DNA polymerase X from Deinococcus radiodurans possesses a structure‐modulated 3′→5′ exonuclease activity involved in radioresistance

Summary Recently a family X DNA polymerase (PolX Dr ) was identified in the radioresistant bacterium Deinococcus radiodurans . Knockout cells show a delay in double‐strand break repair (DSBR) and an increased sensitivity to γ‐irradiation. Here we show that PolX Dr possesses 3′→5′ exonuclease activity that stops cutting close to a loop. PolX Dr consists of a DNA polymerase X domain (PolXc) and a Polymerase and Histidinol Phosphatase (PHP) domain. Deletion of the PHP domain abolishes only the structure‐modulated but not the canonical 3′→5′ exonuclease activity. Thus, the exonuclease resides in the PolXc domain, but the structure‐specificity requires additionally the PHP domain. Mutation of two conserved glycines in the PolXc domain leads to a specific loss of the structure‐modulated exonuclease activity but not the exonuclease activity in general. The PHP domain itself does not show any activity. PolX Dr is the first family X DNA polymerase that harbours an exonuclease activity. The wild‐type protein, the glycine mutant and the two domains were expressed separately in Δ polX Dr cells. The wild‐type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of γ‐irradiated cells. Taken together our in vivo results suggest that both PolX Dr domains play important roles in DSBR in D. radiodurans .