Figure 3.

USP37 localizes to replication forks and promotes fork stability. (A) USP37-associated proteins identified by BioID-USP37 (Yeh et al., 2015). Proteins showing enrichment over control are shown. Significance as determined by the original study is indicated. Note that not all known interactors scored as significant. (B) T98G cells synchronized in G1 or S-phase after release from serum-withdrawal were subject to biochemical fractionation. The fractions were probed for the indicated proteins. (C) iPOND immunoblot detecting USP37 and other fork components at nascent DNA in unperturbed HCT116 cells and in cells treated with 4mM HU for 2 hours. No click serves as negative control. (D) Analysis of iPOND-mass spec data from (Dungrawala et al, 2015). Protein abundance for components of the replisome, replication stress response proteins and USP37 are shown. For protein complexes the mean abundance of all components is shown. (E) Immunoblot analysis of the indicated proteins in the chromatin fraction with and without replication stress. (F) DNA fiber assay measuring fork restart by adding 4mM HU for 3hrs between Idu and Cldu pulses then calculating the percentage of stalled forks (Idu alone). The bar graph represents the mean and sd percent stalled forks, n>100 forks per condition. Statistical significance was determined by one-way ANOVA with Dunnet’s multiple comparisons test (**p<0.01, ***p<0.001). (G) DNA fiber assay measuring replication fork resection by following Idu and Cldu pulses with 4mM HU for 3hrs and then normalizing Cldu length to Idu. Red lines signify mean CldU:IdU, n>150 forks per condition. Statistical significance was determined by the Mann-Whitney test (***p<0.0001).