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. 2023 Aug 7;12:e88875. doi: 10.7554/eLife.88875

Figure 6. Hsp31 repairs MG-derived AGE modifications on DNA and proteins.

(A) Schematic representation of the experimental procedure used for DNA and protein deglycation reactions. (B, C) DNA deglycation by Hsp31. 500 µM dNTPs (B) or 150 µM forward and reverse primers (C) were incubated without (UN) or with 2 mM MG for 2 hr. Subsequently, 5 µg Hsp31 paralogs were added and incubated for 3 hr. The samples were subjected for PCR analysis. (D, E) Hsp31 reverts MG modification on proteins. hSod1 (D) and Lysozyme (E) were glycated for 2 hr with 2 mM MG and incubated with Hsp31 paralogs. The glycation status was determined through western analysis against anti-MAGE antibody. 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. BSA was used as a negative control in all experiments. The experiments were performed in three independent biological replicates. (F) The dual role of Hsp31 reduces MG-induced DNA mutations. Respective genes were PCR amplified, and Sanger sequenced from strains treated with 10 mM MG. The number of genetic mutations was plotted on GraphPad prism 5.0 (n=2).

Figure 6—source data 1. Source data for Figure 6B contains raw image of agarose gel.
Figure 6—source data 2. Source data for Figure 6C contains raw image of agarose gel.
Figure 6—source data 3. Source data for Figure 6D contains raw image of western blot.
Figure 6—source data 4. Source data for Figure 6E contains raw image of western blot.
Figure 6—source data 5. Source data for Figure 6F contains multiple sequence alignment highlighting the mutations.

Figure 6.

Figure 6—figure supplement 1. Hsp31C138A fails to repair glycated DNA and proteins.

Figure 6—figure supplement 1.

(A–D) Hsp31C138A mutant lacks deglycase activity. Macromolecules were treated with 2 mM MG for 2 hr and incubated with Hsp31-WT and Hsp31C138A mutant (2 µg each). The glycation status was determined through PCR and western analysis. (E) Individual genes, as indicated, were PCR amplified from various strains incubated with 10 mM MG for 16 hr and Sanger sequenced. The number of genetic mutations was determined and represented here.
Figure 6—figure supplement 1—source data 1. Source data for Figure 6—figure supplement 1A contains raw image of agarose gel.
Figure 6—figure supplement 1—source data 2. Source data for Figure 6—figure supplement 1B contains raw image of agarose gel.
Figure 6—figure supplement 1—source data 3. Source data for Figure 6—figure supplement 1C contains raw image of western blot.
Figure 6—figure supplement 1—source data 4. Source data for Figure 6—figure supplement 1D contains raw image of western blot.