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-deficien t 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 polycistro nic operon with the preceding cistrons (orf105a and orf105b). Predicted ami no acid sequences of orf105a and orf105b showed substantial similarity to t he competence-damage inducible protein (cinA gene product) from Streptococc us pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the ge nomic DNA of strain rec30, the mutation site in the strain was identified a s 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. rad iodurans in E. coli without My obvious toxicity or death. The gamma-ray res istance 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 vec tor plasmid. This result is consistent with evidence that RecA proteins fro m many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from stra in rec30 did not complement E, coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair. (C) 1999 Else vier Science B.V. All rights reserved.