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. 2011 Mar 25;286(12):10017-26.
doi: 10.1074/jbc.M110.176636. Epub 2011 Feb 1.

Non-B DNA-forming sequences and WRN deficiency independently increase the frequency of base substitution in human cells

Albino Bacolla  1 , Guliang Wang, Aklank Jain, Nadia A Chuzhanova, Regina Z Cer, Jack R Collins, David N Cooper, Vilhelm A Bohr, Karen M Vasquez
Affiliations

Non-B DNA-forming sequences and WRN deficiency independently increase the frequency of base substitution in human cells

Albino Bacolla et al. J Biol Chem. .

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.

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Figures

FIGURE 1.
FIGURE 1.
Overview of the experimental strategy. A, map of the parental pSP189 shuttle vector. The DNA sequences cloned upstream of the supF gene between the EcoRI (E) and XhoI (X) restriction sites are shown. X with a line through it denotes an inactivated XhoI site introduced following cloning. B, representative triplex and Z-DNA structures formed by the cloned inserts in pMEXy, pGG32y, and pSCG14. Only one of four possible isomers is shown, viz. the 3′-YRR conformer, in which the 3′ purine-rich strand of the purine·pyrimidine tract with mirror repeat symmetry engages in the triplex structure. The potential triplex structures formed by pMEXr are the same as for pMEXy. Bullets, Watson-Crick base-pairs; thick lines, Hoogsteen base-pairs; italics, Z-DNA region. C, schematic of the cloverleaf structure and sequence composition, including commonly modified bases, of the su−tRNATyr, the product of the supF gene. Bold type, invariably conserved residues in tRNAs; p, phosphate group at the 5′ end. D, scheme of the chromosomal lacZ gene in the indicator E. coli strain MBM7070, showing the presence (not to scale) of the relevant TAG amber stop codon (top). Upon transformation of MBM7070 cells with the supF-containing plasmids (A), initiation of lacZ gene transcription (arrow), activated by exogenous IPTG, yields fully translated mRNA products in which amber stop codons are read by su−tRNATyr molecules (middle). The encoded β-galactosidase (bottom) hydrolyzes the culture media supplemented with 5-bromo-4-chloro-indoyl-β-galactoside (X-gal), which upon condensation yields 5-bromo-4-chloro-indigo, conferring a blue color upon bacterial colonies. E, representative Western blot and quantitation of the WRN and PCNA proteins expressed in U2OS whole cell extracts. WT, cells harboring the integrated pshSCR plasmid, containing a scrambled shRNA insert; WRN-KD, WRN knockdown cells harboring the integrated pshWRN plasmid containing a WRN-targeting shRNA insert. The graph shows the fraction of WRN protein expressed in WRN-KD relative to WT cells normalized to the amount of PCNA (means ± S.D. from four independent experiments).
FIGURE 2.
FIGURE 2.
Mutation frequencies and spectra in WT and WRN-KD cells. A, fractions of white (mutant) colonies to the total number of colonies for the indicated plasmids (x axis) isolated from WT cells (black) and WRN-KD cells (gray). B, statistical z-test pair-wise comparisons of the data from A. Solid lines, p < 0.001; dashed line, 0.01 < p < 0.05. C, as in A for WT cells; 374 mutant (white) colonies were obtained from a total of 915,386 colonies. The mutation frequencies were 1.4 × 10−4 ± 1.0 × 10−5 (mean, S.E.) for pCEX, 3.2 × 10−4 ± 5.8 × 10−5 for pMEXy, 3.0 × 10−4 ± 5.0 × 10−5 for pMEXr, 8.8 × 10−4 ± 2.2 × 10−4 for pGG32y, and 1.2 × 10−3 ± 1.6 × 10−4 for pSCG14; ***, p < 0.001; *, p = <0.001- 0.023; pair-wise comparisons between pGG32y (or pSCG14) and pCEX (top line), pMEXy (middle line), and pMEXr (bottom line) are shown. D, mutation spectra of selected mutant colonies. Black, single nucleotide changes in the supF gene; white, deletions disrupting the supF gene.
FIGURE 3.
FIGURE 3.
SupF-inactivating single base changes. Sequence composition of the supF gene (below) and base changes (above) found in all sequenced mutant plasmids isolated from WT and WRN-KD U2OS cells. Δ, nucleotide deleted; underlining, double-base substitution; double underlining, GA·TC dinucleotides.
FIGURE 4.
FIGURE 4.
Oxidative damage and single base changes. A, concentration of 8-oxodG divided by the concentration of dG in genomic DNA of WT and WRN-KD cells (means ± S.D. from three DNA purifications). B, schematic diagram for hole migration showing the abstraction of an electron (e−) (step 1) by a radical species in the solvent (not shown) from a base downstream of the target G in an NGNN sequence, forming a radical cation (hole). The hole migrates to the upstream guanine (step 2) through the reorganization of the outer electron cloud involving base stacking between the interacting bases. A drop in the ionization potential traps the hole at the oxidized guanine, which through multiple steps (step 3) (including DNA replication and repair) eventually gives rise to a mutation. C, correlation between single base changes in the supF gene and base stacking at NGNN sequences. The y axis shows the number of mutations (mean ± S.E.) at G residues (from Fig. 3; C residues were also counted with 3′ → 5′ flanking sequences) in NGNN/NNCN sequences. The x axis shows the average free energy contribution [−ΔG(ν)] to nearest neighbor base stacking in single-stranded DNA for the NG, GN, and NN dinucleotides within NGNN (from Table 4 of Ref. 61) with ϵi = 2]; Y, T or C; H, T, C or A; G, a G residue at either the first, third, or fourth position within NGNN. Inset, site energy (eV) for the nucleobase G in 5′-NGN-3′ (from Table 2 of Ref. 55). HGAH = YGAY+YGAA+AGAY. D, the fractions of single nucleotide variants from a lung cancer genome (53) at each of the 64 possible NGNN/NNCN tetranucleotide sequences (compared with the reference human genome assembly hg19) to the number of NGNN/NNCN sequences found within 1 kb (Fkb) of each mutation site were calculated and averaged for the 19 CpG-containing sequences (4.46 ± 1.63 × 10−3) and the 45 non-CpG-containing sequence combinations (2.84 ± 1.02 × 10−3) (p = 0.00051). For the data normalized to the genome-wide number of NGNN/NNCN sequences (Fgw), the respective fractions were 5.23 ± 2.03 × 10−5 (CpG) and 3.36 ± 1.25 × 10−5 (non-CpG); p = 0.00032). E, the Fkb data (D) for the 45 non-CpG-containing sequences (y axis) were plotted against the average base stacking energies for the NGNN sequences (x axis), as determined from Ref. (see C); r = 0.64; p < 0.001. A significant correlation was also obtained when the Fgw fractions were analyzed. Inset, the Fgw data for the combined NGGG/CCCN, NGGN/NCCN, NGAN/NTCN, NGCN/NGCN, and NGTN/NACN were plotted against the ionization potential values of the upstream G (Ref. 54). r = −0.94; p = 0.018.
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