Recombination-restarted replication makes inverted chromosome fusions at inverted repeats - PubMed (original) (raw)

. 2013 Jan 10;493(7431):246-9.

doi: 10.1038/nature11676. Epub 2012 Nov 25.

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Recombination-restarted replication makes inverted chromosome fusions at inverted repeats

Ken'Ichi Mizuno et al. Nature. 2013.

Abstract

Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S-phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination or microhomology-primed re-initiation. Here we ascertain the consequences of replication with a fork restarted by homologous recombination in fission yeast. We identify a new mechanism of chromosomal rearrangement through the observation that recombination-restarted forks have a considerably high propensity to execute a U-turn at small inverted repeats (up to 1 in 40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer, and to non-recurrent CNVs in genomic disorders.

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Figures

Figure 1

Figure 1

Alternative mechanism for inverted chromosomal fusion. A. Cartoon of double and single RTS1 constructs. Replication origins (ars) on Chromosome 3, their distance from ura4 and predicted efficiencies are shown. Open circle indicates centromere 3. Concave blue and dark boxes represent RTS1 or 3×Ter2/3 as indicated. Yellow arrows/boxes represent ura4 sequences and green box represents his3 sequences. Black arrow indicates IRs of the ura4 sequences. Open triangle shows 14 bps interrupting sequence at the palindrome centre. Red bars represent probes. B indicates _Bgl_II restriction site. Sizes of initial and predicted dicentric chromosome _Bgl_II fragments generated by replication template exchange are shown for each relevant strain. B. A representative southern blot of double and single RTS1 strains with arrest off or arrest on. Genomic DNA was digested with _Bgl_II and probed with pB. C. Quantitation of rearranged fragment in B. Mean value and standard deviation of the values are calculated from at least three independent experiments.

Figure 2

Figure 2

Rearrangement frequency is dependent on the repeat size and interrupting sequence size. A. Cartoon of constructs with varying repeat size. P(2400), P(0) noIR and intermediates P(W),are indicated as in Figure 1A. W represents the size of the whole palindrome in bps. X shows the size of the ura4 fragment creating the IR. Grey box indicates heterologous sequence (V). The sum of X and V is always 1200 bps. Probes pB is indicated as red bar. B. Southern blot analyses of P(W) strains for arrest off or arrest on. Genomic DNA was digested with _Bgl_II and probed with pB. C. Quantitation of rearranged fragment in B. Average values and standard deviation are calculated from at least three independent experiments. D. Cartoon of constructs varying interrupting sequence [P(1200)IS(Y)] indicated as in Figure 2A. Y and W represent the size of interrupting sequence and the whole palindrome in bps, respectively. E. Southern blot analysis of P(1200)IS(Y) strains for arrest off or arrest on. Southern blot was performed as described in Figure 1B. F. Quantitation of rearranged fragment in E as described in Figure 1C. Mean value and standard deviation of the values are calculated from at least three independent experiments.

Figure 3

Figure 3

Fidelity of HR-restarted fork improves with distance. A. Cartoon of constructs varying distance from restart site [P(1200)D(Z)]indicated as in Figure 2A. The whole palindrome is 1214 bps. Z indicates distance of the palindrome from RTS1 in kb. B. Southern blot analysis of P(1200)D(Z) strains for arrest off or arrest on. Southern blot was performed as described in Figure 1B. C. Quantitation of rearranged fragment in E as described in Figure 1C. Mean value and standard deviation of the values are calculated from at least three independent experiments.

Figure 4

Figure 4

U-turn at palindrome centre is major mechanism for inverted fusion in double RTS1. A. Southern blot analyses of RuiuR, Rpal1R and Tpal1R for arrest off or arrest on. Genomic DNA was digested with _Bgl_II and probed with pB or pU3 - see Figure 1A. Note that the majority of the rearrangement in Rpal1R detected by probe pU3 is acentric 6.6kb. B. Model for error-prone progression of a recombination-restarted replication fork. Oval, blue concave, and yellow box represent replication origin ARS, obstacle and repeat sequences, respectively. When a replication fork collapses, homologous recombination restarts the collapsed fork. However, the restarted fork is non-canonical and error-prone, causing GCRs at IRs due to executing a U-turn.

References

    1. Lupski JR. Genomic disorders: structural features of the genome can lead to DNA rearrangements and human disease traits. Trends in genetics: TIG. 1998;14:417–422. - PubMed
    1. Liu P, Carvalho CM, Hastings P, Lupski JR. Mechanisms for recurrent and complex human genomic rearrangements. Curr Opin Genet Dev. 2012;22:211–220. - PMC - PubMed
    1. Mondello C, Smirnova A, Giulotto E. Gene amplification, radiation sensitivity and DNA double-strand breaks. Mutat Res. 2010;704:29–37. - PubMed
    1. Lee JA, Carvalho CM, Lupski JR. A DNA replication mechanism for generating nonrecurrent rearrangements associated with genomic disorders. Cell. 2007;131:1235–1247. - PubMed
    1. Hastings PJ, Ira G, Lupski JR. A microhomology-mediated break-induced replication model for the origin of human copy number variation. PLoS Genet. 2009;5:e1000327. - PMC - PubMed

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