Characterization of a DNA damage-inducible membrane protein kinase from Deinococcus radiodurans and its role in bacterial radioresistance and DNA strand break repair.
Deinococcus radiodurans mutant lacking pyrroloquinoline-quinone (PQQ) synthesis shows sensitivity to γ-rays and impairment of DNA double strand break repair. The genome of this bacterium encodes five putative proteins having multiple PQQ binding motifs. The deletion mutants of corresponding genes were generated, and their response to DNA damage was monitored. Only the Δdr2518 mutant exhibited higher sensitivity to DNA damage. Survival of these cells decreased by 3-log cycle both at 6 kGy γ-rays and 1200 Jm(-2) UV (254 nm) radiation, and 2.5-log cycle upon 14 days desiccation at 5% humidity. The Δdr2518 mutant showed complete inhibition of DSB repair until 24 h PIR and disappearance of a few phosphoproteins. The Δdr2518pqqE:cat double mutant showed γ-ray sensitivity similar to Δdr2518 indicating functional interaction of these genes in D. radiodurans. DR2518 contains a eukaryotic type Ser/Thr kinase domain and structural topology suggesting stress responsive transmembrane protein. Its autokinase activity in solution was stimulated by nearly threefold with PQQ and twofold with linear DNA, but not with circular plasmid DNA. More than 15-fold increase in dr2518 transcription and several-fold enhanced in vivo phosphorylation of DR2518 were observed in response to γ irradiation. These results suggest that DR2518 as a DNA damage-responsive protein kinase plays an important role in radiation resistance and DNA strand break repair in D. radiodurans.
Rajpurohit
Yogendra S
YS
Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400 085, India.
Misra
Hari S
HS
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004269:DNA, Bacterial / Q000096:biosynthesis; D034301:Deinococcus / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D005720:Gamma Rays; D017353:Gene Deletion; D045542:PQQ Cofactor / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011836:Radiation Tolerance
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D045542:PQQ Cofactor