Fig. 1: DONSON and FANCM operate in separate pathways to promote replication traverse. | Nature Communications

Fig. 1: DONSON and FANCM operate in separate pathways to promote replication traverse.

From: DONSON and FANCM associate with different replisomes distinguished by replication timing and chromatin domain

Fig. 1

a HeLa cells were treated with siRNA against DONSON or FANCM or both. They were exposed to Dig-TMP/UVA and incubated with CldU, then IdU. Fibers were prepared and patterns displayed by immunofluorescence against the analogues and immunoquantum detection (Q dot 655, in red) for Dig-tagged ICLs. Representative patterns are shown. b Quantitation of pattern distribution. Fibers with ICL encounters: NT = 417; siDONSON = 432; siFANCM = 417; siDONSON + siFANCM = 385, from three independent replicates. Data are mean ± s.d. c IP immunoblot of chromatin proteins from cells expressing GFP (panels 1, 2) or GFP-DONSON (panels 3, 4) exposed to UVA (−) or TMP/UVA (+). The identity of the proteins is indicated. The amounts of PSF1 and CDC45 in the two samples were quantitated. Representative blot (n = 3). Data are mean ± s.d. d PLA test of the influence of ATR inhibition on GFP-DONSON interactions with pMCM2S108, MCM2, and MCM5. Number of nuclei: PLA between GFP-DONSON and pMCM2 in cells treated with UVA = 58, TMP/UVA = 94, TMP/UVA + ATRi = 55; PLA between GFP-DONSON and MCM2 in UVA = 95, TMP/UVA = 89, TMP/UVA + ATRi = 93; PLA between GFP-DONSON and MCM5 in UVA = 88, TMP/UVA = 79, TMP/UVA+ATRi = 73; from three biological replicates. Data are mean ± s.e.m. e PLA assessing the influence of ATR inhibition on GFP-DONSON interactions with CDC45 and PSF1. Scored nuclei of PLA between GFP-DONSON and CDC45 in UVA = 70, TMP/UVA = 71, TMP/UVA + ATRi = 73; scored nuclei of PLA between GFP-DONSON and PSF1 in UVA = 71, TMP/UVA = 64, TMP/UVA + ATRi = 77; from three biological replicates. Data are mean ± s.e.m. f Influence of ATR inhibition on the PLA between GFP-DONSON and Dig-tagged ICLs. Scored nuclei: Vehicle = 72, ATRi = 87, three biological replicates. Data are mean ± s.e.m. For replication pattern frequency experiments and Western blotting image analysis (a, c), a two-sided unpaired t test was used to calculate P-values. For PLA experiments (d–f), a two-sided Mann–Whitney rank-sum test was used to determine if differences were significant. NS: not significant: P > 0.05. Source data are provided as a Source Data file.

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