Hrq1 protein levels are stabilized following cisplatin exposure by mutating the predicted Hrq1 ubiquitylated lysine residues to arginine.

Abstract

(A) Schematic of Hrq1 with lysines residues that are predicted to be ubiquitylated (K164, K219, K221, K872) or conserved between Hrq1 and RecQL4 (K366, K839, K938) is shown. The helicase domain is indicated in red (287–496 aa, InterPro). (B) Hrq1 protein levels are stabilized in Hrq1-7KR mutant. Cycloheximide chase experiments were performed in Hrq1-3xHA or Hrq1-7KR-6xHA expressing cells. Equal number of cells were collected every 30 minutes for 120 minutes. The experiment was performed in duplicate and a representative image is shown. (C) Overexpression or stabilization of Hrq1 leads to increased recombination following cisplatin treatment (35 μg/ml, 16 hours). Schematic of direct repeat recombination (DRR) assay, where recombinants are measured by formation of LEU+ recombinants (graphed). In this assay, two leu2 heteroalleles are disrupted by insertion of an EcoR1 or BsteII restriction sites, respectively, with an intervening URA3 gene. Restoration of LEU+ can occur by Rad51-dependent gene conversion (GC) measured by URA3+ LEU2+ recombinants whereas Rad51-independent single-strand annealing (SSA) is measured by URA3- LEU2+ recombinants. Nine independent colonies were measured for each experiment and the median value from two experiments (horizontal bar) were plotted (Colony count in S3 and S4 Data). ** represents pS5 Data). * represents p<0.05, ** represents p<0.01.</p