Function and biochemical characterization of RecJ in Deinococcus radiodurans.
The single-stranded DNA-specific nuclease RecJ is found in most bacteria where it is involved in the RecFOR double-stranded break (DSBs) repair pathway. DSBs repair mainly occurs via the RecFOR pathway in Deinococcus radiodurans, a well-known radiation-resistant bacterium. A recJ null mutant was constructed to investigate the role of recJ in D. radiodurans. recJ inactivation caused growth defects and sensitivity to high temperatures. However, the radiation resistance of the recJ mutant was only moderately decreased. The full-length D. radiodurans RecJ (DrRecJ) protein was expressed and purified to further characterize its biochemical properties. DrRecJ possessed a Mn(2+) concentration-dependent nuclease activity where the optimal Mn(2+) concentration was 0.1mM. DrRecJ had a similar activity profile after adding 10mM Mg(2+) to reactions with different Mn(2+) concentrations, indicating that Mn(2+) is a RecJ regulator. Escherichia coli RecJ has no activity on 5' ssDNA tails shorter than 6-nt, but DrRecJ could effectively degrade DNA with a 4-nt 5' ssDNA tail, suggesting that DrRecJ may have a wider range of DNA substrates. Moreover, SSB in D. radiodurans stimulated the DrRecJ exonuclease activity, whereas DdrB inhibited it and provided protection to ssDNA. Overall, our results indicate that recJ is a nonessential gene in D. radiodurans and that the activity of DrRecJ is regulated by Mn(2+) and SSB-DdrB.
Key Laboratory of Chinese Ministry of Agriculture for Nuclear-Agricultural Sciences, Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou, China.
Jiandong
J
Jiao
Liangyan
L
Wang
Wenrong
W
Xia
Mingfeng
M
Li
Hongxing
H
Sun
Guangzhi
G
Xu
Bing
B
Tian
Yuejin
Y
Hua
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D034301:Deinococcus / Q000166:cytology / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D004789:Enzyme Activation / Q000187:drug effects / Q000528:radiation effects; D005090:Exodeoxyribonucleases / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D055786:Gene Knockout Techniques; D008345:Manganese / Q000494:pharmacology; D011836:Radiation Tolerance / Q000235:genetics
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D008345:Manganese; D005090:Exodeoxyribonucleases; C070765:recJ protein, Bacteria