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. 2014 Jun 12;7(5):1640-1648.
doi: 10.1016/j.celrep.2014.04.053. Epub 2014 May 29.

Break-induced replication is a source of mutation clusters underlying kataegis

Cynthia J Sakofsky  1 , Steven A Roberts  2 , Ewa Malc  3 , Piotr A Mieczkowski  3 , Michael A Resnick  2 , Dmitry A Gordenin  4 , Anna Malkova  5
Affiliations

Break-induced replication is a source of mutation clusters underlying kataegis

Cynthia J Sakofsky et al. Cell Rep. .

Abstract

Clusters of simultaneous multiple mutations can be a source of rapid change during carcinogenesis and evolution. Such mutation clusters have been recently shown to originate from DNA damage within long single-stranded DNA (ssDNA) formed at resected double-strand breaks and dysfunctional replication forks. Here, we identify double-strand break (DSB)-induced replication (BIR) as another powerful source of mutation clusters that formed in nearly half of wild-type yeast cells undergoing BIR in the presence of alkylating damage. Clustered mutations were primarily formed along the track of DNA synthesis and were frequently associated with additional breakage and rearrangements. Moreover, the base specificity, strand coordination, and strand bias of the mutation spectrum were consistent with mutations arising from damage in persistent ssDNA stretches within unconventional replication intermediates. Altogether, these features closely resemble kataegic events in cancers, suggesting that replication intermediates during BIR may be the most prominent source of mutation clusters across species.

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Figures

Figure 1
Figure 1. DSB repair by BIR
(A) Model of BIR. Dotted lines: newly synthesized DNA. (B) Experimental system to study BIR in yeast. The percent occurrence of the main DSB repair outcomes and their relevant phenotypes are indicated with total isolates scored in parenthesis. Asterisks: statistically significant increase of half-crossovers in MMS versus no-MMS. See Supplemental Experimental Procedures and Table S1 for details.
Figure 2
Figure 2. Mutagenesis associated with BIR in the presence of 1.5mM MMS
(A) The structure of one representative outcome (ALM_31). Left: ethidium bromide-stained PFGE gel. Middle and right: Southern blot analysis of PFGE gel using ADE1- and ADE3- specific probes, respectively. (B) Coverage of Illumina sequencing reads for BIR event (ALM_31) is increased 2x centromere distal to MAT (positions >194180bp) as compared to the parental strain. (C) MMS-induced mutations (blue lines) in ALM_31. Enlarged: mutation cluster on the track of BIR. (D) Clustered mutations in Ade+ Leu- BIR isolates. Positions of mutated bases (colored circles) are depicted along the chromosome III reference. (E and F) Select BIR-associated mutation clusters with mutations located in recipient (E) or donor (F) chromosomes.
Figure 3
Figure 3. Mutagenesis associated with BIR in the presence of 6mM MMS
(A) Chromosome structures of representative BIR outcomes. Upper panel: ethidium bromide stained PFGE gel. Middle and lower panels: Southern blot analysis with ADE1- or ADE3- specific probes, respectively. (B) Coverage of Illumina sequencing reads derived from representative Ade+ Leu- isolates (2x and 3x: fold increases as compared with the parental strain). See Supplemental Experimental Procedures for details. (C) Clustered mutations on chromosome III in Ade+ Leu- isolates indicated as in Figure 2D. (D) Distribution of mutations between the recipient and donor chromosomes for the representative isolates indicated in (C). Complete information about cluster structure and formation is given in Figures S1-S4.
Figure 4
Figure 4. Model of BIR-induced cluster formation
DNA lesions (red stars) from MMS are shown in ssDNA formed by resection and BIR synthesis. Red squares: mutations from TLS bypass. Scissors: Resolution of HJ-like structure. Details of resolution are unknown. Yellow rectangle: damage in dsDNA. One possible scenario of lagging strand synthesis is shown. See text and Supplemental Figures S2, S3 and S4 for details.
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