Hrq1 function and regulation in ICL repair is distinct from intrastrand crosslink repair.

Abstract

(A) Hrq1 functions in a different pathway as Rev1 and Ubc13 to repair ICL. The indicated yeast strains were five-fold serial diluted onto YPD medium containing DMSO and/or YPD medium containing the indicated amount of MMC. The plates were photographed after 2 days of incubation at 30°C in the dark. (B) Hrq1 protein levels does not decreased upon 100 μg/ml MMC treatment. Exponentially growing cells with Hrq1-9xMYC were incubated with cycloheximide in the presence or absence of 100 μg/ml MMC and/or 0.1% DMSO. Protein extracts were analyzed by western blot for Hrq1 protein levels (α-MYC) or a loading control, Kar2 (α-Kar2) at the indicated time points. Quantification from three separate experiment is shown, with mean and SEM graphed (Raw densitometry data in Sheets S-U in S1 Data). (C) Hrq1 functions in the same pathway as Rad5 and Ubc13 to repair intrastrand adducts. The indicated yeast strains were five-fold serial diluted onto YPD medium before being treated with the indicated dosages of UV-C. The plates were photographed after 2 days of incubation at 30°C in the dark. (D) Hrq1 functions in different pathway as Rev1 to repair intrastrand adducts. The indicated yeast strains were five-fold serial diluted onto YPD medium before being treated with the indicated dosage of UV-C. The plates were photographed after 2 days of incubation at 30°C in the dark. (E) Hrq1 protein levels decreased following UV-C treatment. Exponentially growing Hrq1-9xMYC cells were incubated with cycloheximide then treated with indicated dosage of UV-C. Protein was extracted similarly to (B). Quantification is from three separate experiments with mean and SEM graphed (Raw densitometry data in Sheets V-X in S1 Data). (TIF)</p