ABSTRACT
DNA replication-associated mutations are repaired by two components: polymerase proofreading and mismatch repair. The mutation consequences of disruption to both repair components in humans are not well studied. We sequenced cancer genomes from children with inherited biallelic mismatch repair deficiency (bMMRD). High-grade bMMRD brain tumors exhibited massive numbers of substitution mutations (>250/Mb), which was greater than all childhood and most cancers (>7,000 analyzed). All ultra-hypermutated bMMRD cancers acquired early somatic driver mutations in DNA polymerase É or δ. The ensuing mutation signatures and numbers are unique and diagnostic of childhood germ-line bMMRD (P < 10(-13)). Sequential tumor biopsy analysis revealed that bMMRD/polymerase-mutant cancers rapidly amass an excess of simultaneous mutations (â¼600 mutations/cell division), reaching but not exceeding â¼20,000 exonic mutations in <6 months. This implies a threshold compatible with cancer-cell survival. We suggest a new mechanism of cancer progression in which mutations develop in a rapid burst after ablation of replication repair.
Subject(s)
Base Pair Mismatch , Brain Neoplasms/genetics , DNA Mismatch Repair , DNA Replication/genetics , DNA Repair , DNA-Directed DNA Polymerase/genetics , Exons , Germ-Line Mutation , Humans , Microsatellite InstabilityABSTRACT
R-loops, transcriptionally-induced RNA:DNA hybrids, occurring at repeat tracts (CTG)n, (CAG)n, (CGG)n, (CCG)n and (GAA)n, are associated with diseases including myotonic dystrophy, Huntington's disease, fragile X and Friedreich's ataxia. Many of these repeats are bidirectionally transcribed, allowing for single- and double-R-loop configurations, where either or both DNA strands may be RNA-bound. R-loops can trigger repeat instability at (CTG)·(CAG) repeats, but the mechanism of this is unclear. We demonstrate R-loop-mediated instability through processing of R-loops by HeLa and human neuron-like cell extracts. Double-R-loops induced greater instability than single-R-loops. Pre-treatment with RNase H only partially suppressed instability, supporting a model in which R-loops directly generate instability by aberrant processing, or via slipped-DNA formation upon RNA removal and its subsequent aberrant processing. Slipped-DNAs were observed to form following removal of the RNA from R-loops. Since transcriptionally-induced R-loops can occur in the absence of DNA replication, R-loop processing may be a source of repeat instability in the brain. Double-R-loop formation and processing to instability was extended to the expanded C9orf72 (GGGGCC)·(GGCCCC) repeats, known to cause amyotrophic lateral sclerosis and frontotemporal dementia, providing the first suggestion through which these repeats may become unstable. These findings provide a mechanistic basis for R-loop-mediated instability at disease-associated repeats.
