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DNA polymerase X from Deinococcus radiodurans possesses a structure‐modulated 3′→5′ exonuclease activity involved in radioresistance
Author(s) -
Blasius Melanie,
Shevelev Igor,
Jolivet Edmond,
Sommer Suzanne,
Hübscher Ulrich
Publication year - 2006
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1111/j.1365-2958.2006.05077.x
Subject(s) - deinococcus radiodurans , exonuclease , biology , dna polymerase , dna repair , klenow fragment , dna polymerase ii , mutant , microbiology and biotechnology , polymerase , radioresistance , exonuclease iii , dna , genetics , gene , escherichia coli , polymerase chain reaction , cell culture , reverse transcriptase
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 .