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.
Institute of Veterinary Biochemistry and Molecular Biology, University of Zürich-Irchel, Winterthurerstrasse 190, CH 8057 Zürich, Switzerland.
Melanie
M
Blasius
Igor
I
Shevelev
Edmond
E
Jolivet
Suzanne
S
Sommer
Ulrich
U
Hübscher
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