Figure 5. In vitro deglycation of DNA and proteins, and genotoxic sensitivity in the absence of yeast DJ-1 orthologs.

(A) Schematic representation of the experimental procedure followed for DNA and protein deglycation reactions. (B, C) Hsp31 paralogs deglycate DNA. 500 µM dNTPs (B) or 150 µM forward and reverse primers (C) were incubated without (UN) or with 2 mM GO for 2 hr, followed by 3 hr incubation with 5 µg Hsp31 paralogs. The samples were examined for deglycation through PCR. (D, E) Yeast DJ-1 members repair glycated proteins. 2 µg of purified protein hSod1 (D) or Lysozyme (E) were treated without (UN) or with 2 mM GO for 2 hr, and the reactions were further incubated for 3 hr with Hsp31 paralogs. Anti-CML antibody was used to determine glycation levels. BSA was used as a negative control in all experiments. 10 kb DNA ladder was used as a marker (M) for DNA gels, and the Ponceau S stain indicates the equal loading of protein samples. (F) Hsp31 paralogs attenuate genetic mutations. Individual genes were PCR amplified, and Sanger sequenced from the isolated genome of strains treated with 15 mM GO. The number of genetic mutations was calculated and plotted on GraphPad prism 5.0 (n=2). (G) Growth phenotypic analysis. Cells grown until the mid-log phase were spotted on plates containing 0.01% MMS or 15 mM GO or 0.01% MMS and 15 mM GO. The plates were incubated at 30 °C and imaged at 36 hr. (H) Western analysis of RNR3 levels. WT and ΔQ grown till the mid-log phase were supplemented with 0.01% MMS, and 15 mM GO in the culture media and further incubated for 3 hr. Subsequently, RNR3 levels were probed using an anti-HA antibody. (I) RAD52 foci formation. Cells were grown until the mid-exponential phase and were treated without (-) or with 0.03% MMS and 15 mM GO for 1 hr. Subsequently, cells were imaged using a confocal microscope (Olympus FV3000). Representative images have a 10 µm scale. All the experiments were performed in three independent biological replicates.

Figure 5—figure supplement 1. GO-associated macromolecular deglycation, mutation frequency profile, and genotoxic damage.

(A, B) Deglycation of DNA by C138A mutants of Hsp31 paralogs. dNTPs (500 µM) and DNA oligonucleotides (150 µM) were incubated for 2 hr in the absence and presence of 2 mM GO, followed by deglycation with Hsp31 paralogs and C138A mutants for 3 hr. (C, D) C138 amino acid residue is crucial for protein deglycase activity. 2 µg glycated hSod1 and Lysozyme were individually supplemented with 5 µg of Hsp31 paralogs (WT), or mutant paralogs (C138A) and subjected to western analysis to determine the glycation status (E) Respective genes were PCR amplified from cells treated with 15 mM GO for 16 hr and Sanger sequenced. The number of genetic mutations from various genes is represented here. (F) Phenotypic analysis under DNA damaging agents. The cells harvested from the mid-log phase were spotted on plates containing 0.01% MMS or 150 mM HU. Plates were incubated at 30 °C and imaged at 36 hr. (G) Quantitation of RAD52 foci formation. Foci of individual strains were quantitated from three independent biological replicates, statically analysed through paired t-test and plotted on Prism GraphPad 5.0.