DNA polymerase X from Deinococcus radiodurans possesses a structure-modulated 3'-->5' exonuclease activity involved in radioresistance.
Recently a family X DNA polymerase (PolXDr) was identified in the radioresistant bacterium Deinococcus radiodurans. Knockout cells show a delay in double-strand break repair (DSBR) and an increased sensitivity to gamma-irradiation. Here we show that PolXDr possesses 3'-->5' exonuclease activity that stops cutting close to a loop. PolXDr 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. PolXDr 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 DeltapolXDr cells. The wild-type protein could restore the radiation resistance, whereas intriguingly the mutant proteins showed a significant negative effect on survival of gamma-irradiated cells. Taken together our in vivo results suggest that both PolXDr domains play important roles in DSBR in D. radiodurans.
Blasius
Melanie
M
Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, CH 8057 Zürich, Switzerland.
Shevelev
Igor
I
Jolivet
Edmond
E
Sommer
Suzanne
S
Hübscher
Ulrich
U
D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000378:metabolism / Q000528:radiation effects; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034301:Deinococcus / Q000201:enzymology / Q000235:genetics / Q000528:radiation effects*; D005092:Exonucleases / Q000737:chemistry / Q000378:metabolism*; D005720:Gamma Rays; D006642:Histidinol-Phosphatase / Q000235:genetics / Q000378:metabolism; D011836:Radiation Tolerance
D004269:DNA, Bacterial; D004247:DNA; C109534:DNA polymerase X; D004259:DNA-Directed DNA Polymerase; D005092:Exonucleases; D006642:Histidinol-Phosphatase