Fig 2: Protein domain architecture and important motifs in human RPA and UNG. (A) Domain structure and orientation of the RPA trimer (RPA1, RPA2, RPA3) bound to ssDNA. DNA-binding (RPA1-A,B,C; RPA2-D) and protein-binding (RPA1-F; RPA2-WH) domains are indicated. (B) Sequence and important motifs in the ∼90 aa N-terminal regulatory domain of UNG2. Binding motifs for PCNA (PIP-box), RPA and cell-cycle regulated phosphorylation sites are indicated. The UNG1 isoforms also contain residues 45–313, including the RPA-binding helix.

Fig 3: Prenatal exposure to SiO2 NPs promoted cell apoptosis by interfering with the DSB repair in female offspring oocytes. A) Representative immunofluorescence images of chromosome spreads of oocytes in meiosis prophase I in fetal ovaries at E17.5, including zygotene, pachytene, and diplotene stages. At zygotene, the synapsis of homologous chromosomes takes place, with complete but unpaired SCP3‐positive elements accompanied by the presence of γH2AX throughout the nucleoplasm. Continuous SCP3 in short stretches of the synaptonemal complex (SCs) indicates that the oocytes are at pachytene. γH2AX is gradually decreased around the pachytene stage. At diplotene, SCs components disintegrated. Anti‐SCP3 (red) was used to indicate SCs, and Anti‐γH2AX (green) was used to label unrepaired DNA damage. Scale bar, 10 µm. B) The distribution of offspring's oocytes across various sub‐stages of meiotic prophase I at E17.5 following exposure to PBS, SiO2 NPs, and SiO2‐COOH NPs treatment (n = 335 to 393 oocytes per group). C) Average fluorescence intensity of γH2AX in oocytes at the pachytene stage (n = 31 oocytes for PBS group, n = 20 oocytes for SiO2 NPs group, n = 33 oocytes for SiO2‐COOH NPs group). D) Immunofluorescence images depicted cytospreading of oocytes at the pachytene, diplotene, and dictyate stages in ovaries of 1 dpp female offspring from PBS, SiO2 NPs, and SiO2‐COOH NPs treatment groups. oocytes enter the dictyate stage with decondensed chromatin. SCs were shown in red (Anti‐SCP3) and unrepaired DNA damage was shown in green (Anti‐γH2AX). Scale bar, 10 µm. E) The distribution of offspring's oocytes across various sub‐stages of meiotic prophase I at 1 dpp following exposure to PBS, SiO2 NPs, and SiO2‐COOH NPs treatment groups (n = 692 to 772 oocytes per group). F) Percentages of γH2AX‐positive oocytes at the diplotene stage (n = 339 to 447 oocytes per group). G) Immunofluorescence staining of SCP3 (red) and RPA2 (green) in oocytes from female fetuses at E17.5 from each group. Scale bar, 10 µm. H) Graphs show quantification of RPA2 foci numbers per cell at the zygotene, pachytene, and diplotene stages (n = 185 to 199 oocytes per group). I) Representative TUNEL‐stained images of ovarian sections of 3 dpp female offspring from PBS, SiO2 NPs, and SiO2‐COOH NPs treatment groups. Green fluorescence indicates TUNEL‐positive signals, and blue fluorescence indicates cell nuclei. Scale bar, 100 µm. J) Mean of TUNEL‐positive nuclei per section (n = 8 sections per group). K) Representative H&E‐stained images of ovarian sections from 3 dpp and 10 dpp female offspring from PBS, SiO2 NPs and SiO2‐COOH NPs treatment groups. Scale bar, 100 µm. L) Mean of primordial follicles in ovarian sections of 3 dpp female offspring from PBS, SiO2 NPs, and SiO2‐COOH NPs treatment groups (n = 3 mice per group). M) Number of follicles in various stages of 10 dpp female offspring from PBS, SiO2 NPs, and SiO2‐COOH NPs treatment dams (n = 3 mice per group). *p < 0.05, **p < 0.01, ***p < 0.001. Data are presented as mean ± s.d. p value was determined by unpaired two‐tailed Student's t‐test between the two groups.

Fig 4: Model showing targeting of UNG to ssDNA regions in replication forks and transcription loops. (A) Recruitment of UNG2 to post-replicative U:A repair is facilitated by binding of the N-terminal PIP-box to PCNA (nascent strands in red). Correspondingly, recruitment of UNG2 to mutagenic, deaminated cytosines in ssDNA template in front of the replicative polymerases (illustrated in lagging strand only) is mediated by binding of the N-terminal helix to the flexible WH domain of RPA2. Targeting to RPA-bound ssDNA in locally melted dsDNA outside of replication forks is also indicated. CMG complex; replicative helicase complex (Cdc45/Mcm2–7/GINS). (B) Hypothetical model illustrating repair of uracil generated by cytosine deamination in replicative ssDNA. White box illustrates known and suspect (HMCES) RPA2-WH -binding proteins. During unperturbed replication, the majority of replicative ssDNA is formed at the lagging strand, which would face the highest risk of cytosine deamination. If not removed prior to encounter by POLD, this would be 100% mutagenic. Similarly, uracil excision from the ssDNA template and fill-in by TLS polymerases would be highly error-prone (red box). These mutagenic events are counteracted by UNG2, which excises the uracil, and by HMCES, which crosslinks to the AP site and blocks TLS. Blocked replication induces RPA2-WH -dependent recruitment of SMARCAL1, which promotes fork reversal and migration of the AP site into dsDNA ahead of the fork (light green box, right). Prior to further processing, crosslinked HMCES is degraded by the DNA-structure specific protease SPRTN or by proteasomal degradation, thereby facilitating error-free pre-replicative BER. Alternatively, RPA2-WH recruits RAD52 to induce template switching, allowing dGMP insertion across the AP site by employing the nascent leading strand as template (light green box, left). HJ resolution would then allow post-replicative BER. (C) RPA-mediated targeting of AID and UNG to ssDNA regions at transcription sites (e.g. variable and switch regions of Ig loci in B cells).

Fig 5: Expression of HR-associated proteins, including PARP-1, γH2AX and RAD51, significantly correlated with the prognosis of 123 patients with STS. A. Representative images of immunohistochemistry (IHC) staining (positive and negative expression) for PARP-1, γH2AX, RAD51, and RPA32 in samples from patients with fibrosarcoma (FS), synovium sarcoma (SS), leiomyosarcoma (LMS), liposarcoma (LS), and rhabdomyosarcoma (RMS), respectively. (400×, positive: brown). B. Bar chart showing the number of patients with positive and negative staining for PARP-1, γH2AX, RAD51, and RPA32 IHC. C-F. Log-rank tests showed that the patients in the high RAD51 expression group had better event-free survival (EFS; events were defined as local recurrence, distant metastasis, lung metastasis, and death; p < 0.0001), while patients in the high PARP-1 and γH2AX expression groups had worse EFS (p < 0.001); RPA32 expression was not associated with the prognosis of the STS patients (p = 0.9584). The IHC scores for staining extent were evaluated as follows: 0: 0% of cells showing staining, 1: < 5% of cells showing staining, 2: 5- 50% of cells showing staining, and 3: over 50% of cells showing staining. Staining intensity was scored as 0: negative, 1: weak, 2: intermediate, and 3: strong. The final score was determined as the sum of both the extent and intensity scores. Based on the final score, patients were divided into low (scores 0 and 2) and high (3- 6) expression groups. EFS: event-free survival; FS: fibrosarcoma; SS: synovial sarcoma; LMS: leiomyosarcoma; LS: liposarcoma; RMS: rhabdomyosarcoma.