UV induced mutation frequencies resulting from TLS through CPDs in the cII gene in BBMEFs expressing ATPase defective K577A WRN, 3’→5’ exonuclease defective E84A WRN, ATPase defective K274A WRNIP1 or combinations of these mutant proteins.

UV induced (5 J/m2) mutational spectra resulting from TLS through CPDs by Polη in the cII Gene in BBMEFs expressing K577A WRN, K274A WRNIP1, E84A WRN K274A WRNIP1, or K577A WRN K274A WRNIP1.

(A) Mutational spectra in BBMEFs co-depleted for WRN and Polθ and expressing K577A WRN are shown above the sequence; mutational spectra in BBMEFs co-depleted for WRNP1 and Polθ and expressing K274A WRNIP1 are shown below the sequence. Novel hot spots restricted to K577A WRN are indicated in red lettering and novel hot spots restricted to K274A WRNIP1 are depicted in blue lettering. Green lettering indicates novel shared hot spots that appear in cells expressing either of these mutant proteins. (B) Mutational spectra in BBMEFs co-depleted for WRN and WRNIP1, treated with Polθ inhibitor ART558 (Polθi), and expressing both E84A WRN and K274A WRNIP1 are shown above the sequence, and expressing both K577A WRN and K274A WRNIP1 are shown below the sequence. Novel hot spots that appear in BBMEFs expressing combination of these mutant proteins are demarcated by violet lettering. The designations for the other mutational changes in (A) and (B) are: X, deletions; underlines, tandem mutations.

UV induced (5 J/m2) mutation frequencies resulting from TLS opposite (6-4) photoproducts in the cII Gene in BBMEFs expressing E84A WRN, K577A WRN, K274A WRNIP1, or combinations of these mutant proteins.

(A) UV mutations resulting from TLS opposite (6-4) PPs were examined in a BBMEF cell line expressing a CPD photolyase and photoreactivated with UVA (360 nm) light for 3h. Mutation frequencies and SEM were calculated from 3-4 independent experiments. UV induced mutation frequency(last column) resulting from TLS through (6-4) PPs was calculated by subtracting the spontaneous mutation frequency (14.6 x 10-5) from the mutation frequency in UV irradiated cells. (B) Diagrammatic representation of elevation in error-proneness conferred by E84A WRN, K577A WRN, K274 WRNIP1, or by their combinations upon TLS opposite (6-4) PPs by Polη and Polι. The figure depicts the elevation in UV induced mutation frequencies resulting from TLS opposite (6-4) PPs that occurs in BBMEFs expressing these WRN or WRNIP1 mutant proteins.

UV induced (5 J/m2) mutational spectra resulting from TLS opposite (6-4) PPs by Polη and Polι in the cII gene in BBMEFs expressing K577A WRN, K274A WRNIP1, or E84A WRN K274A WRNIP1.

(A) Mutational spectra in BBMEFs depleted for WRN and expressing K577A WRN are shown above the sequence; mutational spectra in BBMEFs depleted for WRNIP1 and expressing K274A WRNIP1 are shown below the sequence. Novel hot spots restricted to K577A WRN are indicated by red lettering, and novel hot spots restricted to K274A WRNIP1 are indicated in blue lettering. Green lettering indicates novel shared hot spots that appear in cells expressing either of these mutant proteins. Hot spots 1 and 5 in WT cells are also present in cells expressing either of these mutant proteins. However, hot spots 2 and 3 present in WT cells are present only in cells expressing K577A WRN. (B) Mutational spectra in BBMEFs depleted for WRNIP1 and expressing K274A WRNIP1 are shown above the sequence; and mutational spectra in BBMEFs co-depleted for WRN and WRNIP1 and expressing E84A WRN and K274A WRNIP1 are shown below the sequence. Novel hot spots that appear in BBMEFs expressing E84A WRN, K274A WRNIP1 are indicated by violet lettering.

Mutation frequencies and nucleotides inserted opposite a thymine glycol carried on the leading strand DNA template of a duplex plasmid in wild type human fibroblasts or WRN -/- fibroblasts and expressing WRN and/or WRNIP1 mutant proteins

Defects in WRN and WRNIP1 ATPase activities elevate G misinsertions opposite εdA by Polι.

(A) Effects of K274A WRNIP1, E84A WRN, K577A WRN mutations and their combinations on the error-proneness of TLS opposite εdA by Polι. Mutation frequencies and nts inserted opposite εdA carried on the leading strand template of a duplex plasmid in WT HFs or WRN-/- HFs expressing WRN and/or WRNIP1 mutant proteins. aNumbers in parenthesis show the total number of mutations. bThese data have been published previously (Yoon et al. 2024) and are shown here for comparison. (B) Diagrammatic representation of A, G, C, or T insertions opposite εdA by Polι that occur in WRN-/- HFs expressing E84A WRN or expressing E84A, K577A WRN together with K274A WRNIP1.

Roles of WRN ATPase, WRNIP1 ATPase, and WRN 3’→5’ exonuclease activities in the high fidelity of TLS by Y-family Pols.

(A) (i and ii) WRN and WRNIP1 ATPases restrain nt misincorporations by Polη opposite CPDs; (iii) WRN exonuclease removes nt misinsertions opposite CPDs by the Polη multiprotein ensemble. (B) (i and ii) WRN and WRNIP1 ATPases restrain nt misincorporations by Polη or Polι opposite (6-4) PPs. (iii) WRN exonuclease removes nt misinsertions opposite (6-4) PPs by the Polη or Polι multiprotein ensemble. (C) (i) WRN and WRNIP1 ATPases restrain G misinsertions by Polι opposite εdA. (ii) WRN exonuclease removes nt misinsertions opposite εdA by the Polι multiprotein ensemble. (D) (i) WRNIP1 ATPase promotes (↓) WRN exonuclease function in the removal of Polκ misinsertions at the Tg lesion. (ii) WRN exonuclease removes nt misinsertions by the Polκ multiprotein ensemble at the Tg lesion.

TLS frequencies opposite a Tg lesion carried on the leading strand DNA template of a duplex plasmid in WT HFs or WRN-/- HFs expressing WRN and/or WRNIP1 mutant proteins

TLS frequencies opposite an εdA lesion carried on the leading strand DNA template of a duplex plasmid in WT HFs or WRN-/- HFs expressing WRN and/or WRNIP1 mutant proteins

siRNA knockdown efficiency of WRNIP1 and stable expression of WT and mutant WRN and WRNIP1 proteins

(A) (i) Schematic representation of WRNIP1 protein. The positions of UBZ, siRNA target site, the core ATPase domain, and the position of K274A mutation in this domain are indicated

(ii) The sequence of the conserved Walker A motif containing the ATP binding deficient K577A mutation in WRN or K274A mutation in WRNIP1 is shown.

(iii) Western blot analyses of the efficiency of WRNIP1 knockdown in HFs and BBMEFs.

(B) Western blot analyses of stable expression of WT and mutant WRN proteins in WRN-/- HFs (left) and BBMEFs (right).

(C) Western blot analyses of stable expression of WT and mutant WRNIP1 proteins in WT HFs (left) and BBMEFs (right).

(D) Western blot analyses of stable expression of combinations of WRN and WRNIP1 mutant proteins in WRN-/- HFs (left) and BBMEFs (right).

Defects in WRN or WRNIP1 ATPase activity do not impair RF progression through UV lesions

(A) Schematic of DNA fiber assay and representative images of stretched DNA fibers in UV irradiated WRN-/- HFs expressing WT WRN or mutant WRN/WRNIP1 proteins.

(B) Quantitative analyses of RF progression through UV lesions represented as mean CldU/IdU ratio based on ∼400 DNA fibers from four independent experiments. Error bars indicate SD. Student’s two-tailed t test p values: ns, not significant; ** p<0.01.

(C) Distribution of CldU/IdU ratios in % of replication tracts measured in WRN-/- HFs expressing WT WRN or mutant WRN/WRNIP1 proteins. The mean CldU/IdU ratios for these data are shown in (B).

Accumulation of K274A WRNIP1 or K577A WRN into UV induced replication foci

(A) UV induced foci in WT HFs expressing Flag WT WRNIP1 or Flag K274A WRNIP1.

(B) UV induced foci in WRN-/- HFs expressing WT WRN or K577A WRN. In (A) and (B), error bars indicate SD.

Imposition of error-proneness on Polη TLS through CPDs by defects in WRN ATPase, WRNIP1 ATPase, WRN 3’→5’ exonuclease or by their combinations

(A) UV induced mutation frequencies resulting from TLS through CPDs by Polη in the cII gene in BBMEFs expressing E84A WRN, K577A WRN, K274A WRNIP1 or combinations of these mutant proteins. The contribution of Polη to UV induced mutation frequency in BBMEFs defective in WRN ATPase, WRNIP1 ATPase, WRN exonuclease, or in combinations of these activities, is indicated within the bar diagram. The simultaneous absence of WRN ATPase and exonuclease activities and WRNIP1 ATPase activity imposes a mutation frequency of ∼90 x 10-5 upon error-free TLS through CPDs by Polη (Figure S4A, last bar).

(B) Verification that the increase in UV induced mutation frequencies seen in the absence of WRN exonuclease, WRN ATPase, or WRNIP1 ATPase activities in (A) results from the error-proneness imposed upon Polη by the absence of these activities.

(C) Verification that the increase in UV induced mutation frequencies seen in the simultaneous absence of both WRN exonuclease and WRNIP1 ATPase activities or in the absence of both the WRN and WRNIP1 ATPases activities in (A) results from the error-proneness imposed upon Polη by the absence of these activities.

UV induced mutational spectra resulting from TLS through CPDs by Polη in the cII gene in BBMEFs expressing K577A WRN or E84A, K577A WRN

UV induced mutational spectra in BBMEFs co-depleted for WRN and Polθ and expressing K577A WRN are shown above the sequence and expressing E84A, K577A WRN are shown below the sequence.

UV induced mutational spectra resulting from TLS opposite (6-4) PPs by Polη and Polι in the cII gene in BBMEFs expressing both K577A WRN and K274A WRNIP1, or K577A WRN, or E84A, K577A WRN

(A) Mutational spectra in BBMEFs co-depleted for WRN and WRNIP1 and co-expressing K577A WRN and K274A WRNIP1. Red and Blue letters demarcate novel hot spots that appear in cells expressing K577A WRN vs. K274A WRNIP1, respectively; violet lettering demarcates hot spots that appear in cells expressing K577A WRN and K274A WRNIP1 together. Green lettering indicates a hot spot that appears in cells expressing either K577A WRN or K274A WRNIP1.

(B) Mutational spectra in BBMEFs depleted for WRN and expressing K577A WRN or E84A, K577A WRN. Hot spots in E84A, K577A WRN (shown below the sequence) resemble those in K577A WRN (shown above the sequence). Violet lettering indicates a minor novel hot spot in E84A, K577A WRN.