Abstract
Analyses of chromosomal aberrations in human genetic disorders have revealed that inverted repeat sequences (IRs) often co-localize with endogenous chromosomal instability and breakage hotspots. Approximately 80% of all IRs in the human genome are short (<100bp), yet the mutagenic potential of such short cruciform-forming sequences has not been characterized. Here, we find that short IRs are enriched at translocation breakpoints in human cancer and stimulate the formation of DNA double-strand breaks (DSBs) and deletions in mammalian and yeast cells. We provide evidence for replication-related mechanisms of IR-induced genetic instability and a novel XPF cleavage-based mechanism independent of DNA replication. These discoveries implicate short IRs as endogenous sources of DNA breakage involved in disease etiology and suggest that these repeats represent a feature of genome plasticity that may contribute to the evolution of the human genome by providing a means for diversity within the population.
Highlights
Genetic analyses of cancer-related genetic instability events have detected regions of the human genome that are hypersusceptible to breakage, which can lead to the deregulation of oncogenes and/or inactivation of tumor suppressors (Popescu, 2003)
This study fills a gap in our understanding of the role of short inverted repeat sequences (IRs) in genomic instability in mammals by providing evidence that cruciforms formed at short IRs (≤30 bp) can stimulate double-strand breaks (DSBs) by stalling DNA replication forks and/or by activating enzymes (i.e. ERCC1-XPF) that cleave the structures, causing deletions. These findings provide a mechanistic explanation for the co-localization between short IRs and human cancer breakpoints, and support the hypothesis that non-B DNA is involved in genetic instability, disease etiology, and evolution
Cruciform formation on the plasmid was confirmed by T7 endonuclease I cleavage (Figure S1). pU+ and the control pUCON were introduced into mammalian COS-7 cells and screened for mutations 48 h post-transfection. pU+ stimulated mutations ~3-fold above that of pUCON (9.2×10−3 vs. 2.9×10−3: P
Summary
Genetic analyses of cancer-related genetic instability events have detected regions of the human genome that are hypersusceptible to breakage, which can lead to the deregulation of oncogenes and/or inactivation of tumor suppressors (Popescu, 2003). Many such regions contain sequences that can adopt alternative conformations (i.e. non-B DNA), and several of these conformations have been shown to be sources of genetic instability (Kurahashi et al, 2004; Nasar et al, 2000; Wang and Vasquez, 2006), yet the underlying mechanisms are not clear. The mutagenic potential of short IRs has not been well characterized
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