Loss of DNA replication control is a potent inducer of gene amplification

Abstract

Eukaryotic cells use numerous mechanisms to ensure that no segment of their DNA is inappropriately re-replicated, but the importance of this stringent control on genome stability has not been tested. Here we show that re-replication in Saccharomyces cerevisiae can strongly induce the initial step of gene amplification, increasing gene copy number from one to two or more. The resulting amplicons consist of large internal chromosomal segments that are bounded by Ty repetitive elements and are intrachromosomally arrayed at their endogenous locus in direct head-to-tail orientation. These re-replication-induced gene amplifications are mediated by nonallelic homologous recombination between the repetitive elements. We suggest that re-replication may be a contributor to gene copy number changes, which are important in fields such as cancer biology, evolution, and human genetics.

Fig. 1

Re-replication greatly stimulates gene amplification. (A) Induction of re-replication from ARS317. Strains containing an ade3-2p copy number reporter cassette integrated at Chr IV_567kb were arrested in G₂/M phase and treated with galactose to trigger re-initiation of ARS317. aCGH analysis of re-replication (>2C) is shown for Chr IV. All other chromosomes maintained a 2C copy number. Top: YJL6974, a non–re-replicating strain with ARS317 in cassette. Middle: YJL6555, a re-replicating strain with no ARS317 in cassette. Bottom: YJL6558, a re-replicating strain with ARS317 in cassette. (B) ARS317 re-replication stimulates gene amplification. Left: Frequency of one-half to one-eighth red-sectored colonies (mean ± SEM, n = 2 to 7 induction replicates; table S1) after 0 or 3 hours of galactose induction of YJL6974, YJL6555, and YJL6558. Right: Amplification frequency estimated by multiplying sector frequency by the fraction of sectored colonies displaying reporter cassette amplification (tables S2 and S4). (C) Red sectors induced by re-replication display gene amplifications. Thirty-five red sectors derived from YJL6558 were analyzed by aCGH and classified on the basis of the copy number profile of Chr IV. A schematic of Chr IV (top) shows the positions of Ty elements (triangles), the centromere (circle), and the ade3-2p cassette (black bar).

Fig. 2

Structure of gene amplifications induced by re-replication. (A) Amplicons remain on the endogenous chromosome. Pulsed-field gel electrophoresis–separated chromosomes from 24 sectors analyzed in Fig. 1C (table S2) were visualized by ethidium bromide staining (left), then probed for ADE3, which detects both ade3 on Chr VII and ade3-2p in the reporter cassette (right). YJL6558 (the parental strain), YJL7100, YJL7106, YJL7113, and YJL7118 did not contain amplifications. The Chr IV doublet pattern for YJL7098 and YJL7099 is consistent with the partial loss of amplification from the population. (B) Amplicons are tandemly arrayed in loco in direct head-to-tail orientation. Schematics of an unamplified amplicon and three possible orientations for amplicons tandemly duplicated in loco are shown. Predicted PCR junction fragments (10) 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 YJL6558 and 19 of the 20 amplified strains displayed in (A).

Fig. 3

Role of nonallelic homologous recombination in RRIGA. (A) Schematic of hybrid recombinant Ty elements (fig. S4) found at the interamplicon junction of four isolates with segmental amplifications on Chr IV from 515 to 650 kb. (B) Re-replication–induced sectoring is dependent on HR and not NHEJ. YJL7443 (dnl4Δ) and YJL7452 (rad52Δ) are otherwise isogenic with re-replicating strain YJL6558. Re-replication–induced sectoring frequency (mean ±SEM, n = 2 to 7 induction replicates; table S1) was analyzed as described for Fig. 1B. (C) Re-replication–induced gene amplification is dependent on HR and not NHEJ. A representative aCGH analysis of Chr IV for 24 red sectors derived from YJL7443 (dnl4Δ) and 48 derived from YJL7452 (rad52Δ) is shown (table S6). (D) How re-replication might stimulate NAHR. Arrowheads, nonallelic or hybrid recombinant repetitive element; arrows, amplified segments.