Development of thermotolerance requires interaction between polymerase-beta and heat shock proteins

Abstract

Although heat shock proteins (HSP) are well known to contribute to thermotolerance, they only play a supporting role in the phenomenon. Recently, it has been reported that heat sensitivity depends on heat-induced DNA double-strand breaks (DSB), and that thermotolerance also depends on the suppression of DSB formation. However the critical elements involved in thermotolerance have not yet been fully identified. Heat produces DSB and leads to cell death through denaturation and dysfunction of heat-labile repair proteins such as DNA polymerase-beta (Pol beta). Here the authors show that thermotolerance was partially suppressed in Pol beta(-/-) mouse embryonic fibroblasts (MEF) when compared to the wild-type MEF, and was also suppressed in the presence of the HSP inhibitor, KNK437, in both cell lines. Moreover, the authors found that heat-induced gamma H2AX was suppressed in the thermotolerant cells. These results suggest that Pol beta at least contributes to thermotolerance through its reactivation and stimulation by Hsp27 and Hsp70. In addition, it appears possible that fewer DSB were formed after a challenging heat exposure because preheat-induced Hsp27 and Hsp70 can rescue or restore other, as yet unidentified, heat-labile proteins besides Pol beta. The present novel findings provide strong evidence that Pol beta functions as a critical element involved in thermotolerance and exerts an important role in heat-induced DSB.

Figure 1

Heat shock protein (HSP) accumulation and binding to polymerase‐β (Polβ) in the nucleus. H1299 cells were treated at various intervals after a preheating treatment (45.5°C, 5 min). (a) Thermotolerance ratio (TTR). (b) HSP bands in Western blot analysis. Lane 1, untreated control samples. Lanes 2–6, samples at various periods after a heat treatment alone (45.5°C, 5 min): lane 2, 1.5 h; lane 3, 3 h; lane 4, 6 h; lane 5, 12 h; lane 6, 24 h. (c) Density of HSP bands with Western blot analysis. (j) () Hsp27. () Hsp70. (d) Co‐immunoprecipitation experiments with nuclear extracts. Lane 1, non‐heating treatment; lane 2, 3 h after a preheating treatment alone (45.5°C, 5 min); lane 3, challenging heat treatment (45.5°C, 20 min) at 3 h after a preheating treatment (45.5°C, 5 min); lane 4, challenging heat treatment alone (45.5°C, 20 min).

Figure 2

The effect of polymerase‐β (Polβ) on thermotolerance. Polβ^+/+ and Polβ^−/– mouse embryonic fibroblasts (MEF) were heated (45.5°C) after various times at intervals (0, 6, 12, or 24 h) after a preheating treatment (45.5°C, 5 min). (a) Survival curves. Dotted lines show survival curves at 45.5°C for different heating periods without preheating. Error bars represent ±SD. (b) Thermotolerance ratio (TTR). () Polβ^+/+ MEFs. () Polβ^−/– MEFs. *P < 0.05 and **P < 0.01, by Student's t‐test between Polβ^+/+ and Polβ^−/– MEF, respectively.

Figure 3

The effect of the heat shock protein (HSP) inhibitor, KNK437 on thermotolerance in polymerase‐β (Polβ)^+/+ and Polβ^−/– mouse embryonic fibroblasts (MEF). Polβ^+/+ and Polβ^−/– MEF were heated (45.5°C) at 12 h after a preheating treatment (45.5°C, 5 min) with KNK437. (a) Survival curves. Error bars represent ±SD. (,,) Cells were not conditioned by a preheating treatment. (,,) Cells were conditioned by a preheating treatment. Left panel: Polβ^+/+ MEF. Right panel: Polβ^−/– MEF. (,) 0 µM KNK437. (,) 100 µM KNK437. (,) 300 µM KNK437. (b) Thermotolerance ratios. () Polβ^+/+ MEF. () Polβ^−/– MEF.

Figure 4

Relative γH2AX intensity. Polymerase‐β (Polβ)^+/+ and Polβ^−/– mouse embryonic fibroblasts (MEF) were heated (45.5°C, 20 min) at 12 h after a preheating exposure (45.5°C, 5 min) with or without KNK437. () Polβ^+/+ MEF. () Polβ^−/– MEF.

Figure 5

A model for involvement of polymerase‐β (Polβ) in heat‐induced double‐strand breaks (DSB). Heat induces base modifications through free radical species. Polβ are heat sensitive compared with incision enzymes for excision repair. Heat indirectly induces nicks through inhibition of base excision repair. DSB appears when nicks form in close proximity to each other on both strands through a cell cycle, and a nick is converted into DSB at a DNA replication fork during the S‐phase. When cells are preconditioned, Polβ is protected or reactivated through the interaction with heat shock protein (HSP) and fewer DSB are generated.