Replication Stress Induces Genome-wide Copy Number Changes in Human Cells that Resemble Polymorphic and Pathogenic Variants

Abstract

Copy number variants (CNVs) are an important component of genomic variation in humans and other mammals. Similar de novo deletions and duplications, or copy number changes (CNCs), are now known to be a major cause of genetic and developmental disorders and to arise somatically in many cancers. A major mechanism leading to both CNVs and disease-associated CNCs is meiotic unequal crossing over, or nonallelic homologous recombination (NAHR), mediated by flanking repeated sequences or segmental duplications. Others appear to involve nonhomologous end joining (NHEJ) or aberrant replication suggesting a mitotic cell origin. Here we show that aphidicolin-induced replication stress in normal human cells leads to a high frequency of CNCs of tens to thousands of kilobases across the human genome that closely resemble CNVs and disease-associated CNCs. Most deletion and duplication breakpoint junctions were characterized by short (<6 bp) microhomologies, consistent with the hypothesis that these rearrangements were formed by NHEJ or a replication-coupled process, such as template switching. This is a previously unrecognized consequence of replication stress and suggests that replication fork stalling and subsequent error-prone repair are important mechanisms in the formation of CNVs and pathogenic CNCs in humans.

Hydroxyurea induces de novo copy number variants in human cells

“…HU-induced deletions and duplications were generally large, ranging in size from 1 kb to 35.7 Mb, with a median size of 132 kb (median deletion size, 126 kb; median duplication size, 166 kb). Consistent with our previous report (18), APH-induced deletions and duplications were similar, ranging from 1 kb to 80.3 Mb, with a median size of 165 kb (median deletion size, 163 kb; median duplication size, 191 kb). These sizes are similar to those seen in spontaneous CNVs arising in untreated control cells, which ranged from 19 kb to 1.5 Mb, with a median size of 187 kb (median deletion size, 196 kb; median duplication size, 166 kb).…”

“…The sizes and breakpoint junction sequences of HU-induced CNVs are consistent with the nonrecurrent class of de novo pathogenic CNVs (37-41), a large class of normal human CNVs (5-7, 42), and APH-induced CNVs (17,18,43) (Fig. S4).…”

“…Because meiotic cells do not replicate chromosomes, and nonhomologous repair is down-regulated during meiosis (45), constitutional human CNVs similar to those detected here are predicted to originate in mitotic cells in or leading to the germline (18). The mitotic cell origin hypothesis has important implications for the risk involved in CNV formation.…”

“…However, nonrecurrent CNVs are observed throughout the genome in regions lacking such segmental duplications, and the mechanisms and risk factors involved in their formation are poorly understood. We previously demonstrated that low doses of the DNA polymerase inhibitor aphidicolin (APH) induced de novo CNVs at a high frequency (17,18). These CNVs resembled both normal polymorphic and de novo, nonrecurrent CNVs seen in humans in size and structure.…”

Mechanism of complex gross chromosomal rearrangements: A commentary on Concomitant microduplications of MECP2 and ATRX in male patients with severe mental retardation

“…Indeed, the administration of DNA polymerase inhibitors in cell cultures induces deletions and duplications that have junctions with microhomology. 9 In this context, MMBIR appears to provide a sophisticated explanation for the DNA repair pathway that leads to the complex junctions observed in GCRs. Double-strandbreaks during replication are generally repaired via a homology-dependent pathway whereby DNA ends seek out homologous sequences and then invades the corresponding DNA duplexes with the aid of the RAD51 and RAD52 and commence DNA synthesis (Figure 1, left).…”