Abstract 2732: Short inverted repeats are hot spots for genetic instability in mammalian cells

Abstract

Abstract Analyses of chromosomal aberrations in genetic disorders such as leukemia and lymphoma have provided compelling evidence that segments of the human genome are prone to breakage that lead to DNA rearrangements. Many of these hot spot areas contain sequences that have the potential to form non-B DNA such as Z-DNA, H-DNA or cruciforms. Although research has shown that large palindromes are mutagenic in mammalian cells, the instability of short inverted repeat sequences (30 bp), which are mapped near breakpoints and quite abundant in the eukaryotic genome, has not been investigated. In order to study the effects of short cruciform-forming sequences on genetic instability, we subcloned short AT- and CG-rich inverted repeats into a lacZ’ mutation-reporter shuttle vector. The cruciform structures that formed at the palindromic sequences were probed using S1 nuclease to confirm the presence of the non-B DNA structure. Cruciform-induced mutagenesis was measured in mammalian COS-7 cells via transfection then blue-white screening in DH5α cells. Ligation-mediated PCR provided direct evidence that cruciform-forming sequences induced double-stranded breaks in mammalian cells. In vitro replication assays using SV40- HeLa cell-free extracts were employed to establish the connection between replication and cruciform-induced genetic instability. Results from our study demonstrate for the first time that cruciform structures that form at short inverted repeats are mutagenic in mammalian cells. The majority of the mutants induced by cruciforms were large-scale deletions that spanned the inverted repeat sequence. Analyses of junctions revealed that >80% of the mutants contained microhomologies which are characteristic of error-prone nonhomologous end-joining repair. This suggests that cruciform structure is cleaved by replication-independent, structure-specific nucleases in mammalian cells that result in DNA double-stranded breaks at positions directly adjacent to the inverted repeat. These results indicate that cruciform-forming sequences may play a role in genetic instability and contribute to human diseases associated with gene translocations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2732. doi:10.1158/1538-7445.AM2011-2732