Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30.
Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.
Narumi
I
I
Biotechnology Laboratory, Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, 1233 Watanuki, Takasaki, Japan. narumi@taka.jaeri.go.jp
Satoh
K
K
Kikuchi
M
M
Funayama
T
T
Kitayama
S
S
Yanagisawa
T
T
Watanabe
H
H
Yamamoto
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004260:DNA Repair / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics / Q000528:radiation effects; D004926:Escherichia coli / Q000235:genetics / Q000528:radiation effects; D005720:Gamma Rays; D005798:Genes, Bacterial / Q000528:radiation effects; D005816:Genetic Complementation Test; D006095:Gram-Positive Cocci / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics* / Q000528:radiation effects; D011938:Rec A Recombinases / Q000096:biosynthesis / Q000235:genetics* / Q000528:radiation effects
D004269:DNA, Bacterial; D011938:Rec A Recombinases