The Mechanism of DNA damage and chromothripsis from chromosome segregation errors
Alexander Spektor1,2, Cheng-Zhong Zhang1,2, Yingying Zhang1, Neil Umbreit1,2, Maya Feldman1,4, Arifa Ahsan1,4, Matthew Meyerson1,2,3 and David Pellman1,2,3,4*
1Dana-Farber Cancer Institute, 2Harvard Medical School, 3Broad Institute of Harvard and MIT, 4Howard Hughes Medical Institute
Tumorigenesis and resistance to therapy are generally believed to arise through a classical Darwinian process, with gradual, multigenerational accrual of mutations.However, recent cancer genome sequencing suggests that many cancers may accumulate a large number of mutations rapidly, perhaps during the course of a single cell cycle. The most dramatic example of such “punctuated equilibrium-type” rapid genome evolution is chromothripsis, a novel mutational process characterized by massive chromosome rearrangements and a unique pattern of DNA copy number variations, all curiously restricted to one or a few chromosomes. The mechanism(s) leading to chromothripsis have been unclear, but our group previously proposed that it could result through physical isolation of chromosomes in abnormal nuclear structures called micronuclei1. Micronuclei are deficient in key nuclear functions such as nuclear envelope stability, DNA replication, and transcription, and because of these abnormalities develop DNA damage upon entry into S-phase. By combining live-cell imaging with single-cell whole genome sequencing in a method called Look-seq, we recently showed that micronucleation can lead to a large number of rearrangements on the missegregated chromosome within one cell cycle, in some cases recapitulating all the hallmarks of chromothripsis2. Here we describe our latest efforts to elucidate the mechanism of chromothripsis in detail, by examining the timing and cell cycle dependence of events that lead to DNA breakage and chromosome reassembly. Our results indicate that chromosome rearrangements and chromothripsis can occur even in the absence of nuclear envelope rupture during interphase, but may require reincorporation of the micronucleated chromosome into one of the daughter cell nuclei. We are also using CRIPSR/Cas9 gene knockouts in combination with our Look-seq approach to identify the genetic requirements for chromothripsis.Together, these experiments advance our understanding of the ways by which chromosome segregation errors shape cancer genomes.
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