Abstract
Although alternative DNA secondary structures (non-B DNA) can induce genomic rearrangements, their associated mutational spectra remain largely unknown. The helicase activity of WRN, which is absent in the human progeroid Werner syndrome, is thought to counteract this genomic instability. We determined non-B DNA-induced mutation frequencies and spectra in human U2OS osteosarcoma cells and assessed the role of WRN in isogenic knockdown (WRN-KD) cells using a supF gene mutation reporter system flanked by triplex- or Z-DNA-forming sequences. Although both non-B DNA and WRN-KD served to increase the mutation frequency, the increase afforded by WRN-KD was independent of DNA structure despite the fact that purified WRN helicase was found to resolve these structures in vitro. In U2OS cells, ∼70% of mutations comprised single-base substitutions, mostly at G·C base-pairs, with the remaining ∼30% being microdeletions. The number of mutations at G·C base-pairs in the context of NGNN/NNCN sequences correlated well with predicted free energies of base stacking and ionization potentials, suggesting a possible origin via oxidation reactions involving electron loss and subsequent electron transfer (hole migration) between neighboring bases. A set of ∼40,000 somatic mutations at G·C base pairs identified in a lung cancer genome exhibited similar correlations, implying that hole migration may also be involved. We conclude that alternative DNA conformations, WRN deficiency and lung tumorigenesis may all serve to increase the mutation rate by promoting, through diverse pathways, oxidation reactions that perturb the electron orbitals of neighboring bases. It follows that such "hole migration" is likely to play a much more widespread role in mutagenesis than previously anticipated.
Highlights
From the ‡Department of Molecular Carcinogenesis, Science Park-Research Division, The University of Texas, M
We measured mutation frequencies and spectra in a plasmid reporter system (Fig. 1A) in which the supF gene was preceded by sequences capable of forming either triplex structures of increasing thermal stability or a Z-DNA conformation (Fig. 1B)
These plasmids were transfected into human osteosarcoma U2OS cells containing an integrated plasmid encoding either a control shRNA (WT) or a targeting shRNA designed to stably knockdown WRN (WRN-KD) [48]
Summary
Plasmids—Plasmids pCEX, pMEXy, pMEXr, pGG32y, and pSCG14 are derivatives of plasmid pSP189 in which inserts (Fig. 1A) were cloned between the EcoRI and XhoI restriction sites. Mutation Screening—To determine supF reporter-gene mutation frequencies and spectra, plasmids were extracted from WT and WRN-KD cells and transformed in the indicator E. coli strain MBM7070. The mutations induced by non-B DNA conformations were assessed by comparing the mutation frequencies and spectra obtained with sequences cloned upstream of the supF gene (Fig. 1A), which included: 1) a control sequence (pCEX) unable to form any known non-B DNA structure; 2) two sequences from the human c-MYC promoter (pMEXy and pMEXr) capable of adopting metastable triplex structures (Fig. 1B); and 3) two sequences (pGG32y and pSCG14) predicted to fold into stable triplex and Z-DNA conformations (Fig. 1B), respectively. Databases and Statistical Analyses—A data set of 54,422 germline missense and nonsense mutations was retrieved from the Human Gene Mutation Database [52] These mutations, as well as those mutations identified in the WT and WRN-KD cells, were recategorized according to their occurrence in the 10 possible alternative dinucleotide pairs. Differences in the numbers of transversions and transitions generated were assessed by means of McNemar’s test between two correlated proportions
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