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: Characterization of nucleotide excision repair (NER) pathway in VEH- and EDC-MMSCs. A mRNA levels of Xpb, Xpc, Xpf, Ddb1, Ddb2, and Xpd in VEH- and EDC-MMSCs isolated from 5-month-old rats. 18S was used to normalize the expression data. B Representative gel and protein levels of XPA, XPB, XPC, XPF, DDB1, XPD, XPG, and DDB2 in VEH- and EDC-MMSCs isolated from 5-month-old rats. Data were normalized by the amount of β-actin protein levels. C IHC images (×20 magnification, insets are at ×40 magnification) of XPA and XPC in myometrial tissues from 5-month-old Eker rats treated neonatally with VEH or EDC. Scale bar 200 µm. Data are shown as mean ± S.E.M. from triplicate data. *p < 0.05, **p < 0.01, Student’s t test. EDC endocrine-disrupting chemical, MMSCs myometrial stem cells, XP xeroderma pigmentosum, DDB1/2 DNA damage-binding protein 1/2

Fig 4: 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 5: Xeroderma pigmentosum complementation group F (XPF)-positive cells in the observation group.