Regulatory Proteins Of RecA In Deinococcus Radiodurans

This study is part of an effort to understand the radioresistant mechanism in Deinococcus radiodurans. D. radiodurans is well known as one of the most radioresistant organisms and survives acute exposures to γ-ray radiation that exceed 15 kGy without lethality or induced mutation. It has been revealed that its remarkable radioresistance is mainly due to its strong DNA repair ability. This process was thought to be RecA-dependent because RecA protein expression is DNA damage inducible and D. radiodurans lost the radioresistant ability greatly after the disruption of recA. However, little has been known about how RecA functions in recombination pathways, in parallel to what has been described in E. coli, partly due to lack of RecB, C, E, J, O and T proteins homologous to the ones in E. coli. The regulation of RecA in D. radiodurans is still elusive, akhough PprI (or IreE) was suggested playing a critical role in RecA induction. Frequent recombination events initiated by the RecA in stationary growing bacteria and under non-stressed conditions would be deleterious to the genomes. A tight negative regulatory control may be needed. Two lexA analogs (draO344 and dra0074) and a recX (drl310) analog were found in D. radiodurans, which involved in the negative regulation of RecA in E. coli and many other bacteria. Studies on them are helpful in understanding RecA regulation and radioresistant mechanism in D. radiodurans.In E. coli, RecA is induced by DNA damage as a co-protease to facilitate self-cleavage of the LexA. The cleavage of LexA triggers DNA damage response because LexA normally represses most of the DNA damage-inducible genes including recA. D. radiodurans have two LexA analogs and the peptide sequence assay showed that possess conserved LexA domains, including a variant helix-tum-helix DNA-binding motif in N-terminal domain, conserved cleavage site (Ala-Gly), C-terminal domain involved in the cleavage reaction as a proteinase, and autocleavage activity promoted by RecA.However, Narumi et al. proved that D. radiodurans LexA protein (draO344) is not involved in RecA induction following y irradiation. It is intriguing to find the function of another LexA protein (dra0074) in the regulation of RecA in the highly radioresistant bacterium. We constructed a null mutant of gene dra0074 to study its function. Results of the transcriptional and immunoblot assays showed that gene dra0074 is not involved in RecA induction, indicating that recA expression needs no LexA in D. radiodurans. Two-dimensional (2-D) gel electrophoresis further confirmed that the gene is irrelevant to the DNA repair induced by ionizing radiation, but is involved in other metabolic process in D. radiodurans.RecX is a regulator found in many bacteria. Its coding region is located on the same coding strand downstream of recA and the two genes are cotranscribed usually. It was suggested that RecX could regulate the activity of RecA and reduces the effects of RecA overexpression mostly. There is also a recX homologue (drl310) in D. radiodurans located far away recA and its role hasn't been studied to day. In this work, we inactivated and overexpressed recX gene in D. radiodurans, and further assay reveal that D. radiodurans RecX protein not .only negatively regulates RecA expression but also inhibits the recombination and coprotease activities of RecA in the stationary phase cells, showed dual negative regulatory mechanisms of RecX to the RecA functions in D. radiodurans. Under non-stress conditions, RecX was benefit to the stability of D. radiodurans genome. This finding sheds more light on the RecA regulation in D. radiodurans.Reactive oxygen species (ROS) scavenging" is an important contributor to the prevention mechanism because the lethal effect of IR is known to be oxygen enhanced in the presence of oxygen by the generation of oxygen radicals and hydrogen peroxide that damage cell membranes, proteins, and nucleic acids. The absence of RecX resulted in the elevation of the antioxidarit activities in D. radiodurans, and further assays showed that RecX has no effect the enzyme activity but a negative effect on the expression of SOD and CAT. Although it is not clear that whether RecX mediated antioxidant regulation is an indirect result of RecX mediation of RecA activity, RecA, CAT, and SOD are all...