Functional Analysis Of SbcC And SbcD Genes In DNA Repair Of Deinococcus Radiodurans

Deinococcus radiodurans has an extraordinary resistance to ionizing radiation (IR) and a wide range of other DNA-damaging conditions, including ultraviolet irradiation, oxidizing agents and desiccation. Ionizing radiation induces DNA double-stranded breaks (DSBs) that are the most lethal form of DNA damage. The radiation resistance phenotype of D. radiodurans is largely contributed by its extraordinary capacity for DSBs repair. But the molecular mechanisms underlying this phenotype remain poorly understood.Based on analysis of the genome sequence of D. radiodurans, functional homologs of essentially all of the DNA repair genes identified in D. radiodurans R1 have been identified in other prokaryotic species. Compared to the genes taking part in the recombinational repair in E. coli, the RecBC, RecET and SbcB recombinases are missing. Therefore, the RecBC and RecET pathways do not occur at all in D. radiodurans. Instead, only the SbcCD and RecFOR pathways are present potentially to participate in DSBs repair. However, the authenticated function of SbcCD and RecFOR in D. radiodurans is still unknown.Based on amino acid sequence analysis, the SbcD and SbcC proteins are highly conserved in all organisms and belong to the Mre11-Rad50 protein family identified in all kingdoms of life. In eukaryotes, Mre11 and Rad50 are the orthologs of SbcD and SbcC, respectively. Mre11 and Rad50 together with Nbs1 constitute the Mre11-Rad50-Nbs1 (MRN) complex. The MRN complex , which resides at the crossroads of DNA repair and checkpoint signaling, rapidly forms prominent foci at damage sites following DSB induction. This complex carries out the initial processing of the DSB ends. Mutations in the genes that encode components of this complex result in DNA-damage hypersensitivity, genomic instability, telomere shortening, and aberrant meiosis. Therefore, the SbcCD complex might play a key role in homologous recombination (HR) to repair DSBs and other DNA damages in D. radiodurans.In this study, drSbcD (DR1921) and drSbcC (DR1922) were chosen to investigate their genetic functions in vivo, enzyme characteristics in vitro, and the changes of the global transcriptome profiles by the bioinformatic method and biochemical and molecular biological method. The status of drSbcCD complex in DNA metabolism and repair would be reestimated. The main work and its results are appended following:1. The null mutant D was constructed via the drsbcD gene was disrupted by direct reverse-orientation insertional mutagenesis technology. The disrupted drsbcD gene remarkably increases the cells' sensitivity to various types of DNA damaging agents, such as ionizing radiation, ultraviolet irradiation, hydrogen peroxide, and mitomycin C. Because of the difference of DNA damage mechanisms, the categories of the induced DNA damages were varied. So drSbcD is an important protein to repair many different kinds of DNA damage. Under the normal condition, mutant D grows slowly and its biomass is declined. It means that drsbcD gene is essential for cell growth. The kinetics of reassembly of broken chromosomes after radiation of wildtype R1 and mutant D were compared by PFGE. The result shows that disruption of the drsbcD gene displays a large delay in the restoration of an intact genome following IR. In sum, the drSbcD protein plays a key role in both growth and DNA repair in this organism, especially in repair of DSBs generated after cellular exposure to IR. These results demonstrate that the drSbcD protein plays an important role in maintaining genome stability in D. radiodurans.2. The drSbcC and drSbcD genes were cloned and overexpressed in Escherichia coli cells respectively. The nearly homogeneous drSbcC and drSbcD proteins were purified and reconstituted to form a stable complex in vitro. The drSbcCD complex has an ATP-independent 3' to 5' exonuclease activity, and it also has an ATP-independent endonuclease activity. The drSbcCD complex has a secondary structure-specific endonuclease activity. It can cleave the 3' flap DNA substrate and the hairpin DNA. The drSbcCD complex has a 3' to 5' exonuclease activity when the DNA termini are blocked by the proteins. These results suggest that the drSbcCD complex could recognize the DNA fragments in abnormal structure, and its nuclease activities play important roles in DNA metabolism and DNA repair process.3. The microarray method was used to investigate the changes of the global transcriptome profile under normal or IR stress condition. The global transcriptome profile response to drsbcD deletion showed that about 54-gene changed at least 2-fold in mutant D. Among them, 76 percent of up-regulated genes were located within chromosome DRC. Some of these genes were coded as transposases and resolvases. This result means that drsbcD gene can regulate transposation. Furthermore, under the stress condition of 6k Gy IR, the profile expression patterns of many genes (e.g., dnlJ,gyrB,recA,recD,recN,ssb,uvrA,uvrB,uvrD,recG,ddrB, ddrC, ddrD and pprA) were similar to acute gamma - irradiation and prolonged desiccation. This means that under stress condition drSbcD protein can bind to DNA damage sites and release the inhibitive position. Maybe the insertion sequences accelerate the HR processing to reconstruct the intact genome.Taken together, the drSbcD and drSbcC proteins can reconstruct the drSbcCD complex by themselves. The drSbcCD complex act as a multi-functional factor in maintaining the genome stability and contributing the extraordinary ability to D. radiodurans.