Induced frequency is frequency after 3 hr re-replication minus frequency after 0 hr re-replication.
Figure 6.
RRIGA proceeds most efficiently when the re-initiating origin lies within the amplicon.
A) Schematic comparing SSA models for RRIGA when the re-initiating origin is within the amplicon versus when the origin is outside. The latter case requires long range strand resection back beyond the re-initiating origin in order to expose homologous sequences for NAHR. B) Schematic showing relocation of the RA3 element to change its position relative to the re-initiating origin (ARS317, light blue line). The re-replication profile is shown above. C) Induced amplification frequencies (mean ± SEM, n = 3) for strains with the indicated amplicon boundaries. Induced amplification frequency calculated as described for Figure 5B.