Fig 2: ERCC1-XPF and MSH2-MSH3 are required for Z-DNA-induced mutagenesis in human cells.a Schematic of blue-white mutation assay (adapted from Vasquez et al.61). b Mutation frequencies for WT, XPF-deficient, siRNA control, and siRNA-depleted MSH2 human cell lines. Student’s t-test was used to calculate statistical values. c Representative ChIP analysis performed on (pUCON and pUCG14) plasmids in WT and XPF-deficient human cells using antibodies against NER (top panel) and MMR (middle panel) proteins, and in MSH2-depleted cells (bottom panel). IgG and H3 served as negative and positive controls, respectively. d Quantification of ChIP analysis comparing the Z-DNA-forming sequence to the control B-DNA region (Z/C), normalized to input. Data in b, d are presented as means ± SEM of triplicate experiments, and values of each repeat are shown as dot plots on the bars. See also Supplementary Figs. 3–7, and Supplementary Table 1.

Fig 3: Proposed mechanism for Z-DNA-induced genetic instability in eukaryotes.During DNA metabolic processes, the CG repeat sequence is unwrapped from histones (blue circles) and negative supercoiling is generated, which stimulates Z-DNA formation. The structure of Z-DNA is recognized as “damage” by the MSH2-MSH3 complex, signaling repair. The ERCC1-XPF complex is recruited to the site for cleavage near the Z-DNA-forming region (processing of the B-Z junction on the right side of Z-DNA is shown in the figure; however, similar processing could also occur at the other B-Z junction on the left, marked as a scissor), resulting in DSBs in an attempt to repair the “damage”. The breaks may be processed in an error-free fashion, or the breaks may be processed in an error-generating fashion resulting in genomic instability in the form of large deletions and translocations, which may contribute to disease etiology.

Fig 4: ERCC1-XPF cleaves near the Z-DNA-forming region.a S1 nuclease assay schematic. b Top panel: SYBR Gold-stained agarose gel demonstrating S1 cleavage at single-stranded regions corresponding to the B-Z junctions on the Z-DNA-forming plasmid (Z), resulting in ~780 bp fragment (red arrow) that is absent in the control B-DNA plasmid (C). Bottom panel: higher exposure of S1 cleavage product. [NOC, nicked open circular; L, linear; SC, supercoiled species]. c PCR primer extension assay schematic using plasmid DNA in human XPF-proficient or XPF-deficient WCE. Purified DNA was used as a template for a primer extension assay using either the left or right primer (green arrows). d PCR products were separated on a 1.5% agarose gel revealing extra cleavage products (red arrows) from the Z-DNA plasmid in XPF-proficient WCE. Plasmid DNA only (P) served as a negative control, and EcoRI (E), restriction at the Z-DNA-forming insert served as a positive control, resulting in ~160 or ~180 bp products (black arrow). Lane 1: template only; lane 2: left primer; and lane 3: right primer. See also Supplementary Table 1. Extension products of >1000 bp resulted from primer “run off” from the template. Shorter extension products resulting from cleavage on plasmids in WCE are indicated by red arrows.

Fig 5: Rad10-Rad1 and Msh2-Msh3 are required for Z-DNA-induced genetic instability in S. cerevisiae.a B-DNA (left panel) and left-handed Z-DNA flanked by two regions of B-DNA (right panel) B-Z junctions are labeled (red arrows). b Schematic of the YAC fragility assay. c Mutation rates calculated as rate of FOAR for WT, rad1?, rad10?, msh2?, and msh3? strains containing control B-DNA or Z-DNA-forming YACs [Student’s t-test was used to calculate a P value. *P < 0.001]. Data are presented as means ± SEM of triplicate experiments, and values of each repeat are shown as dot plots on the bars. d Mutation spectra of FOAR clones from control B-DNA or Z-DNA-forming YACs in the WT strain were determined by sequencing and PCR, and mutants were categorized as either point mutation (PM)/small deletion within the URA3 gene or complete loss of the right arm of the chromosome as a result of a DSB60. “Adjusted FOAR rate and spectra” was calculated by multiplying the percentage of each category (calculated from 30 mutants) by the total FOAR ratio. See also Supplementary Figs. 1 and 2, and Supplementary Tables 1 and 2.