The chromosome breakage-fusion-bridge (BFB) cycle is a mutational process that produces gene amplification and genome instability. Signatures of BFB cycles can be observed in cancer genomes alongside chromothripsis, another catastrophic mutational phenomenon. Here, we explain this association by elucidating a mutational cascade, downstream of the single cell division error of chromosome bridge formation, that rapidly generates extreme genomic complexity.  We show that actomyosin forces are required for initial bridge breakage and mutagenesis, following which chromothripsis accumulates with aberrant interphase replication of bridge DNA.  This is then followed by an unexpected burst of DNA replication in the next mitosis, generating extensive DNA damage.  During this second cell division, broken bridge chromosomes frequently mis-segregate and form micronuclei, promoting additional chromothripsis. We further show that this mutational cascade generates the continuing evolution and sub-clonal heterogeneity characteristic of many human cancers.

Chromosome bridges were generated by the following procedures: (1) TRF2-DN induction with 0.1 μg/ml doxycycline for 14-16 hours; (2) Cas9 induction with 1 μg/ml doxycycline for 14-16 hours in cells constitutively expressing sgRNA targeting the Chr4 subtelomere (see details below); (3) topoisomerase II inhibition with 100 nM ICRF-193; (4) partial depletion of condensin by transfection of 1 nM SMC2 ON-TARGETplus siRNA SMARTpool L-006836-01-0005 (Dharmacon) using Lipofectamine 3000. Cell synchronization and induction of micronuclei by nocodazole block and washout was performed as described cells were treated with 100 ng/ml nocodazole for 6 hours, followed by mitotic-shakeoff.

Long-term live-imaging and correlative single-cell whole-genome sequencing (“Look-Seq”) was performed as previously described to isolate cells of interest. Cell lysis and whole-genome amplification was then performed using the REPLI-g Single Cell kit (Qiagen), and the amplified DNA was purified, sheared to ~500 bp size, and processed with a Library Preparation Kit (KAPA) for multiplexed next-generation sequencing. Copy number and structural variant analysis was then performed for each sample as described in the accompanying manuscript.

For long-term evolution experiments, the Look-Seq procedure was used with the following modifications. Chromosome bridges were formed in RPE-1 cells with doxycycline-inducible CRISPR-Cas9 constituitively expressing sgRNA targeted to a subtelomeric region of Chr4. After bridge breakage, single cells were isolated into 96-well culture plates and then grown into populations. Cells were then taken from the populations for karyotyping and bulk sequencing with the Chromium Single Cell CNV kit (10X Genomics). Sequencing was done on the NovaSeq platform (Illumina). Reads were aligned to the GRCh38 genome and demultiplexed using the CellRanger software (10X Genomics). We then obtained haplotype-specific read depth for each germline heterozygous site using GATK ASEReadCounter (Broad Institute). These provided counts were used to generate plots of haplotype copy number using the provided scripts.

Raw WGS sequencing data for these samples are available via the Sequence Read Archive (SRA) under BioProject PRJNA602546.