Identification and disruption analysis of the recN gene in the extremely radioresistant bacterium Deinococcus radiodurans.
We isolated a radiosensitive mutant strain, KR4128, from a wild-type strain of Deinococcus radiodurans, which is known as a extremely radioresistant bacterium. The gene that restore the defect of the mutant in DNA repair was cloned, and it turned out to be the homolog of the recN gene of Escherichia coli. The recN gene encoded a protein of 58 kDa, and, in its N-terminal region, a potential ATP binding domain was conserved as expected for a prokaryotic RecN protein. An analysis of sequence of the mutant recN gene revealed a G:C to T:A transversion near the 3' end of the coding region. This alteration causes an ochre mutation, and results in the truncation of 47 amino acids from the C-terminal region of the RecN protein. The null mutant of recN gene was constructed by insertional mutagenesis, and it showed substantial sensitivities to various types of DNA damaging agents, indicating that a single defect in the recN gene can directly affect the DNA damage resistant phenotype in D. radiodurans. The recN locus of KR4128 was also disrupted and the disruptant indicated the sensitivity that was indistinguishable from its progenitor. The result indicate that the transversion in the recN gene of KR4128 cells causes a complete loss of function of the RecN protein and thus the C-terminal region of the RecN protein includes domain essential to its function.
Funayama
T
T
Biotechnology Laboratory, Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Japan. funayama@taka.jaeri.go.jp
Narumi
I
I
Kikuchi
M
M
Kitayama
S
S
Watanabe
H
H
Yamamoto
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D004260:DNA Repair / Q000235:genetics / Q000528:radiation effects; D004262:DNA Restriction Enzymes; D003851:Deoxyribonucleases / Q000235:genetics* / Q000302:isolation & purification; D005798:Genes, Bacterial; D006095:Gram-Positive Cocci / Q000235:genetics* / Q000528:radiation effects; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011836:Radiation Tolerance / Q000235:genetics*
D001426:Bacterial Proteins; D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes; C047879:RecN protein, Bacteria