Recurrent DNA Break Clusters Regulated by Polymerase Theta Are Essential for Replication Stress Induced Copy Number Variation
Copy number variation (CNV) is a form of genetic alteration that has been strongly implicated in the pathogenesis of a wide range of neurological disorders. Replication stress is a common cause of CNV. Despite the prevailing model that CNV arises from DNA double strand breaks (DSBs), there has been no assay that directly perturbs presumed DSB sources and measures CNV output. In this article, we identified a subset of recurrent DNA break clusters (RDC) as a causal factor for CNV formation. In murine neural progenitor cells under replication stress, mapping the formation of CNVs revealed their exclusive location in RDC regions that contain actively transcribed genes. CRISPR/Cas9-mediated transcriptional suppression demonstrated that CNV formation depends on transcription-mediated RDCs, but is independent of elevated replication timing. We found that polymerase theta (Pol ?), a  protector against CNV formation,  plays a critical but context dependent role upstream of RDC. Chemically inhibiting the polymerase activity of Pol ? reduced end filling and micro-homology-mediated joining in XRCC4/P53-deficient cells. Conversely, Pol ? inhibition led to elevated DSB density at RDC-containing loci in wild-type neural stem and progenitor cells, suggesting its role in preventing transcription-replication conflict. These results underscore the role of RDCs in driving genome heterogeneity, and suggest their potential involvement in the pathogenesis of various conditions including neuropsychiatric disorders and cancer.